Expression quantitative trait loci and genetic regulatory network analysis reveals that Gabra2 is involved in stress responses in the mouse.

PubMed

Dai, Jiajuan; Wang, Xusheng; Chen, Ying; Wang, Xiaodong; Zhu, Jun; Lu, Lu

2009-11-01

Previous studies have revealed that the subunit alpha 2 (Gabra2) of the gamma-aminobutyric acid receptor plays a critical role in the stress response. However, little is known about the gentetic regulatory network for Gabra2 and the stress response. We combined gene expression microarray analysis and quantitative trait loci (QTL) mapping to characterize the genetic regulatory network for Gabra2 expression in the hippocampus of BXD recombinant inbred (RI) mice. Our analysis found that the expression level of Gabra2 exhibited much variation in the hippocampus across the BXD RI strains and between the parental strains, C57BL/6J, and DBA/2J. Expression QTL (eQTL) mapping showed three microarray probe sets of Gabra2 to have highly significant linkage likelihood ratio statistic (LRS) scores. Gene co-regulatory network analysis showed that 10 genes, including Gria3, Chka, Drd3, Homer1, Grik2, Odz4, Prkag2, Grm5, Gabrb1, and Nlgn1 are directly or indirectly associated with stress responses. Eleven genes were implicated as Gabra2 downstream genes through mapping joint modulation. The genetical genomics approach demonstrates the importance and the potential power of the eQTL studies in identifying genetic regulatory networks that contribute to complex traits, such as stress responses.

A nonparametric mean-variance smoothing method to assess Arabidopsis cold stress transcriptional regulator CBF2 overexpression microarray data.

PubMed

Hu, Pingsha; Maiti, Tapabrata

2011-01-01

Microarray is a powerful tool for genome-wide gene expression analysis. In microarray expression data, often mean and variance have certain relationships. We present a non-parametric mean-variance smoothing method (NPMVS) to analyze differentially expressed genes. In this method, a nonlinear smoothing curve is fitted to estimate the relationship between mean and variance. Inference is then made upon shrinkage estimation of posterior means assuming variances are known. Different methods have been applied to simulated datasets, in which a variety of mean and variance relationships were imposed. The simulation study showed that NPMVS outperformed the other two popular shrinkage estimation methods in some mean-variance relationships; and NPMVS was competitive with the two methods in other relationships. A real biological dataset, in which a cold stress transcription factor gene, CBF2, was overexpressed, has also been analyzed with the three methods. Gene ontology and cis-element analysis showed that NPMVS identified more cold and stress responsive genes than the other two methods did. The good performance of NPMVS is mainly due to its shrinkage estimation for both means and variances. In addition, NPMVS exploits a non-parametric regression between mean and variance, instead of assuming a specific parametric relationship between mean and variance. The source code written in R is available from the authors on request.

A Nonparametric Mean-Variance Smoothing Method to Assess Arabidopsis Cold Stress Transcriptional Regulator CBF2 Overexpression Microarray Data

PubMed Central

Hu, Pingsha; Maiti, Tapabrata

2011-01-01

Microarray is a powerful tool for genome-wide gene expression analysis. In microarray expression data, often mean and variance have certain relationships. We present a non-parametric mean-variance smoothing method (NPMVS) to analyze differentially expressed genes. In this method, a nonlinear smoothing curve is fitted to estimate the relationship between mean and variance. Inference is then made upon shrinkage estimation of posterior means assuming variances are known. Different methods have been applied to simulated datasets, in which a variety of mean and variance relationships were imposed. The simulation study showed that NPMVS outperformed the other two popular shrinkage estimation methods in some mean-variance relationships; and NPMVS was competitive with the two methods in other relationships. A real biological dataset, in which a cold stress transcription factor gene, CBF2, was overexpressed, has also been analyzed with the three methods. Gene ontology and cis-element analysis showed that NPMVS identified more cold and stress responsive genes than the other two methods did. The good performance of NPMVS is mainly due to its shrinkage estimation for both means and variances. In addition, NPMVS exploits a non-parametric regression between mean and variance, instead of assuming a specific parametric relationship between mean and variance. The source code written in R is available from the authors on request. PMID:21611181

Transcriptome Analysis of Early Responsive Genes in Rice during Magnaporthe oryzae Infection.

PubMed

Wang, Yiming; Kwon, Soon Jae; Wu, Jingni; Choi, Jaeyoung; Lee, Yong-Hwan; Agrawal, Ganesh Kumar; Tamogami, Shigeru; Rakwal, Randeep; Park, Sang-Ryeol; Kim, Beom-Gi; Jung, Ki-Hong; Kang, Kyu Young; Kim, Sang Gon; Kim, Sun Tae

2014-12-01

Rice blast disease caused by Magnaporthe oryzae is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early response genes in rice, we utilized the transcriptome analysis approach using a 300 K tilling microarray to rice leaves infected with compatible and incompatible M. oryzae strains. Prior to the microarray experiment, total RNA was validated by measuring the differential expression of rice defense-related marker genes (chitinase 2, barwin, PBZ1, and PR-10) by RT-PCR, and phytoalexins (sakuranetin and momilactone A) with HPLC. Microarray analysis revealed that 231 genes were up-regulated (>2 fold change, p < 0.05) in the incompatible interaction compared to the compatible one. Highly expressed genes were functionally characterized into metabolic processes and oxidation-reduction categories. The oxidative stress response was induced in both early and later infection stages. Biotic stress overview from MapMan analysis revealed that the phytohormone ethylene as well as signaling molecules jasmonic acid and salicylic acid is important for defense gene regulation. WRKY and Myb transcription factors were also involved in signal transduction processes. Additionally, receptor-like kinases were more likely associated with the defense response, and their expression patterns were validated by RT-PCR. Our results suggest that candidate genes, including receptor-like protein kinases, may play a key role in disease resistance against M. oryzae attack.

Stress Sensors and Signal Transducers in Cyanobacteria

PubMed Central

Los, Dmitry A.; Zorina, Anna; Sinetova, Maria; Kryazhov, Sergey; Mironov, Kirill; Zinchenko, Vladislav V.

2010-01-01

In living cells, the perception of environmental stress and the subsequent transduction of stress signals are primary events in the acclimation to changes in the environment. Some molecular sensors and transducers of environmental stress cannot be identified by traditional and conventional methods. Based on genomic information, a systematic approach has been applied to the solution of this problem in cyanobacteria, involving mutagenesis of potential sensors and signal transducers in combination with DNA microarray analyses for the genome-wide expression of genes. Forty-five genes for the histidine kinases (Hiks), 12 genes for serine-threonine protein kinases (Spks), 42 genes for response regulators (Rres), seven genes for RNA polymerase sigma factors, and nearly 70 genes for transcription factors have been successfully inactivated by targeted mutagenesis in the unicellular cyanobacterium Synechocystis sp. PCC 6803. Screening of mutant libraries by genome-wide DNA microarray analysis under various stress and non-stress conditions has allowed identification of proteins that perceive and transduce signals of environmental stress. Here we summarize recent progress in the identification of sensory and regulatory systems, including Hiks, Rres, Spks, sigma factors, transcription factors, and the role of genomic DNA supercoiling in the regulation of the responses of cyanobacterial cells to various types of stress. PMID:22294932

Identification and expression analysis of cold and freezing stress responsive genes of Brassica oleracea.

PubMed

Ahmed, Nasar Uddin; Jung, Hee-Jeong; Park, Jong-In; Cho, Yong-Gu; Hur, Yoonkang; Nou, Ill-Sup

2015-01-10

Cold and freezing stress is a major environmental constraint to the production of Brassica crops. Enhancement of tolerance by exploiting cold and freezing tolerance related genes offers the most efficient approach to address this problem. Cold-induced transcriptional profiling is a promising approach to the identification of potential genes related to cold and freezing stress tolerance. In this study, 99 highly expressed genes were identified from a whole genome microarray dataset of Brassica rapa. Blast search analysis of the Brassica oleracea database revealed the corresponding homologous genes. To validate their expression, pre-selected cold tolerant and susceptible cabbage lines were analyzed. Out of 99 BoCRGs, 43 were differentially expressed in response to varying degrees of cold and freezing stress in the contrasting cabbage lines. Among the differentially expressed genes, 18 were highly up-regulated in the tolerant lines, which is consistent with their microarray expression. Additionally, 12 BoCRGs were expressed differentially after cold stress treatment in two contrasting cabbage lines, and BoCRG54, 56, 59, 62, 70, 72 and 99 were predicted to be involved in cold regulatory pathways. Taken together, the cold-responsive genes identified in this study provide additional direction for elucidating the regulatory network of low temperature stress tolerance and developing cold and freezing stress resistant Brassica crops. Copyright © 2014 Elsevier B.V. All rights reserved.

Mechanical Stress Induces Biotic and Abiotic Stress Responses via a Novel cis-Element

PubMed Central

Walley, Justin W; Coughlan, Sean; Hudson, Matthew E; Covington, Michael F; Kaspi, Roy; Banu, Gopalan; Harmer, Stacey L; Dehesh, Katayoon

2007-01-01

Plants are continuously exposed to a myriad of abiotic and biotic stresses. However, the molecular mechanisms by which these stress signals are perceived and transduced are poorly understood. To begin to identify primary stress signal transduction components, we have focused on genes that respond rapidly (within 5 min) to stress signals. Because it has been hypothesized that detection of physical stress is a mechanism common to mounting a response against a broad range of environmental stresses, we have utilized mechanical wounding as the stress stimulus and performed whole genome microarray analysis of Arabidopsis thaliana leaf tissue. This led to the identification of a number of rapid wound responsive (RWR) genes. Comparison of RWR genes with published abiotic and biotic stress microarray datasets demonstrates a large overlap across a wide range of environmental stresses. Interestingly, RWR genes also exhibit a striking level and pattern of circadian regulation, with induced and repressed genes displaying antiphasic rhythms. Using bioinformatic analysis, we identified a novel motif overrepresented in the promoters of RWR genes, herein designated as the Rapid Stress Response Element (RSRE). We demonstrate in transgenic plants that multimerized RSREs are sufficient to confer a rapid response to both biotic and abiotic stresses in vivo, thereby establishing the functional involvement of this motif in primary transcriptional stress responses. Collectively, our data provide evidence for a novel cis-element that is distributed across the promoters of an array of diverse stress-responsive genes, poised to respond immediately and coordinately to stress signals. This structure suggests that plants may have a transcriptional network resembling the general stress signaling pathway in yeast and that the RSRE element may provide the key to this coordinate regulation. PMID:17953483

Akt/FOXO3a signaling modulates the endothelial stress response through regulation of heat shock protein 70 expression.

PubMed

Kim, Hyo-Soo; Skurk, Carsten; Maatz, Henrike; Shiojima, Ichiro; Ivashchenko, Yuri; Yoon, Suk-Won; Park, Young-Bae; Walsh, Kenneth

2005-06-01

To identify new antiapoptotic targets of the PI3K-Akt signaling pathway in endothelial cells, adenovirus-mediated Akt1 gene transfer and oligonucleotide microarrays were used to examine Akt-regulated transcripts. DNA microarray analysis revealed that HSP70 expression underwent the greatest fold activation of 12,532 transcripts examined in human umbilical vein endothelial cells (HUVEC) transduced with constitutively active Akt1. Akt1 gene transfer increased HSP70 transcript expression by 24.8-fold as determined by quantitative PCR and promoted a dose-dependent up-regulation of HSP70 protein as determined by Western immunoblot analysis. Gene transfer of FOXO3a, a downstream target of Akt in endothelial cells, significantly suppressed both basal and stress-induced HSP70 protein expression. FOXO3a induced caspase-9-dependent apoptosis in HUVEC, and cotransduction with Ad-HSP70 rescued endothelial cells from FOXO3a-induced apoptosis under basal and stress conditions. Our results identify HSP70 as a new antiapoptotic target of Akt-FOXO3a signaling in endothelial cells that controls viability through modulation of the stress-induced intrinsic cell death pathway.

Development and assessment of whole-genome oligonucleotide microarrays to analyze an anaerobic microbial community and its responses to oxidative stress.

PubMed

Scholten, Johannes C M; Culley, David E; Nie, Lei; Munn, Kyle J; Chow, Lely; Brockman, Fred J; Zhang, Weiwen

2007-06-29

The application of DNA microarray technology to investigate multiple-species microbial communities presents great challenges. In this study, we reported the design and quality assessment of four whole genome oligonucleotide microarrays for two syntroph bacteria, Desulfovibrio vulgaris and Syntrophobacter fumaroxidans, and two archaeal methanogens, Methanosarcina barkeri, and Methanospirillum hungatei, and their application to analyze global gene expression in a four-species microbial community in response to oxidative stress. In order to minimize the possibility of cross-hybridization, cross-genome comparison was performed to assure all probes unique to each genome so that the microarrays could provide species-level resolution. Microarray quality was validated by the good reproducibility of experimental measurements of multiple biological and analytical replicates. This study showed that S. fumaroxidans and M. hungatei responded to the oxidative stress with up-regulation of several genes known to be involved in reactive oxygen species (ROS) detoxification, such as catalase and rubrerythrin in S. fumaroxidans and thioredoxin and heat shock protein Hsp20 in M. hungatei. However, D. vulgaris seemed to be less sensitive to the oxidative stress as a member of a four-species community, since no gene involved in ROS detoxification was up-regulated. Our work demonstrated the successful application of microarrays to a multiple-species microbial community, and our preliminary results indicated that this approach could provide novel insights on the metabolism within microbial communities.

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells

PubMed Central

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects. PMID:27547033

DNA Microarray Highlights Nrf2-Mediated Neuron Protection Targeted by Wasabi-Derived Isothiocyanates in IMR-32 Cells.

PubMed

Trio, Phoebe Zapanta; Fujisaki, Satoru; Tanigawa, Shunsuke; Hisanaga, Ayami; Sakao, Kozue; Hou, De-Xing

2016-01-01

6-(Methylsulfinyl)hexyl isothiocyanate (6-MSITC), 6-(methylthio)hexyl isothiocyanate (6-MTITC), and 4-(methylsulfinyl)butyl isothiocyanate (4-MSITC) are isothiocyanate (ITC) bioactive compounds from Japanese Wasabi. Previous in vivo studies highlighted the neuroprotective potential of ITCs since ITCs enhance the production of antioxidant-related enzymes. Thus, in this present study, a genome-wide DNA microarray analysis was designed to profile gene expression changes in a neuron cell line, IMR-32, stimulated by these ITCs. Among these ITCs, 6-MSITC caused the expression changes of most genes (263), of which 100 genes were upregulated and 163 genes were downregulated. Gene categorization showed that most of the differentially expressed genes are involved in oxidative stress response, and pathway analysis further revealed that Nrf2-mediated oxidative stress pathway is the top of the ITC-modulated signaling pathway. Finally, real-time polymerase chain reaction (PCR) and Western blotting confirmed the gene expression and protein products of the major targets by ITCs. Taken together, Wasabi-derived ITCs might target the Nrf2-mediated oxidative stress pathway to exert neuroprotective effects.

Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera

PubMed Central

2014-01-01

Background WRKY transcription factors are one of the largest families of transcriptional regulators in plants. WRKY genes are not only found to play significant roles in biotic and abiotic stress response, but also regulate growth and development. Grapevine (Vitis vinifera) production is largely limited by stressful climate conditions such as cold stress and the role of WRKY genes in the survival of grapevine under these conditions remains unknown. Results We identified a total of 59 VvWRKYs from the V. vinifera genome, belonging to four subgroups according to conserved WRKY domains and zinc-finger structure. The majority of VvWRKYs were expressed in more than one tissue among the 7 tissues examined which included young leaves, mature leaves, tendril, stem apex, root, young fruits and ripe fruits. Publicly available microarray data suggested that a subset of VvWRKYs was activated in response to diverse stresses. Quantitative real-time PCR (qRT-PCR) results demonstrated that the expression levels of 36 VvWRKYs are changed following cold exposure. Comparative analysis was performed on data from publicly available microarray experiments, previous global transcriptome analysis studies, and qRT-PCR. We identified 15 VvWRKYs in at least two of these databases which may relate to cold stress. Among them, the transcription of three genes can be induced by exogenous ABA application, suggesting that they can be involved in an ABA-dependent signaling pathway in response to cold stress. Conclusions We identified 59 VvWRKYs from the V. vinifera genome and 15 of them showed cold stress-induced expression patterns. These genes represented candidate genes for future functional analysis of VvWRKYs involved in the low temperature-related signal pathways in grape. PMID:24755338

Translational arrest due to cytoplasmic redox stress delays adaptation to growth on methanol and heterologous protein expression in a typical fed-batch culture of Pichia pastoris.

PubMed

Edwards-Jones, Bryn; Aw, Rochelle; Barton, Geraint R; Tredwell, Gregory D; Bundy, Jacob G; Leak, David J

2015-01-01

We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR.

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways

PubMed Central

Kanika, Nirmala; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J.; Melman, Arnold; Davies, Kelvin

2010-01-01

Objectives To investigate the role that oxidative stress plays in the development of diabetic cystopathy. Materials and methods Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month-old diabetic rats was carried out using microarray analysis. Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. The activity of protein degradation pathways was assessed using western blot analysis. Results Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10−10). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. Conclusions Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function. PMID:21518418

An efficient method to identify differentially expressed genes in microarray experiments

PubMed Central

Qin, Huaizhen; Feng, Tao; Harding, Scott A.; Tsai, Chung-Jui; Zhang, Shuanglin

2013-01-01

Motivation Microarray experiments typically analyze thousands to tens of thousands of genes from small numbers of biological replicates. The fact that genes are normally expressed in functionally relevant patterns suggests that gene-expression data can be stratified and clustered into relatively homogenous groups. Cluster-wise dimensionality reduction should make it feasible to improve screening power while minimizing information loss. Results We propose a powerful and computationally simple method for finding differentially expressed genes in small microarray experiments. The method incorporates a novel stratification-based tight clustering algorithm, principal component analysis and information pooling. Comprehensive simulations show that our method is substantially more powerful than the popular SAM and eBayes approaches. We applied the method to three real microarray datasets: one from a Populus nitrogen stress experiment with 3 biological replicates; and two from public microarray datasets of human cancers with 10 to 40 biological replicates. In all three analyses, our method proved more robust than the popular alternatives for identification of differentially expressed genes. Availability The C++ code to implement the proposed method is available upon request for academic use. PMID:18453554

An expression database for roots of the model legume Medicago truncatula under salt stress

PubMed Central

2009-01-01

Background Medicago truncatula is a model legume whose genome is currently being sequenced by an international consortium. Abiotic stresses such as salt stress limit plant growth and crop productivity, including those of legumes. We anticipate that studies on M. truncatula will shed light on other economically important legumes across the world. Here, we report the development of a database called MtED that contains gene expression profiles of the roots of M. truncatula based on time-course salt stress experiments using the Affymetrix Medicago GeneChip. Our hope is that MtED will provide information to assist in improving abiotic stress resistance in legumes. Description The results of our microarray experiment with roots of M. truncatula under 180 mM sodium chloride were deposited in the MtED database. Additionally, sequence and annotation information regarding microarray probe sets were included. MtED provides functional category analysis based on Gene and GeneBins Ontology, and other Web-based tools for querying and retrieving query results, browsing pathways and transcription factor families, showing metabolic maps, and comparing and visualizing expression profiles. Utilities like mapping probe sets to genome of M. truncatula and In-Silico PCR were implemented by BLAT software suite, which were also available through MtED database. Conclusion MtED was built in the PHP script language and as a MySQL relational database system on a Linux server. It has an integrated Web interface, which facilitates ready examination and interpretation of the results of microarray experiments. It is intended to help in selecting gene markers to improve abiotic stress resistance in legumes. MtED is available at http://bioinformatics.cau.edu.cn/MtED/. PMID:19906315

An expression database for roots of the model legume Medicago truncatula under salt stress.

PubMed

Li, Daofeng; Su, Zhen; Dong, Jiangli; Wang, Tao

2009-11-11

Medicago truncatula is a model legume whose genome is currently being sequenced by an international consortium. Abiotic stresses such as salt stress limit plant growth and crop productivity, including those of legumes. We anticipate that studies on M. truncatula will shed light on other economically important legumes across the world. Here, we report the development of a database called MtED that contains gene expression profiles of the roots of M. truncatula based on time-course salt stress experiments using the Affymetrix Medicago GeneChip. Our hope is that MtED will provide information to assist in improving abiotic stress resistance in legumes. The results of our microarray experiment with roots of M. truncatula under 180 mM sodium chloride were deposited in the MtED database. Additionally, sequence and annotation information regarding microarray probe sets were included. MtED provides functional category analysis based on Gene and GeneBins Ontology, and other Web-based tools for querying and retrieving query results, browsing pathways and transcription factor families, showing metabolic maps, and comparing and visualizing expression profiles. Utilities like mapping probe sets to genome of M. truncatula and In-Silico PCR were implemented by BLAT software suite, which were also available through MtED database. MtED was built in the PHP script language and as a MySQL relational database system on a Linux server. It has an integrated Web interface, which facilitates ready examination and interpretation of the results of microarray experiments. It is intended to help in selecting gene markers to improve abiotic stress resistance in legumes. MtED is available at http://bioinformatics.cau.edu.cn/MtED/.

Genome-wide analysis of the heat stress response in Zebu (Sahiwal) cattle.

PubMed

Mehla, Kusum; Magotra, Ankit; Choudhary, Jyoti; Singh, A K; Mohanty, A K; Upadhyay, R C; Srinivasan, Surendran; Gupta, Pankaj; Choudhary, Neelam; Antony, Bristo; Khan, Farheen

2014-01-10

Environmental-induced hyperthermia compromises animal production with drastic economic consequences to global animal agriculture and jeopardizes animal welfare. Heat stress is a major stressor that occurs as a result of an imbalance between heat production within the body and its dissipation and it affects animals at cellular, molecular and ecological levels. The molecular mechanism underlying the physiology of heat stress in the cattle remains undefined. The present study sought to evaluate mRNA expression profiles in the cattle blood in response to heat stress. In this study we report the genes that were differentially expressed in response to heat stress using global scale genome expression technology (Microarray). Four Sahiwal heifers were exposed to 42°C with 90% humidity for 4h followed by normothermia. Gene expression changes include activation of heat shock transcription factor 1 (HSF1), increased expression of heat shock proteins (HSP) and decreased expression and synthesis of other proteins, immune system activation via extracellular secretion of HSP. A cDNA microarray analysis found 140 transcripts to be up-regulated and 77 down-regulated in the cattle blood after heat treatment (P<0.05). But still a comprehensive explanation for the direction of fold change and the specific genes involved in response to acute heat stress still remains to be explored. These findings may provide insights into the underlying mechanism of physiology of heat stress in cattle. Understanding the biology and mechanisms of heat stress is critical to developing approaches to ameliorate current production issues for improving animal performance and agriculture economics. © 2013 Elsevier B.V. All rights reserved.

Development of a cDNA microarray to identify gene expression of Puccinellia tenuiflora under saline-alkali stress.

PubMed

Wang, Yucheng; Yang, Chuanping; Liu, Guifeng; Jiang, Jing

2007-08-01

Puccinellia tenuiflora is the main grass species growing in the saline-alkali soil of the Songnen plain in northeastern China, suggesting it has a high tolerance to saline stress. In this study, cDNA microarrays containing 1067 clones of P. tenuiflora were constructed to investigate gene expression patterns resulting from saline-alkali (NaHCO(3)) stress. RNA was extracted from P. tenuiflora treated with 400 mmol L(-1) NaHCO(3) for 6, 12, 24 and 48 h. Untreated (no saline-alkali stress) samples were used as control. A total of 95 transcripts were differentially regulated under the conditions studied, and 38, 35, 25 and 49 genes were differentially expressed with NaHCO(3) stress for 6, 12, 24 and 48h, respectively. Among these, approximately 40% were putative novel or functionally unknown genes, and the remainder function in photosynthesis, cell rescue, defense, transport, metabolism, transcription regulation and protein destination, etc. Analysis of the P. tenuiflora genes demonstrated the complexity of, and differences in, gene expression patterns resulting from different NaHCO(3) stress times. The genetic relationship between P. tenuiflora and other plants was investigated by BlastN analysis. The results showed nearly 20% of the expressed sequence tags from P. tenuiflora shared significant similarities with rice Oryza sativa, an important food crop. The close genetic relationship between these two species suggests that P. tenuiflora may be a good plant model for studying saline-alkali tolerance mechanisms in O. sativa.

Molecular Phylogenetic and Expression Analysis of the Complete WRKY Transcription Factor Family in Maize

PubMed Central

Wei, Kai-Fa; Chen, Juan; Chen, Yan-Feng; Wu, Ling-Juan; Xie, Dao-Xin

2012-01-01

The WRKY transcription factors function in plant growth and development, and response to the biotic and abiotic stresses. Although many studies have focused on the functional identification of the WRKY transcription factors, much less is known about molecular phylogenetic and global expression analysis of the complete WRKY family in maize. In this study, we identified 136 WRKY proteins coded by 119 genes in the B73 inbred line from the complete genome and named them in an orderly manner. Then, a comprehensive phylogenetic analysis of five species was performed to explore the origin and evolutionary patterns of these WRKY genes, and the result showed that gene duplication is the major driving force for the origin of new groups and subgroups and functional divergence during evolution. Chromosomal location analysis of maize WRKY genes indicated that 20 gene clusters are distributed unevenly in the genome. Microarray-based expression analysis has revealed that 131 WRKY transcripts encoded by 116 genes may participate in the regulation of maize growth and development. Among them, 102 transcripts are stably expressed with a coefficient of variation (CV) value of <15%. The remaining 29 transcripts produced by 25 WRKY genes with the CV value of >15% are further analysed to discover new organ- or tissue-specific genes. In addition, microarray analyses of transcriptional responses to drought stress and fungal infection showed that maize WRKY proteins are involved in stress responses. All these results contribute to a deep probing into the roles of WRKY transcription factors in maize growth and development and stress tolerance. PMID:22279089

Molecular phylogenetic and expression analysis of the complete WRKY transcription factor family in maize.

PubMed

Wei, Kai-Fa; Chen, Juan; Chen, Yan-Feng; Wu, Ling-Juan; Xie, Dao-Xin

2012-04-01

The WRKY transcription factors function in plant growth and development, and response to the biotic and abiotic stresses. Although many studies have focused on the functional identification of the WRKY transcription factors, much less is known about molecular phylogenetic and global expression analysis of the complete WRKY family in maize. In this study, we identified 136 WRKY proteins coded by 119 genes in the B73 inbred line from the complete genome and named them in an orderly manner. Then, a comprehensive phylogenetic analysis of five species was performed to explore the origin and evolutionary patterns of these WRKY genes, and the result showed that gene duplication is the major driving force for the origin of new groups and subgroups and functional divergence during evolution. Chromosomal location analysis of maize WRKY genes indicated that 20 gene clusters are distributed unevenly in the genome. Microarray-based expression analysis has revealed that 131 WRKY transcripts encoded by 116 genes may participate in the regulation of maize growth and development. Among them, 102 transcripts are stably expressed with a coefficient of variation (CV) value of <15%. The remaining 29 transcripts produced by 25 WRKY genes with the CV value of >15% are further analysed to discover new organ- or tissue-specific genes. In addition, microarray analyses of transcriptional responses to drought stress and fungal infection showed that maize WRKY proteins are involved in stress responses. All these results contribute to a deep probing into the roles of WRKY transcription factors in maize growth and development and stress tolerance.

Gene expression profiling in gill tissues of White spot syndrome virus infected black tiger shrimp Penaeus monodon by DNA microarray.

PubMed

Shekhar, M S; Gomathi, A; Gopikrishna, G; Ponniah, A G

2015-06-01

White spot syndrome virus (WSSV) continues to be the most devastating viral pathogen infecting penaeid shrimp the world over. The genome of WSSV has been deciphered and characterized from three geographical isolates and significant progress has been made in developing various molecular diagnostic methods to detect the virus. However, the information on host immune gene response to WSSV pathogenesis is limited. Microarray analysis was carried out as an approach to analyse the gene expression in black tiger shrimp Penaeus monodon in response to WSSV infection. Gill tissues collected from the WSSV infected shrimp at 6, 24, 48 h and moribund stage were analysed for differential gene expression. Shrimp cDNAs of 40,059 unique sequences were considered for designing the microarray chip. The Cy3-labeled cRNA derived from healthy and WSSV-infected shrimp was subjected to hybridization with all the DNA spots in the microarray which revealed 8,633 and 11,147 as up- and down-regulated genes respectively at different time intervals post infection. The altered expression of these numerous genes represented diverse functions such as immune response, osmoregulation, apoptosis, nucleic acid binding, energy and metabolism, signal transduction, stress response and molting. The changes in gene expression profiles observed by microarray analysis provides molecular insights and framework of genes which are up- and down-regulated at different time intervals during WSSV infection in shrimp. The microarray data was validated by Real Time analysis of four differentially expressed genes involved in apoptosis (translationally controlled tumor protein, inhibitor of apoptosis protein, ubiquitin conjugated enzyme E2 and caspase) for gene expression levels. The role of apoptosis related genes in WSSV infected shrimp is discussed herein.

Characterization of regulatory pathways in Xylella fastidiosa: genes and phenotypes controlled by algU.

PubMed

Shi, Xiang Yang; Dumenyo, C Korsi; Hernandez-Martinez, Rufina; Azad, Hamid; Cooksey, Donald A

2007-11-01

Many virulence genes in plant bacterial pathogens are coordinately regulated by "global" regulatory genes. Conducting DNA microarray analysis of bacterial mutants of such genes, compared with the wild type, can help to refine the list of genes that may contribute to virulence in bacterial pathogens. The regulatory gene algU, with roles in stress response and regulation of the biosynthesis of the exopolysaccharide alginate in Pseudomonas aeruginosa and many other bacteria, has been extensively studied. The role of algU in Xylella fastidiosa, the cause of Pierce's disease of grapevines, was analyzed by mutation and whole-genome microarray analysis to define its involvement in aggregation, biofilm formation, and virulence. In this study, an algU::nptII mutant had reduced cell-cell aggregation, attachment, and biofilm formation and lower virulence in grapevines. Microarray analysis showed that 42 genes had significantly lower expression in the algU::nptII mutant than in the wild type. Among these are several genes that could contribute to cell aggregation and biofilm formation, as well as other physiological processes such as virulence, competition, and survival.

Effects of chronic restraint stress on body weight, food intake, and hypothalamic gene expressions in mice.

PubMed

Jeong, Joo Yeon; Lee, Dong Hoon; Kang, Sang Soo

2013-12-01

Stress affects body weight and food intake, but the underlying mechanisms are not well understood. We evaluated the changes in body weight and food intake of ICR male mice subjected to daily 2 hours restraint stress for 15 days. Hypothalamic gene expression profiling was analyzed by cDNA microarray. Daily body weight and food intake measurements revealed that both parameters decreased rapidly after initiating daily restraint stress. Body weights of stressed mice then remained significantly lower than the control body weights, even though food intake slowly recovered to 90% of the control intake at the end of the experiment. cDNA microarray analysis revealed that chronic restraint stress affects the expression of hypothalamic genes possibly related to body weight control. Since decreases of daily food intake and body weight were remarkable in days 1 to 4 of restraint, we examined the expression of food intake-related genes in the hypothalamus. During these periods, the expressions of ghrelin and pro-opiomelanocortin mRNA were significantly changed in mice undergoing restraint stress. Moreover, daily serum corticosterone levels gradually increased, while leptin levels significantly decreased. The present study demonstrates that restraint stress affects body weight and food intake by initially modifying canonical food intake-related genes and then later modifying other genes involved in energy metabolism. These genetic changes appear to be mediated, at least in part, by corticosterone.

Translational Arrest Due to Cytoplasmic Redox Stress Delays Adaptation to Growth on Methanol and Heterologous Protein Expression in a Typical Fed-Batch Culture of Pichia pastoris

PubMed Central

Edwards-Jones, Bryn; Aw, Rochelle; Barton, Geraint R.; Tredwell, Gregory D.; Bundy, Jacob G.; Leak, David J.

2015-01-01

Results We have followed a typical fed-batch induction regime for heterologous protein production under the control of the AOX1 promoter using both microarray and metabolomic analysis. The genetic constructs involved 1 and 3 copies of the TRY1 gene, encoding human trypsinogen. In small-scale laboratory cultures, expression of the 3 copy-number construct induced the unfolded protein response (UPR) sufficiently that titres of extracellular trypsinogen were lower in the 3-copy construct than with the 1-copy construct. In the fed-batch-culture, a similar pattern was observed, with higher expression from the 1-copy construct, but in this case there was no significant induction of UPR with the 3-copy strain. Analysis of the microarray and metabolomic information indicates that the 3-copy strain was undergoing cytoplasmic redox stress at the point of induction with methanol. In this Crabtree-negative yeast, this redox stress appeared to delay the adaptation to growth on methanol and supressed heterologous protein production, probably due to a block in translation. Conclusion Although redox imbalance as a result of artificially imposed hypoxia has previously been described, this is the first time that it has been characterised as a result of a transient metabolic imbalance and shown to involve a stress response which can lead to translational arrest. Without detailed analysis of the underlying processes it could easily have been mis-interpreted as secretion stress, transmitted through the UPR. PMID:25785713

Oxidative stress status accompanying diabetic bladder cystopathy results in the activation of protein degradation pathways.

PubMed

Kanika, Nirmala D; Chang, Jinsook; Tong, Yuehong; Tiplitsky, Scott; Lin, Juan; Yohannes, Elizabeth; Tar, Moses; Chance, Mark; Christ, George J; Melman, Arnold; Davies, Kelvin D

2011-05-01

• To investigate the role that oxidative stress plays in the development of diabetic cystopathy. • Comparative gene expression in the bladder of non-diabetic and streptozotocin (STZ)-induced 2-month- old diabetic rats was carried out using microarray analysis. • Evidence of oxidative stress was investigated in the bladder by analyzing glutathione S-transferase activity, lipid peroxidation, and carbonylation and nitrosylation of proteins. • The activity of protein degradation pathways was assessed using Western blot analysis. • Analysis of global gene expression showed that detrusor smooth muscle tissue of STZ-induced diabetes undergoes significant enrichment in targets involved in the production or regulation of reactive oxygen species (P = 1.27 × 10(-10)). The microarray analysis was confirmed by showing that markers of oxidative stress were all significantly increased in the diabetic bladder. • It was hypothesized that the sequelae to oxidative stress would be increased protein damage and apoptosis. • This was confirmed by showing that two key proteins involved in protein degradation (Nedd4 and LC3B) were greatly up-regulated in diabetic bladders compared to controls by 12.2 ± 0.76 and 4.4 ± 1.0-fold, respectively, and the apoptosis inducing protein, BAX, was up-regulated by 6.76 ± 0.76-fold. • Overall, the findings obtained in the present study add to the growing body of evidence showing that diabetic cystopathy is associated with oxidative damage of smooth muscle cells, and results in protein damage and activation of apoptotic pathways that may contribute to a deterioration in bladder function. © 2010 THE AUTHORS; BJU INTERNATIONAL © 2010 BJU INTERNATIONAL.

Alteration of Gene Expression, DNA Methylation, and Histone Methylation in Free Radical Scavenging Networks in Adult Mouse Hippocampus following Fetal Alcohol Exposure.

PubMed

Chater-Diehl, Eric J; Laufer, Benjamin I; Castellani, Christina A; Alberry, Bonnie L; Singh, Shiva M

2016-01-01

The molecular basis of Fetal Alcohol Spectrum Disorders (FASD) is poorly understood; however, epigenetic and gene expression changes have been implicated. We have developed a mouse model of FASD characterized by learning and memory impairment and persistent gene expression changes. Epigenetic marks may maintain expression changes over a mouse's lifetime, an area few have explored. Here, mice were injected with saline or ethanol on postnatal days four and seven. At 70 days of age gene expression microarray, methylated DNA immunoprecipitation microarray, H3K4me3 and H3K27me3 chromatin immunoprecipitation microarray were performed. Following extensive pathway analysis of the affected genes, we identified the top affected gene expression pathway as "Free radical scavenging". We confirmed six of these changes by droplet digital PCR including the caspase Casp3 and Wnt transcription factor Tcf7l2. The top pathway for all methylation-affected genes was "Peroxisome biogenesis"; we confirmed differential DNA methylation in the Acca1 thiolase promoter. Altered methylation and gene expression in oxidative stress pathways in the adult hippocampus suggests a novel interface between epigenetic and oxidative stress mechanisms in FASD.

Microarray Analysis of Tomato Plants Exposed to the Nonviruliferous or Viruliferous Whitefly Vector Harboring Pepper golden mosaic virus

PubMed Central

Musser, Richard O.; Hum-Musser, Sue M.; Gallucci, Matthew; DesRochers, Brittany; Brown, Judith K.

2014-01-01

Abstract Plants are routinely exposed to biotic and abiotic stresses to which they have evolved by synthesizing constitutive and induced defense compounds. Induced defense compounds are usually made, initially, at low levels; however, following further stimulation by specific kinds of biotic and abiotic stresses, they can be synthesized in relatively large amounts to abate the particular stress. cDNA microarray hybridization was used to identify an array of genes that were differentially expressed in tomato plants 15 d after they were exposed to feeding by nonviruliferous whiteflies or by viruliferous whiteflies carrying Pepper golden mosaic virus (PepGMV) ( Begomovirus, Geminiviridae ). Tomato plants inoculated by viruliferous whiteflies developed symptoms characteristic of PepGMV, whereas plants exposed to nonviruliferous whitefly feeding or nonwounded (negative) control plants exhibited no disease symptoms. The microarray analysis yielded over 290 spotted probes, with significantly altered expression of 161 putative annotated gene targets, and 129 spotted probes of unknown identities. The majority of the differentially regulated “known” genes were associated with the plants exposed to viruliferous compared with nonviruliferous whitefly feeding. Overall, significant differences in gene expression were represented by major physiological functions including defense-, pathogen-, photosynthesis-, and signaling-related responses and were similar to genes identified for other insect–plant systems. Viruliferous whitefly-stimulated gene expression was validated by real-time quantitative polymerase chain reaction of selected, representative candidate genes (messenger RNA): arginase, dehydrin, pathogenesis-related proteins 1 and -4, polyphenol oxidase, and several protease inhibitors. This is the first comparative profiling of the expression of tomato plants portraying different responses to biotic stress induced by viruliferous whitefly feeding (with resultant virus infection) compared with whitefly feeding only and negative control nonwounded plants exposed to neither. These results may be applicable to many other plant–insect–pathogen system interactions. PMID:25525099

Assessing the impact of Benzo[a]pyrene on Marine Mussels: Application of a novel targeted low density microarray complementing classical biomarker responses

PubMed Central

Sforzini, Susanna; Arlt, Volker M.; Barranger, Audrey; Dallas, Lorna J.; Oliveri, Caterina; Aminot, Yann; Pacchioni, Beniamina; Millino, Caterina; Lanfranchi, Gerolamo; Readman, James W.; Moore, Michael N.; Viarengo, Aldo; Jha, Awadhesh N.

2017-01-01

Despite the increasing use of mussels in environmental monitoring and ecotoxicological studies, their genomes and gene functions have not been thoroughly explored. Several cDNA microarrays were recently proposed for Mytilus spp., but putatively identified partial transcripts have rendered the generation of robust transcriptional responses difficult in terms of pathway identification. We developed a new low density oligonucleotide microarray with 465 probes covering the same number of genes. Target genes were selected to cover most of the well-known biological processes in the stress response documented over the last decade in bivalve species at the cellular and tissue levels. Our new ‘STressREsponse Microarray’ (STREM) platform consists of eight sub-arrays with three replicates for each target in each sub-array. To assess the potential use of the new array, we tested the effect of the ubiquitous environmental pollutant benzo[a]pyrene (B[a]P) at 5, 50, and 100 μg/L on two target tissues, the gills and digestive gland, of Mytilus galloprovincialis exposed invivo for three days. Bioaccumulation of B[a]P was also determined demonstrating exposure in both tissues. In addition to the well-known effects of B[a]P on DNA metabolism and oxidative stress, the new array data provided clues about the implication of other biological processes, such as cytoskeleton, immune response, adhesion to substrate, and mitochondrial activities. Transcriptional data were confirmed using qRT-PCR. We further investigated cellular functions and possible alterations related to biological processes highlighted by the microarray data using oxidative stress biomarkers (Lipofuscin content) and the assessment of genotoxicity. DNA damage, as measured by the alkaline comet assay, increased as a function of dose.DNA adducts measurements using 32P-postlabeling method also showed the presence of bulky DNA adducts (i.e. dG-N2-BPDE). Lipofiscin content increased significantly in B[a]P exposed mussels. Immunohistochemical analysis of tubulin and actin showed changes in cytoskeleton organisation. Our results adopting an integrated approach confirmed that the combination of newly developed transcriptomic approcah, classical biomarkers along with chemical analysis of water and tissue samples should be considered for environmental bioimonitoring and ecotoxicological studies to obtain holistic information to assess the impact of contaminants on the biota. PMID:28651000

Microarray analysis of the abscission-related transcriptome in tomato flower abscission zone in response to auxin depletion

USDA-ARS?s Scientific Manuscript database

Abscission, the separation of organs from the parent plant, results in postharvest quality loss in many fresh produce. The process is initiated by changes in the auxin gradient across the abscission zone (AZ), is triggered by ethylene and may be accelerated by postharvest stresses. Although changes ...

Differential gene expression of wheat progeny with contrasting levels of transpiration efficiency.

PubMed

Xue, Gang-Ping; McIntyre, C Lynne; Chapman, Scott; Bower, Neil I; Way, Heather; Reverter, Antonio; Clarke, Bryan; Shorter, Ray

2006-08-01

High water use efficiency or transpiration efficiency (TE) in wheat is a desirable physiological trait for increasing grain yield under water-limited environments. The identification of genes associated with this trait would facilitate the selection for genotypes with higher TE using molecular markers. We performed an expression profiling (microarray) analysis of approximately 16,000 unique wheat ESTs to identify genes that were differentially expressed between wheat progeny lines with contrasting TE levels from a cross between Quarrion (high TE) and Genaro 81 (low TE). We also conducted a second microarray analysis to identify genes responsive to drought stress in wheat leaves. Ninety-three genes that were differentially expressed between high and low TE progeny lines were identified. One fifth of these genes were markedly responsive to drought stress. Several potential growth-related regulatory genes, which were down-regulated by drought, were expressed at a higher level in the high TE lines than the low TE lines and are potentially associated with a biomass production component of the Quarrion-derived high TE trait. Eighteen of the TE differentially expressed genes were further analysed using quantitative RT-PCR on a separate set of plant samples from those used for microarray analysis. The expression levels of 11 of the 18 genes were positively correlated with the high TE trait, measured as carbon isotope discrimination (Delta(13)C). These data indicate that some of these TE differentially expressed genes are candidates for investigating processes that underlie the high TE trait or for use as expression quantitative trait loci (eQTLs) for TE.

Transcriptional regulation of defence genes and involvement of the WRKY transcription factor in arbuscular mycorrhizal potato root colonization.

PubMed

Gallou, Adrien; Declerck, Stéphane; Cranenbrouck, Sylvie

2012-03-01

The establishment of arbuscular mycorrhizal associations causes major changes in plant roots and affects significantly the host in term of plant nutrition and resistance against biotic and abiotic stresses. As a consequence, major changes in root transcriptome, especially in plant genes related to biotic stresses, are expected. Potato microarray analysis, followed by real-time quantitative PCR, was performed to detect the wide transcriptome changes induced during the pre-, early and late stages of potato root colonization by Glomus sp. MUCL 41833. The microarray analysis revealed 526 up-regulated and 132 down-regulated genes during the pre-stage, 272 up-regulated and 109 down-regulated genes during the early stage and 734 up-regulated and 122 down-regulated genes during the late stage of root colonization. The most important class of regulated genes was associated to plant stress and in particular to the WRKY transcription factors genes during the pre-stage of root colonization. The expression profiling clearly demonstrated a wide transcriptional change during the pre-, early and late stages of root colonization. It further suggested that the WRKY transcription factor genes are involved in the mechanisms controlling the arbuscular mycorrhizal establishment by the regulation of plant defence genes.

Bifidobacterium breve B-3 exerts metabolic syndrome-suppressing effects in the liver of diet-induced obese mice: a DNA microarray analysis.

PubMed

Kondo, S; Kamei, A; Xiao, J Z; Iwatsuki, K; Abe, K

2013-09-01

We previously reported that supplementation with Bifidobacterium breve B-3 reduced body weight gain and accumulation of visceral fat in a dose-dependent manner, and improved serum levels of total cholesterol, glucose and insulin in a mouse model of diet-induced obesity. In this study, we investigated the expression of genes in the liver using DNA microarray analysis and q-PCR to reveal the mechanism of these anti-obesity effects in this mouse model. Administration of B. breve B-3 led to regulated gene expression of pathways involved in lipid metabolism and response to stress. The results indicate that these regulations in the liver are related to the anti-metabolic syndrome effects of B. breve B-3.

TUSC2(FUS1)-erlotinib Induced Vulnerabilities in Epidermal Growth Factor Receptor(EGFR) Wildtype Non-small Cell Lung Cancer(NSCLC) Targeted by the Repurposed Drug Auranofin.

PubMed

Xiaobo, Cao; Majidi, Mourad; Feng, Meng; Shao, Ruping; Wang, Jing; Zhao, Yang; Baladandayuthapani, Veerabhadran; Song, Juhee; Fang, Bingliang; Ji, Lin; Mehran, Reza; Roth, Jack A

2016-11-15

Expression of the TUSC2/FUS1 tumor suppressor gene in TUSC2 deficient EGFR wildtype lung cancer cells increased sensitivity to erlotinib. Microarray mRNA expression analysis of TUSC2 inducible lung cancer cells treated with erlotinib uncovered defects in the response to oxidative stress suggesting that increasing reactive oxygen species (ROS) would enhance therapeutic efficacy. Addition of the thioredoxin reductase 1 inhibitor (TXNRD1) auranofin (AF) to NSCLC cells treated with combination of TUSC2 forced expression with erlotinib increased tumor cell apoptosis and inhibited colony formation. TXNRD1 overexpression rescued tumors from AF-TUSC2-erlotinib induced apoptosis. Neutralizing ROS with nordihydroguaiaretic acid (NDGA) abrogated cell death induced by AF-TUSC2-erlotinib, indicating a regulatory role for ROS in the efficacy of the three drug combination. Isobologram-based statistical analysis of this combination demonstrated superior synergism, compared with each individual treatment at lower concentrations. In NSCLC tumor xenografts, tumor growth was markedly inhibited and animal survival was prolonged over controls by AF-TUSC2-erlotinib. Microarray mRNA expression analysis uncovered oxidative stress and DNA damage gene signatures significantly upregulated by AF-TUSC2-erlotinib compared to TUSC2-erlotinib. Pathway analysis showed the highest positive z-score for the NRF2-mediated oxidative stress response. Taken together these findings show that the combination of TUSC2-erlotinib induces additional novel vulnerabilities that can be targeted with AF.

Evaluation of the skin irritation using a DNA microarray on a reconstructed human epidermal model.

PubMed

Niwa, Makoto; Nagai, Kanji; Oike, Hideaki; Kobori, Masuko

2009-02-01

To avoid the need to use animals to test the skin irritancy potential of chemicals and cosmetics, it is important to establish an in vitro method based on the reconstructed human epidermal model. To evaluate skin irritancy efficiently and sensitively, we determined the gene expression induced by a topically-applied mild irritant sodium dodecyl sulfate (SDS) in a reconstructed human epidermal model LabCyte EPI-MODEL (LabCyte) using a DNA microarray carrying genes that were related to inflammation, immunity, stress and housekeeping. The expression and secretion of IL-1alpha in reconstructed human epidermal culture is known to be induced by irritation. We detected the induction of IL-1alpha expression and its secretion into the cell culture medium by treatment with 0.075% SDS for 18 h in LabCyte culture using DNA microarray, quantitative reverse-transcription polymerase chain reaction (RT-PCR) and ELISA. DNA microarray analysis indicated that the expression of 10 of the 205 genes carried on the DNA microarray was significantly induced in a LabCyte culture by 0.05% or 0.075% SDS irritation for 18 h. RT-PCR analysis confirmed that SDS treatment significantly induced the expressions of interleukin-1 receptor antagonist (IL-1RN), FOS-like antigen 1 (FOSL1), heat shock 70 kDa protein 1A (HSPA1) and myeloid differentiation primary response gene (88) (MYD88), as well as the known marker genes for irritation IL-1beta and IL-8 in a LabCyte culture. Our results showed that a DNA microarray is a useful tool for efficiently evaluating mild skin irritation using a reconstructed human epidermal model.

Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

PubMed

Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

2011-08-01

The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research.

Microarray Я US: a user-friendly graphical interface to Bioconductor tools that enables accurate microarray data analysis and expedites comprehensive functional analysis of microarray results.

PubMed

Dai, Yilin; Guo, Ling; Li, Meng; Chen, Yi-Bu

2012-06-08

Microarray data analysis presents a significant challenge to researchers who are unable to use the powerful Bioconductor and its numerous tools due to their lack of knowledge of R language. Among the few existing software programs that offer a graphic user interface to Bioconductor packages, none have implemented a comprehensive strategy to address the accuracy and reliability issue of microarray data analysis due to the well known probe design problems associated with many widely used microarray chips. There is also a lack of tools that would expedite the functional analysis of microarray results. We present Microarray Я US, an R-based graphical user interface that implements over a dozen popular Bioconductor packages to offer researchers a streamlined workflow for routine differential microarray expression data analysis without the need to learn R language. In order to enable a more accurate analysis and interpretation of microarray data, we incorporated the latest custom probe re-definition and re-annotation for Affymetrix and Illumina chips. A versatile microarray results output utility tool was also implemented for easy and fast generation of input files for over 20 of the most widely used functional analysis software programs. Coupled with a well-designed user interface, Microarray Я US leverages cutting edge Bioconductor packages for researchers with no knowledge in R language. It also enables a more reliable and accurate microarray data analysis and expedites downstream functional analysis of microarray results.

Microarray analyses reveal novel targets of exercise-induced stress resistance in the dorsal raphe nucleus

PubMed Central

Loughridge, Alice B.; Greenwood, Benjamin N.; Day, Heidi E. W.; McQueen, Matthew B.; Fleshner, Monika

2013-01-01

Serotonin (5-HT) is implicated in the development of stress-related mood disorders in humans. Physical activity reduces the risk of developing stress-related mood disorders, such as depression and anxiety. In rats, 6 weeks of wheel running protects against stress-induced behaviors thought to resemble symptoms of human anxiety and depression. The mechanisms by which exercise confers protection against stress-induced behaviors, however, remain unknown. One way by which exercise could generate stress resistance is by producing plastic changes in gene expression in the dorsal raphe nucleus (DRN). The DRN has a high concentration of 5-HT neurons and is implicated in stress-related mood disorders. The goal of the current experiment was to identify changes in the expression of genes that could be novel targets of exercise-induced stress resistance in the DRN. Adult, male F344 rats were allowed voluntary access to running wheels for 6 weeks; exposed to inescapable stress or no stress; and sacrificed immediately and 2 h after stressor termination. Laser capture micro dissection selectively sampled the DRN. mRNA expression was measured using the whole genome Affymetrix microarray. Comprehensive data analyses of gene expression included differential gene expression, log fold change (LFC) contrast analyses with False Discovery Rate correction, KEGG and Wiki Web Gestalt pathway enrichment analyses, and Weighted Gene Correlational Network Analysis (WGCNA). Our results suggest that physically active rats exposed to stress modulate expression of twice the number of genes, and display a more rapid and strongly coordinated response, than sedentary rats. Bioinformatics analyses revealed several potential targets of stress resistance including genes that are related to immune processes, tryptophan metabolism, and circadian/diurnal rhythms. PMID:23717271

Microarray Meta-Analysis Focused on the Response of Genes Involved in Redox Homeostasis to Diverse Abiotic Stresses in Rice

PubMed Central

de Abreu Neto, Joao B.; Frei, Michael

2016-01-01

Plants are exposed to a wide range of abiotic stresses (AS), which often occur in combination. Because physiological investigations typically focus on one stress, our understanding of unspecific stress responses remains limited. The plant redox homeostasis, i.e., the production and removal of reactive oxygen species (ROS), may be involved in many environmental stress conditions. Therefore, this study intended to identify genes, which are activated in diverse AS, focusing on ROS-related pathways. We conducted a meta-analysis (MA) of microarray experiments, focusing on rice. Transcriptome data were mined from public databases and fellow researchers, which represented 36 different experiments and investigated diverse AS, including ozone stress, drought, heat, cold, salinity, and mineral deficiencies/toxicities. To overcome the inherent artifacts of different MA methods, data were processed using Fisher, rOP, REM, and product of rank (GeneSelector), and genes identified by most approaches were considered as shared differentially expressed genes (DEGs). Two MA strategies were adopted: first, datasets were separated into shoot, root, and seedling experiments, and these tissues were analyzed separately to identify shared DEGs. Second, shoot and seedling experiments were classed into oxidative stress (OS), i.e., ozone and hydrogen peroxide treatments directly producing ROS in plant tissue, and other AS, in which ROS production is indirect. In all tissues and stress conditions, genes a priori considered as ROS-related were overrepresented among the DEGs, as they represented 4% of all expressed genes but 7–10% of the DEGs. The combined MA approach was substantially more conservative than individual MA methods and identified 1001 shared DEGs in shoots, 837 shared DEGs in root, and 1172 shared DEGs in seedlings. Within the OS and AS groups, 990 and 1727 shared DEGs were identified, respectively. In total, 311 genes were shared between OS and AS, including many regulatory genes. Combined co-expression analysis identified among those a cluster of 42 genes, many involved in the photosynthetic apparatus and responsive to drought, iron deficiency, arsenic toxicity, and ozone. Our data demonstrate the importance of redox homeostasis in plant stress responses and the power of MA to identify candidate genes underlying unspecific signaling pathways. PMID:26793229

Global Microarray Analysis of Alkaliphilic Halotolerant Bacterium Bacillus sp. N16-5 Salt Stress Adaptation

PubMed Central

Yin, Liang; Xue, Yanfen; Ma, Yanhe

2015-01-01

The alkaliphilic halotolerant bacterium Bacillus sp. N16-5 is often exposed to salt stress in its natural habitats. In this study, we used one-colour microarrays to investigate adaptive responses of Bacillus sp. N16-5 transcriptome to long-term growth at different salinity levels (0%, 2%, 8%, and 15% NaCl) and to a sudden salt increase from 0% to 8% NaCl. The common strategies used by bacteria to survive and grow at high salt conditions, such as K+ uptake, Na+ efflux, and the accumulation of organic compatible solutes (glycine betaine and ectoine), were observed in Bacillus sp. N16-5. The genes of SigB regulon involved in general stress responses and chaperone-encoding genes were also induced by high salt concentration. Moreover, the genes regulating swarming ability and the composition of the cytoplasmic membrane and cell wall were also differentially expressed. The genes involved in iron uptake were down-regulated, whereas the iron homeostasis regulator Fur was up-regulated, suggesting that Fur may play a role in the salt adaption of Bacillus sp. N16-5. In summary, we present a comprehensive gene expression profiling of alkaliphilic Bacillus sp. N16-5 cells exposed to high salt stress, which would help elucidate the mechanisms underlying alkaliphilic Bacillus spp. survival in and adaptation to salt stress. PMID:26030352

Systemic Analysis of Heat Shock Response Induced by Heat Shock and a Proteasome Inhibitor MG132

PubMed Central

Kim, Hee-Jung; Joo, Hye Joon; Kim, Yung Hee; Ahn, Soyeon; Chang, Jun; Hwang, Kyu-Baek; Lee, Dong-Hee; Lee, Kong-Joo

2011-01-01

The molecular basis of heat shock response (HSR), a cellular defense mechanism against various stresses, is not well understood. In this, the first comprehensive analysis of gene expression changes in response to heat shock and MG132 (a proteasome inhibitor), both of which are known to induce heat shock proteins (Hsps), we compared the responses of normal mouse fibrosarcoma cell line, RIF- 1, and its thermotolerant variant cell line, TR-RIF-1 (TR), to the two stresses. The cellular responses we examined included Hsp expressions, cell viability, total protein synthesis patterns, and accumulation of poly-ubiquitinated proteins. We also compared the mRNA expression profiles and kinetics, in the two cell lines exposed to the two stresses, using microarray analysis. In contrast to RIF-1 cells, TR cells resist heat shock caused changes in cell viability and whole-cell protein synthesis. The patterns of total cellular protein synthesis and accumulation of poly-ubiquitinated proteins in the two cell lines were distinct, depending on the stress and the cell line. Microarray analysis revealed that the gene expression pattern of TR cells was faster and more transient than that of RIF-1 cells, in response to heat shock, while both RIF-1 and TR cells showed similar kinetics of mRNA expression in response to MG132. We also found that 2,208 genes were up-regulated more than 2 fold and could sort them into three groups: 1) genes regulated by both heat shock and MG132, (e.g. chaperones); 2) those regulated only by heat shock (e.g. DNA binding proteins including histones); and 3) those regulated only by MG132 (e.g. innate immunity and defense related molecules). This study shows that heat shock and MG132 share some aspects of HSR signaling pathway, at the same time, inducing distinct stress response signaling pathways, triggered by distinct abnormal proteins. PMID:21738571

Transcription profile of brewery yeast under fermentation conditions.

PubMed

James, T C; Campbell, S; Donnelly, D; Bond, U

2003-01-01

Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.

Transcriptome profiling of a Saccharomyces cerevisiae mutant with a constitutively activated Ras/cAMP pathway.

PubMed

Jones, D L; Petty, J; Hoyle, D C; Hayes, A; Ragni, E; Popolo, L; Oliver, S G; Stateva, L I

2003-12-16

Often changes in gene expression levels have been considered significant only when above/below some arbitrarily chosen threshold. We investigated the effect of applying a purely statistical approach to microarray analysis and demonstrated that small changes in gene expression have biological significance. Whole genome microarray analysis of a pde2Delta mutant, constructed in the Saccharomyces cerevisiae reference strain FY23, revealed altered expression of approximately 11% of protein encoding genes. The mutant, characterized by constitutive activation of the Ras/cAMP pathway, has increased sensitivity to stress, reduced ability to assimilate nonfermentable carbon sources, and some cell wall integrity defects. Applying the Munich Information Centre for Protein Sequences (MIPS) functional categories revealed increased expression of genes related to ribosome biogenesis and downregulation of genes in the cell rescue, defense, cell death and aging category, suggesting a decreased response to stress conditions. A reduced level of gene expression in the unfolded protein response pathway (UPR) was observed. Cell wall genes whose expression was affected by this mutation were also identified. Several of the cAMP-responsive orphan genes, upon further investigation, revealed cell wall functions; others had previously unidentified phenotypes assigned to them. This investigation provides a statistical global transcriptome analysis of the cellular response to constitutive activation of the Ras/cAMP pathway.

Use of whole genome expression analysis in the toxicity screening of nanoparticles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fröhlich, Eleonore, E-mail: eleonore.froehlich@medunigraz.at; Meindl, Claudia; Wagner, Karin

2014-10-15

The use of nanoparticles (NPs) offers exciting new options in technical and medical applications provided they do not cause adverse cellular effects. Cellular effects of NPs depend on particle parameters and exposure conditions. In this study, whole genome expression arrays were employed to identify the influence of particle size, cytotoxicity, protein coating, and surface functionalization of polystyrene particles as model particles and for short carbon nanotubes (CNTs) as particles with potential interest in medical treatment. Another aim of the study was to find out whether screening by microarray would identify other or additional targets than commonly used cell-based assays formore » NP action. Whole genome expression analysis and assays for cell viability, interleukin secretion, oxidative stress, and apoptosis were employed. Similar to conventional assays, microarray data identified inflammation, oxidative stress, and apoptosis as affected by NP treatment. Application of lower particle doses and presence of protein decreased the total number of regulated genes but did not markedly influence the top regulated genes. Cellular effects of CNTs were small; only carboxyl-functionalized single-walled CNTs caused appreciable regulation of genes. It can be concluded that regulated functions correlated well with results in cell-based assays. Presence of protein mitigated cytotoxicity but did not cause a different pattern of regulated processes. - Highlights: • Regulated functions were screened using whole genome expression assays. • Polystyrene particles regulated more genes than short carbon nanotubes. • Protein coating of polystyrene particles did not change regulation pattern. • Functions regulated by microarray were confirmed by cell-based assay.« less

Analysis of High-Throughput ELISA Microarray Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Daly, Don S.; Zangar, Richard C.

Our research group develops analytical methods and software for the high-throughput analysis of quantitative enzyme-linked immunosorbent assay (ELISA) microarrays. ELISA microarrays differ from DNA microarrays in several fundamental aspects and most algorithms for analysis of DNA microarray data are not applicable to ELISA microarrays. In this review, we provide an overview of the steps involved in ELISA microarray data analysis and how the statistically sound algorithms we have developed provide an integrated software suite to address the needs of each data-processing step. The algorithms discussed are available in a set of open-source software tools (http://www.pnl.gov/statistics/ProMAT).

EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

PubMed Central

Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

2008-01-01

Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

[Differential gene expression in incompatible interaction between Lilium regale Wilson and Fusarium oxysporum f. sp. lilii revealed by combined SSH and microarray analysis].

PubMed

Rao, J; Liu, D; Zhang, N; He, H; Ge, F; Chen, C

2014-01-01

Fusarium wilt, caused by a soilborne pathogen Fusarium oxysporum f. sp. lilii, is the major disease of lily (Lilium L.). In order to isolate the genes differentially expressed in a resistant reaction to F. oxysporum in L. regale Wilson, a cDNA library was constructed with L. regale root during F. oxysporum infection using the suppression subtractive hybridization (SSH), and a total of 585 unique expressed sequence tags (ESTs) were obtained. Furthermore, the gene expression profiles in the incompatible interaction between L. regale and F. oxysporum were revealed by oligonucleotide microarray analysis of 585 unique ESTs comparison to the compatible interaction between a susceptible Lilium Oriental Hybrid 'Siberia' and F. oxysporum. The result of expression profile analysis indicated that the genes encoding pathogenesis-related proteins (PRs), antioxidative stress enzymes, secondary metabolism enzymes, transcription factors, signal transduction proteins as well as a large number of unknown genes were involved in early defense response of L. regale to F. oxysporum infection. Moreover, the following quantitative reverse transcription PCR (QRT-PCR) analysis confirmed reliability of the oligonucleotide microarray data. In the present study, isolation of differentially expressed genes in L. regale during response to F. oxysporum helped to uncover the molecular mechanism associated with the resistance of L. regale against F. oxysporum.

Microarray-based analysis of gene expression in lycopersicon esculentum seedling roots in response to cadmium, chromium, mercury, and lead.

PubMed

Hou, Jing; Liu, Xinhui; Wang, Juan; Zhao, Shengnan; Cui, Baoshan

2015-02-03

The effects of heavy metals in agricultural soils have received special attention due to their potential for accumulation in crops, which can affect species at all trophic levels. Therefore, there is a critical need for reliable bioassays for assessing risk levels due to heavy metals in agricultural soil. In the present study, we used microarrays to investigate changes in gene expression of Lycopersicon esculentum in response to Cd-, Cr-, Hg-, or Pb-spiked soil. Exposure to (1)/10 median lethal concentrations (LC50) of Cd, Cr, Hg, or Pb for 7 days resulted in expression changes in 29 Cd-specific, 58 Cr-specific, 192 Hg-specific and 864 Pb-specific genes as determined by microarray analysis, whereas conventional morphological and physiological bioassays did not reveal any toxicant stresses. Hierarchical clustering analysis showed that the characteristic gene expression profiles induced by Cd, Cr, Hg, and Pb were distinct from not only the control but also one another. Furthermore, a total of three genes related to "ion transport" for Cd, 14 genes related to "external encapsulating structure organization", "reproductive developmental process", "lipid metabolic process" and "response to stimulus" for Cr, 11 genes related to "cellular metabolic process" and "cellular response to stimulus" for Hg, 78 genes related to 20 biological processes (e.g., DNA metabolic process, monosaccharide catabolic process, cell division) for Pb were identified and selected as their potential biomarkers. These findings demonstrated that microarray-based analysis of Lycopersicon esculentum was a sensitive tool for the early detection of potential toxicity of heavy metals in agricultural soil, as well as an effective tool for identifying the heavy metal-specific genes, which should be useful for assessing risk levels due to heavy metals in agricultural soil.

Chemiluminescence microarrays in analytical chemistry: a critical review.

PubMed

Seidel, Michael; Niessner, Reinhard

2014-09-01

Multi-analyte immunoassays on microarrays and on multiplex DNA microarrays have been described for quantitative analysis of small organic molecules (e.g., antibiotics, drugs of abuse, small molecule toxins), proteins (e.g., antibodies or protein toxins), and microorganisms, viruses, and eukaryotic cells. In analytical chemistry, multi-analyte detection by use of analytical microarrays has become an innovative research topic because of the possibility of generating several sets of quantitative data for different analyte classes in a short time. Chemiluminescence (CL) microarrays are powerful tools for rapid multiplex analysis of complex matrices. A wide range of applications for CL microarrays is described in the literature dealing with analytical microarrays. The motivation for this review is to summarize the current state of CL-based analytical microarrays. Combining analysis of different compound classes on CL microarrays reduces analysis time, cost of reagents, and use of laboratory space. Applications are discussed, with examples from food safety, water safety, environmental monitoring, diagnostics, forensics, toxicology, and biosecurity. The potential and limitations of research on multiplex analysis by use of CL microarrays are discussed in this review.

Acquisition of thermotolerant yeast Saccharomyces cerevisiae by breeding via stepwise adaptation.

PubMed

Satomura, Atsushi; Katsuyama, Yoshiaki; Miura, Natsuko; Kuroda, Kouichi; Tomio, Ayako; Bamba, Takeshi; Fukusaki, Eiichiro; Ueda, Mitsuyoshi

2013-01-01

A thermotolerant Saccharomyces cerevisiae yeast strain, YK60-1, was bred from a parental strain, MT8-1, via stepwise adaptation. YK60-1 grew at 40°C, a temperature at which MT8-1 could not grow at all. YK60-1 exhibited faster growth than MT8-1 at 30°C. To investigate the mechanisms how MT8-1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress-responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60-1. Furthermore, nontargeting metabolome analysis showed that YK60-1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8-1. In conclusion, S. cerevisiae MT8-1 acquired thermotolerance by induction of specific stress-responsive genes and enhanced intracellular trehalose levels. © 2013 American Institute of Chemical Engineers.

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

PubMed Central

2013-01-01

Background The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. Results A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. Conclusions This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported. PMID:24168212

mRNA-Seq and microarray development for the Grooved Carpet shell clam, Ruditapes decussatus: a functional approach to unravel host-parasite interaction.

PubMed

Leite, Ricardo B; Milan, Massimo; Coppe, Alessandro; Bortoluzzi, Stefania; dos Anjos, António; Reinhardt, Richard; Saavedra, Carlos; Patarnello, Tomaso; Cancela, M Leonor; Bargelloni, Luca

2013-10-29

The Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis. A database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed. This study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Concurrent Drought Stress and Vascular Pathogen Infection Induce Common and Distinct Transcriptomic Responses in Chickpea

PubMed Central

Sinha, Ranjita; Gupta, Aarti; Senthil-Kumar, Muthappa

2017-01-01

Chickpea (Cicer arietinum); the second largest legume grown worldwide is prone to drought and various pathogen infections. These drought and pathogen stresses often occur concurrently in the field conditions. However, the molecular events in response to that are largely unknown. The present study examines the transcriptome dynamics in chickpea plants exposed to a combination of water-deficit stress and Ralstonia solanacearum infection. R. solanacearum is a potential wilt disease causing pathogen in chickpea. Drought stressed chickpea plants were infected with this pathogen and the plants were allowed to experience progressive drought with 2 and 4 days of R. solanacearum infection called short duration stress (SD stresses) and long duration stress (LD stresses), respectively. Our study showed that R. solanacearum multiplication decreased under SD-combined stress compared to SD-pathogen but there was no significant change in LD-combined stress compared to LD-pathogen. The microarray analysis during these conditions showed that 821 and 1039 differentially expressed genes (DEGs) were unique to SD- and LD-combined stresses, respectively, when compared with individual stress conditions. Three and fifteen genes were common among all the SD-stress treatments and LD-stress treatments, respectively. Genes involved in secondary cell wall biosynthesis, alkaloid biosynthesis, defense related proteins, and osmo-protectants were up-regulated during combined stress. The expression of genes involved in lignin and cellulose biosynthesis were specifically up-regulated in SD-combined, LD-combined, and LD-pathogen stress. A close transcriptomic association of LD-pathogen stress with SD-combined stress was observed in this study which indicates that R. solanacearum infection also exerts drought stress along with pathogen stress thus mimics combined stress effect. Furthermore the expression profiling of candidate genes using real-time quantitative PCR validated the microarray data. The study showed that down-regulation of defense-related genes during LD-combined stress resulted in an increased bacterial multiplication as compared to SD-combined stress. Overall, our study highlights a sub-set of DEGs uniquely expressed in response to combined stress, which serve as potential candidates for further functional characterization to delineate the molecular response of the plant to concurrent drought-pathogen stress. PMID:28382041

Transcriptome Analysis of the Role of GlnD/GlnBK in Nitrogen Stress Adaptation by Sinorhizobium meliloti Rm1021

PubMed Central

Yurgel, Svetlana N.; Rice, Jennifer; Kahn, Michael L.

2013-01-01

Transcriptional changes in the nitrogen stress response (NSR) of wild type S. meliloti Rm1021, and isogenic strains missing both PII proteins, GlnB and GlnK, or carrying a ΔglnD-sm2 mutation were analyzed using whole-genome microarrays. This approach allowed us to identify a number of new genes involved in the NSR and showed that the response of these bacteria to nitrogen stress overlaps with other stress responses, including induction of the fixK2 transcriptional activator and genes that are part of the phosphate stress response. Our data also show that GlnD and GlnBK proteins may regulate many genes that are not part of the NSR. Analysis of transcriptome profiles of the Rm1021 ΔglnD-sm2 strain allowed us to identify several genes that appear to be regulated by GlnD without the participation of the PII proteins. PMID:23516427

cDNA microarray analysis of esophageal cancer: discoveries and prospects.

PubMed

Shimada, Yutaka; Sato, Fumiaki; Shimizu, Kazuharu; Tsujimoto, Gozoh; Tsukada, Kazuhiro

2009-07-01

Recent progress in molecular biology has revealed many genetic and epigenetic alterations that are involved in the development and progression of esophageal cancer. Microarray analysis has also revealed several genetic networks that are involved in esophageal cancer. However, clinical application of microarray techniques and use of microarray data have not yet occurred. In this review, we focus on the recent developments and problems with microarray analysis of esophageal cancer.

Importing MAGE-ML format microarray data into BioConductor.

PubMed

Durinck, Steffen; Allemeersch, Joke; Carey, Vincent J; Moreau, Yves; De Moor, Bart

2004-12-12

The microarray gene expression markup language (MAGE-ML) is a widely used XML (eXtensible Markup Language) standard for describing and exchanging information about microarray experiments. It can describe microarray designs, microarray experiment designs, gene expression data and data analysis results. We describe RMAGEML, a new Bioconductor package that provides a link between cDNA microarray data stored in MAGE-ML format and the Bioconductor framework for preprocessing, visualization and analysis of microarray experiments. http://www.bioconductor.org. Open Source.

MOLECULAR METHODS (E.G., MICROARRAYS) APPLIED TO PLANT GENOMES FOR ASSESSING GENETIC CHANGE AND ENVIRONMENTAL STRESS

EPA Science Inventory

This is a technical document that presents a detailed sample standard operating procedure (S.O.P.) for preparing plant nucleic acid samples for microarray analyses using commercial ¿chips¿ such as those sold by Affymetrix. It also presents the application of a commercially availa...

Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

PubMed

Villeneuve, L; Wang, Rong-Lin; Bencic, David C; Biales, Adam D; Martinović, Dalma; Lazorchak, James M; Toth, Gregory; Ankley, Gerald T

2009-08-01

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

The Longhorn Array Database (LAD): An Open-Source, MIAME compliant implementation of the Stanford Microarray Database (SMD)

PubMed Central

Killion, Patrick J; Sherlock, Gavin; Iyer, Vishwanath R

2003-01-01

Background The power of microarray analysis can be realized only if data is systematically archived and linked to biological annotations as well as analysis algorithms. Description The Longhorn Array Database (LAD) is a MIAME compliant microarray database that operates on PostgreSQL and Linux. It is a fully open source version of the Stanford Microarray Database (SMD), one of the largest microarray databases. LAD is available at Conclusions Our development of LAD provides a simple, free, open, reliable and proven solution for storage and analysis of two-color microarray data. PMID:12930545

Initiation of follicular atresia: gene networks during early atresia in pig ovaries.

PubMed

Zhang, Jinbi; Liu, Yang; Yao, Wang; Li, Qifa; Liu, Hong-Lin; Pan, Zengxiang

2018-05-09

In mammals, more than 99% of ovarian follicles undergo a degenerative process known as atresia. The molecular events involve in atresia initiation remain incompletely understood. The objective of this study was to analyze differential gene expression profiles of medium antral ovarian follicles during early atresia in pig. The transcriptome evaluation was performed on cDNA microarrays using healthy and early atretic follicle samples and was validated by quantitative PCR. Annotation analysis applying current database (sus scrofa 11.1) revealed 450 significantly differential expressed genes between healthy and early atretic follicles. Among them, 142 were significantly up-regulated in early atretic with respect to healthy group and 308 were down-regulated. Similar expression trends were observed between microarray data and qRT-PCR confirmation, which indicated the reliability of the microarray analysis. Further analysis of the differential expressed genes revealed the most significantly affected biological functions during early atresia including blood vessel development, regulation of DNA-templated transcription in response to stress and negative regulation of cell adhesion. The pathway and interaction analysis suggested that atresia initiation associates with 1) a crosstalk of cell apoptosis, autophagy, and ferroptosis rather than change of typical apoptosis markers, 2) dramatic shift of steroidogenic enzymes, 3) deficient glutathione metabolism, and 4) vascular degeneration. The novel gene candidates and pathways identified in the current study will lead to a comprehensive view of the molecular regulation of ovarian follicular atresia and a new understanding of atresia initiation.

Transcriptome analysis identifies genes involved in ethanol response of Saccharomyces cerevisiae in Agave tequilana juice.

PubMed

Ramirez-Córdova, Jesús; Drnevich, Jenny; Madrigal-Pulido, Jaime Alberto; Arrizon, Javier; Allen, Kirk; Martínez-Velázquez, Moisés; Alvarez-Maya, Ikuri

2012-08-01

During ethanol fermentation, yeast cells are exposed to stress due to the accumulation of ethanol, cell growth is altered and the output of the target product is reduced. For Agave beverages, like tequila, no reports have been published on the global gene expression under ethanol stress. In this work, we used microarray analysis to identify Saccharomyces cerevisiae genes involved in the ethanol response. Gene expression of a tequila yeast strain of S. cerevisiae (AR5) was explored by comparing global gene expression with that of laboratory strain S288C, both after ethanol exposure. Additionally, we used two different culture conditions, cells grown in Agave tequilana juice as a natural fermentation media or grown in yeast-extract peptone dextrose as artificial media. Of the 6368 S. cerevisiae genes in the microarray, 657 genes were identified that had different expression responses to ethanol stress due to strain and/or media. A cluster of 28 genes was found over-expressed specifically in the AR5 tequila strain that could be involved in the adaptation to tequila yeast fermentation, 14 of which are unknown such as yor343c, ylr162w, ygr182c, ymr265c, yer053c-a or ydr415c. These could be the most suitable genes for transforming tequila yeast to increase ethanol tolerance in the tequila fermentation process. Other genes involved in response to stress (RFC4, TSA1, MLH1, PAU3, RAD53) or transport (CYB2, TIP20, QCR9) were expressed in the same cluster. Unknown genes could be good candidates for the development of recombinant yeasts with ethanol tolerance for use in industrial tequila fermentation.

Molecular pathological analysis for determining the possible mechanism of piperonyl butoxide-induced hepatocarcinogenesis in mice.

PubMed

Muguruma, Masako; Nishimura, Jihei; Jin, Meilan; Kashida, Yoko; Moto, Mitsuyoshi; Takahashi, Miwa; Yokouchi, Yusuke; Mitsumori, Kunitoshi

2006-12-07

Piperonyl butoxide (PBO), alpha-[2-(2-butoxyethoxy)ethoxy]-4,5-methylene-dioxy-2-propyltoluene, is widely used as a synergist for pyrethrins. In order to clarify the possible mechanism of non-genotoxic hepatocarcinogenesis induced by PBO, molecular pathological analyses consisting of low-density microarray analysis and real-time reverse transcriptase (RT)-PCR were performed in male ICR mice fed a basal powdered diet containing 6000 or 0 ppm PBO for 1, 4, or 8 weeks. The animals were sacrificed at weeks 1, 4, and 8, and the livers were histopathologically examined and analyzed for gene expression using the microarray at weeks 1 and 4 followed by real-time RT-PCR at each time point. Reactive oxygen species (ROS) products were also measured using liver microsomes. At each time point, the hepatocytes of PBO-treated mice showed centrilobular hypertrophy and increased lipofuscin deposition in Schmorl staining. The ROS products were significantly increased in the liver microsomes of PBO-treated mice. In the microarray analysis, the expression of oxidative and metabolic stress-related genes--cytochrome P450 (Cyp) 1A1, Cyp2A5 (week 1 only), Cyp2B9, Cyp2B10, and NADPH-cytochrome P450 oxidoreductase (Por) was over-expressed in mice given PBO at weeks 1 and 4. Fluctuations of these genes were confirmed by real-time RT-PCR in PBO-treated mice at each time point. In additional real-time RT-PCR, the expression of Cyclin D1 gene, key regulator of cell-cycle progression, and Xrcc5 gene, DNA damage repair-related gene, was significantly increased at each time point and at week 8, respectively. These results suggest the possibility that PBO has the potential to generate ROS via the metabolic pathway and to induce oxidative stress, including oxidative DNA damage, resulting in the induction of hepatocellular tumors in mice.

Global transcriptional profiling of Burkholderia pseudomallei under salt stress reveals differential effects on the Bsa type III secretion system

PubMed Central

2010-01-01

Background Burkholderia pseudomallei is the causative agent of melioidosis where the highest reported incidence world wide is in the Northeast of Thailand, where saline soil and water are prevalent. Moreover, recent reports indicate a potential pathogenic role for B. pseudomallei in cystic fibrosis lung disease, where an increased sodium chloride (NaCl) concentration in airway surface liquid has been proposed. These observations raise the possibility that high salinity may represent a favorable niche for B. pseudomallei. We therefore investigated the global transcriptional response of B. pseudomallei to increased salinity using microarray analysis. Results Transcriptome analysis of B. pseudomallei under salt stress revealed several genes significantly up-regulated in the presence of 320 mM NaCl including genes associated with the bsa-derived Type III secretion system (T3SS). Microarray data were verified by reverse transcriptase-polymerase chain reactions (RT-PCR). Western blot analysis confirmed the increased expression and secretion of the invasion-associated type III secreted proteins BipD and BopE in B. pseudomallei cultures at 170 and 320 mM NaCl relative to salt-free medium. Furthermore, salt-treated B. pseudomallei exhibited greater invasion efficiency into the lung epithelial cell line A549 in a manner partly dependent on a functional Bsa system. Conclusions B. pseudomallei responds to salt stress by modulating the transcription of a relatively small set of genes, among which is the bsa locus associated with invasion and virulence. Expression and secretion of Bsa-secreted proteins was elevated in the presence of exogenous salt and the invasion efficiency was enhanced. Our data indicate that salinity has the potential to influence the virulence of B. pseudomallei. PMID:20540813

Analysis of bHLH coding genes using gene co-expression network approach.

PubMed

Srivastava, Swati; Sanchita; Singh, Garima; Singh, Noopur; Srivastava, Gaurava; Sharma, Ashok

2016-07-01

Network analysis provides a powerful framework for the interpretation of data. It uses novel reference network-based metrices for module evolution. These could be used to identify module of highly connected genes showing variation in co-expression network. In this study, a co-expression network-based approach was used for analyzing the genes from microarray data. Our approach consists of a simple but robust rank-based network construction. The publicly available gene expression data of Solanum tuberosum under cold and heat stresses were considered to create and analyze a gene co-expression network. The analysis provide highly co-expressed module of bHLH coding genes based on correlation values. Our approach was to analyze the variation of genes expression, according to the time period of stress through co-expression network approach. As the result, the seed genes were identified showing multiple connections with other genes in the same cluster. Seed genes were found to be vary in different time periods of stress. These analyzed seed genes may be utilized further as marker genes for developing the stress tolerant plant species.

Salty dog, an SLC5 symporter, modulates Drosophila response to salt stress.

PubMed

Stergiopoulos, Konstantinos; Cabrero, Pablo; Davies, Shireen-Anne; Dow, Julian A T

2009-03-03

To regulate their internal environments, organisms must adapt to varying ion levels in their diet. Adult Drosophila were exposed to dietary salt stress, and their physiological, survival, and gene expression responses monitored. Insects continued to feed on NaCl-elevated diet, although levels >4% wt/vol ultimately proved fatal. Affymetrix microarray analysis of flies fed on diet containing elevated NaCl showed a phased response: the earliest response was widespread upregulation of immune genes, followed by upregulation of carbohydrate metabolism as the immune response was downregulated, then finally a switch to amino acid catabolism and inhibition of genes associated with the reproductive axis. Significantly, the online transcriptomic resource FlyAtlas reports that most of the modulated genes are predominantly expressed in hindgut or Malpighian (renal) tubule, implicating these excretory tissues as the major responders to salt stress. Three genes were selected for further study: the SLC5 symporter CG2196, the GLUT transporter CG6484, and the transcription factor sugarbabe (previously implicated in starvation and stress responses). Expression profiles predicted by microarray were validated by quantitative PCR (qPCR); expression was mapped to the alimentary canal by in situ hybridization. CG2196::eYFP overexpression constructs were localized to the basolateral membrane of the Malpighian (renal) tubules, and RNAi against CG2196 improved survival on high-salt diet, even when driven specifically to just principal cells of the Malpighian tubule, confirming both this tissue and this transporter as major determinants of survival upon salt stress. Accordingly, CG2196 was renamed salty dog (salt).

The Microarray Revolution: Perspectives from Educators

ERIC Educational Resources Information Center

Brewster, Jay L.; Beason, K. Beth; Eckdahl, Todd T.; Evans, Irene M.

2004-01-01

In recent years, microarray analysis has become a key experimental tool, enabling the analysis of genome-wide patterns of gene expression. This review approaches the microarray revolution with a focus upon four topics: 1) the early development of this technology and its application to cancer diagnostics; 2) a primer of microarray research,…

Monitoring the expression of maize genes in developing kernels under drought stress using oligo-microarray

USDA-ARS?s Scientific Manuscript database

Preharvest A. flavus infection is usually exacerbated when maize plants suffer drought stress in the late grain-fill stage. However, the field observation suggests that drought-tolerant maize lines displayed less aflatoxin contamination under the stress in comparison with the drought-sensitive maize...

The RdDM Pathway Is Required for Basal Heat Tolerance in Arabidopsis

PubMed Central

Jonak, Claudia

2013-01-01

Heat stress affects epigenetic gene silencing in Arabidopsis. To test for a mechanistic involvement of epigenetic regulation in heat-stress responses, we analyzed the heat tolerance of mutants defective in DNA methylation, histone modifications, chromatin-remodeling, or siRNA-based silencing pathways. Plants deficient in NRPD2, the common second-largest subunit of RNA polymerases IV and V, and in the Rpd3-type histone deacetylase HDA6 were hypersensitive to heat exposure. Microarray analysis demonstrated that NRPD2 and HDA6 have independent roles in transcriptional reprogramming in response to temperature stress. The misexpression of protein-coding genes in nrpd2 mutants recovering from heat correlated with defective epigenetic regulation of adjacent transposon remnants which involved the loss of control of heat-stress-induced read-through transcription. We provide evidence that the transcriptional response to temperature stress, at least partially, relies on the integrity of the RNA-dependent DNA methylation pathway. PMID:23376771

Molecular profiles of pre- and postoperative breast cancer tumours reveal differentially expressed genes.

PubMed

Riis, Margit L H; Lüders, Torben; Markert, Elke K; Haakensen, Vilde D; Nesbakken, Anne-Jorun; Kristensen, Vessela N; Bukholm, Ida R K

2012-01-01

Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun families as well as genes of which the expression has previously been associated with cancer. The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology.

Molecular Profiles of Pre- and Postoperative Breast Cancer Tumours Reveal Differentially Expressed Genes

PubMed Central

Riis, Margit L. H.; Lüders, Torben; Markert, Elke K.; Haakensen, Vilde D.; Nesbakken, Anne-Jorun; Kristensen, Vessela N.; Bukholm, Ida R. K.

2012-01-01

Gene expression studies on breast cancer have generally been performed on tissue obtained at the time of surgery. In this study, we have compared the gene expression profiles in preoperative tissue (core needle biopsies) while tumor is still in its normal milieu to postoperative tissue from the same tumor obtained during surgery. Thirteen patients were included of which eleven had undergone sentinel node diagnosis procedure before operation. Microarray gene expression analysis was performed using total RNA from all the samples. Paired significance analysis of microarrays revealed 228 differently expressed genes, including several early response stress-related genes such as members of the fos and jun families as well as genes of which the expression has previously been associated with cancer. The expression profiles found in the analyses of breast cancer tissue must be evaluated with caution. Different profiles may simply be the result of differences in the surgical trauma and timing of when samples are taken and not necessarily associated with tumor biology. PMID:23227362

Analysis of the Metabolic Pathways Affected by Poly(γ-glutamic Acid) in Arabidopsis thaliana Based on GeneChip Microarray.

PubMed

Xu, Zongqi; Lei, Peng; Feng, Xiaohai; Li, Sha; Xu, Hong

2016-08-17

Plant growth is promoted by poly(γ-glutamic acid) (γ-PGA). However, the molecular mechanism underlying such promotion is not yet well understood. Therefore, we used GeneChip microarrays to explore the effects of γ-PGA on gene transcription in Arabidopsis thaliana. Our results revealed 299 genes significantly regulated by γ-PGA. These differently expressed genes participate mainly in metabolic and cellular processes and in stimuli responses. The metabolic pathways linked to these differently expressed genes were also investigated. A total of 64 of the 299 differently expressed genes were shown to be directly involved in 24 pathways such as brassinosteroid biosynthesis, α-linolenic acid metabolism, phenylpropanoid biosynthesis, and nitrogen metabolism, all of which were influenced by γ-PGA. The analysis demonstrated that γ-PGA promoted nitrogen assimilation and biosynthesis of brassinosteroids, jasmonic acid, and lignins, providing a better explanation for why γ-PGA promotes growth and enhances stress tolerance in plants.

Identification of bovine leukemia virus tax function associated with host cell transcription, signaling, stress response and immune response pathway by microarray-based gene expression analysis

PubMed Central

2012-01-01

Background Bovine leukemia virus (BLV) is associated with enzootic bovine leukosis and is closely related to human T-cell leukemia virus type I. The Tax protein of BLV is a transcriptional activator of viral replication and a key contributor to oncogenic potential. We previously identified interesting mutant forms of Tax with elevated (TaxD247G) or reduced (TaxS240P) transactivation effects on BLV replication and propagation. However, the effects of these mutations on functions other than transcriptional activation are unknown. In this study, to identify genes that play a role in the cascade of signal events regulated by wild-type and mutant Tax proteins, we used a large-scale host cell gene-profiling approach. Results Using a microarray containing approximately 18,400 human mRNA transcripts, we found several alterations after the expression of Tax proteins in genes involved in many cellular functions such as transcription, signal transduction, cell growth, apoptosis, stress response, and immune response, indicating that Tax protein has multiple biological effects on various cellular environments. We also found that TaxD247G strongly regulated more genes involved in transcription, signal transduction, and cell growth functions, contrary to TaxS240P, which regulated fewer genes. In addition, the expression of genes related to stress response significantly increased in the presence of TaxS240P as compared to wild-type Tax and TaxD247G. By contrast, the largest group of downregulated genes was related to immune response, and the majority of these genes belonged to the interferon family. However, no significant difference in the expression level of downregulated genes was observed among the Tax proteins. Finally, the expression of important cellular factors obtained from the human microarray results were validated at the RNA and protein levels by real-time quantitative reverse transcription-polymerase chain reaction and western blotting, respectively, after transfecting Tax proteins into bovine cells and human HeLa cells. Conclusion A comparative analysis of wild-type and mutant Tax proteins indicates that Tax protein exerts a significant impact on cellular functions as diverse as transcription, signal transduction, cell growth, stress response and immune response. Importantly, our study is the first report that shows the extent to which BLV Tax regulates the innate immune response. PMID:22455445

Role of OSGIN1 in mediating smoking-induced autophagy in the human airway epithelium.

PubMed

Wang, Guoqing; Zhou, Haixia; Strulovici-Barel, Yael; Al-Hijji, Mohammed; Ou, Xuemei; Salit, Jacqueline; Walters, Matthew S; Staudt, Michelle R; Kaner, Robert J; Crystal, Ronald G

2017-07-03

Enhanced macroautophagy/autophagy is recognized as a component of the pathogenesis of smoking-induced airway disease. Based on the knowledge that enhanced autophagy is linked to oxidative stress and the DNA damage response, both of which are linked to smoking, we used microarray analysis of the airway epithelium to identify smoking upregulated genes known to respond to oxidative stress and the DNA damage response. This analysis identified OSGIN1 (oxidative stress induced growth inhibitor 1) as significantly upregulated by smoking, in both the large and small airway epithelium, an observation confirmed by an independent small airway microarray cohort, TaqMan PCR of large and small airway samples and RNA-Seq of small airway samples. High and low OSGIN1 expressors have different autophagy gene expression patterns in vivo. Genome-wide correlation of RNAseq analysis of airway basal/progenitor cells showed a direct correlation of OSGIN1 mRNA levels to multiple classic autophagy genes. In vitro cigarette smoke extract exposure of primary airway basal/progenitor cells was accompanied by a dose-dependent upregulation of OSGIN1 and autophagy induction. Lentivirus-mediated expression of OSGIN1 in human primary basal/progenitor cells induced puncta-like staining of MAP1LC3B and upregulation of MAP1LC3B mRNA and protein and SQSTM1 mRNA expression level in a dose and time-dependent manner. OSGIN1-induction of autophagosome, amphisome and autolysosome formation was confirmed by colocalization of MAP1LC3B with SQSTM1 or CD63 (endosome marker) and LAMP1 (lysosome marker). Both OSGIN1 overexpression and knockdown enhanced the smoking-evoked autophagic response. Together, these observations support the concept that smoking-induced upregulation of OSGIN1 is one link between smoking-induced stress and enhanced-autophagy in the human airway epithelium.

EMAAS: An extensible grid-based Rich Internet Application for microarray data analysis and management

PubMed Central

Barton, G; Abbott, J; Chiba, N; Huang, DW; Huang, Y; Krznaric, M; Mack-Smith, J; Saleem, A; Sherman, BT; Tiwari, B; Tomlinson, C; Aitman, T; Darlington, J; Game, L; Sternberg, MJE; Butcher, SA

2008-01-01

Background Microarray experimentation requires the application of complex analysis methods as well as the use of non-trivial computer technologies to manage the resultant large data sets. This, together with the proliferation of tools and techniques for microarray data analysis, makes it very challenging for a laboratory scientist to keep up-to-date with the latest developments in this field. Our aim was to develop a distributed e-support system for microarray data analysis and management. Results EMAAS (Extensible MicroArray Analysis System) is a multi-user rich internet application (RIA) providing simple, robust access to up-to-date resources for microarray data storage and analysis, combined with integrated tools to optimise real time user support and training. The system leverages the power of distributed computing to perform microarray analyses, and provides seamless access to resources located at various remote facilities. The EMAAS framework allows users to import microarray data from several sources to an underlying database, to pre-process, quality assess and analyse the data, to perform functional analyses, and to track data analysis steps, all through a single easy to use web portal. This interface offers distance support to users both in the form of video tutorials and via live screen feeds using the web conferencing tool EVO. A number of analysis packages, including R-Bioconductor and Affymetrix Power Tools have been integrated on the server side and are available programmatically through the Postgres-PLR library or on grid compute clusters. Integrated distributed resources include the functional annotation tool DAVID, GeneCards and the microarray data repositories GEO, CELSIUS and MiMiR. EMAAS currently supports analysis of Affymetrix 3' and Exon expression arrays, and the system is extensible to cater for other microarray and transcriptomic platforms. Conclusion EMAAS enables users to track and perform microarray data management and analysis tasks through a single easy-to-use web application. The system architecture is flexible and scalable to allow new array types, analysis algorithms and tools to be added with relative ease and to cope with large increases in data volume. PMID:19032776

Functional Genomic Analysis of Variation on Beef Tenderness Induced by Acute Stress in Angus Cattle

PubMed Central

Zhao, Chunping; Tian, Fei; Yu, Ying; Luo, Juan; Mitra, Apratim; Zhan, Fei; Hou, Yali; Liu, George; Zan, Linsen; Updike, M. Scott; Song, Jiuzhou

2012-01-01

Beef is one of the leading sources of protein, B vitamins, iron, and zinc in human food. Beef palatability is based on three general criteria: tenderness, juiciness, and flavor, of which tenderness is thought to be the most important factor. In this study, we found that beef tenderness, measured by the Warner-Bratzler shear force (WBSF), was dramatically increased by acute stress. Microarray analysis and qPCR identified a variety of genes that were differentially expressed. Pathway analysis showed that these genes were involved in immune response and regulation of metabolism process as activators or repressors. Further analysis identified that these changes may be related with CpG methylation of several genes. Therefore, the results from this study provide an enhanced understanding of the mechanisms that genetic and epigenetic regulations control meat quality and beef tenderness. PMID:22566754

An Introduction to MAMA (Meta-Analysis of MicroArray data) System.

PubMed

Zhang, Zhe; Fenstermacher, David

2005-01-01

Analyzing microarray data across multiple experiments has been proven advantageous. To support this kind of analysis, we are developing a software system called MAMA (Meta-Analysis of MicroArray data). MAMA utilizes a client-server architecture with a relational database on the server-side for the storage of microarray datasets collected from various resources. The client-side is an application running on the end user's computer that allows the user to manipulate microarray data and analytical results locally. MAMA implementation will integrate several analytical methods, including meta-analysis within an open-source framework offering other developers the flexibility to plug in additional statistical algorithms.

Gene expression analysis in rat lungs after intratracheal exposure to nanoparticles doped with cadmium

NASA Astrophysics Data System (ADS)

Coccini, Teresa; Fabbri, Marco; Roda, Elisa; Grazia Sacco, Maria; Manzo, Luigi; Gribaldo, Laura

2011-07-01

Silica nanoparticles (NPs) incorporating cadmium (Cd) have been developed for a range of potential application including drug delivery devices. Occupational Cd inhalation has been associated with emphysema, pulmonary fibrosis and lung tumours. Mechanistically, Cd can induce oxidative stress and mediate cell-signalling pathways that are involved in inflammation.This in vivo study aimed at investigating pulmonary molecular effects of NPs doped with Cd (NP-Cd, 1 mg/animal) compared to soluble CdCl2 (400 μg/animal), in Sprague Dawley rats treated intra-tracheally, 7 and 30 days after administration. NPs of silica containing Cd salt were prepared starting from commercial nano-size silica powder (HiSil™ T700 Degussa) with average pore size of 20 nm and surface area of 240 m2/g. Toxicogenomic analysis was performed by the DNA microarray technology (using Agilent Whole Rat Genome Microarray 4×44K) to evaluate changes in gene expression of the entire genome. These findings indicate that the whole genome analysis may represent a valuable approach to assess the whole spectrum of biological responses to cadmium containing nanomaterials.

Integrating microarray analysis and the soybean genome to understand the soybeans iron deficiency response

PubMed Central

2009-01-01

Background Soybeans grown in the upper Midwestern United States often suffer from iron deficiency chlorosis, which results in yield loss at the end of the season. To better understand the effect of iron availability on soybean yield, we identified genes in two near isogenic lines with changes in expression patterns when plants were grown in iron sufficient and iron deficient conditions. Results Transcriptional profiles of soybean (Glycine max, L. Merr) near isogenic lines Clark (PI548553, iron efficient) and IsoClark (PI547430, iron inefficient) grown under Fe-sufficient and Fe-limited conditions were analyzed and compared using the Affymetrix® GeneChip® Soybean Genome Array. There were 835 candidate genes in the Clark (PI548553) genotype and 200 candidate genes in the IsoClark (PI547430) genotype putatively involved in soybean's iron stress response. Of these candidate genes, fifty-eight genes in the Clark genotype were identified with a genetic location within known iron efficiency QTL and 21 in the IsoClark genotype. The arrays also identified 170 single feature polymorphisms (SFPs) specific to either Clark or IsoClark. A sliding window analysis of the microarray data and the 7X genome assembly coupled with an iterative model of the data showed the candidate genes are clustered in the genome. An analysis of 5' untranslated regions in the promoter of candidate genes identified 11 conserved motifs in 248 differentially expressed genes, all from the Clark genotype, representing 129 clusters identified earlier, confirming the cluster analysis results. Conclusion These analyses have identified the first genes with expression patterns that are affected by iron stress and are located within QTL specific to iron deficiency stress. The genetic location and promoter motif analysis results support the hypothesis that the differentially expressed genes are co-regulated. The combined results of all analyses lead us to postulate iron inefficiency in soybean is a result of a mutation in a transcription factor(s), which controls the expression of genes required in inducing an iron stress response. PMID:19678937

Time-series RNA-seq analysis package (TRAP) and its application to the analysis of rice, Oryza sativa L. ssp. Japonica, upon drought stress.

PubMed

Jo, Kyuri; Kwon, Hawk-Bin; Kim, Sun

2014-06-01

Measuring expression levels of genes at the whole genome level can be useful for many purposes, especially for revealing biological pathways underlying specific phenotype conditions. When gene expression is measured over a time period, we have opportunities to understand how organisms react to stress conditions over time. Thus many biologists routinely measure whole genome level gene expressions at multiple time points. However, there are several technical difficulties for analyzing such whole genome expression data. In addition, these days gene expression data is often measured by using RNA-sequencing rather than microarray technologies and then analysis of expression data is much more complicated since the analysis process should start with mapping short reads and produce differentially activated pathways and also possibly interactions among pathways. In addition, many useful tools for analyzing microarray gene expression data are not applicable for the RNA-seq data. Thus a comprehensive package for analyzing time series transcriptome data is much needed. In this article, we present a comprehensive package, Time-series RNA-seq Analysis Package (TRAP), integrating all necessary tasks such as mapping short reads, measuring gene expression levels, finding differentially expressed genes (DEGs), clustering and pathway analysis for time-series data in a single environment. In addition to implementing useful algorithms that are not available for RNA-seq data, we extended existing pathway analysis methods, ORA and SPIA, for time series analysis and estimates statistical values for combined dataset by an advanced metric. TRAP also produces visual summary of pathway interactions. Gene expression change labeling, a practical clustering method used in TRAP, enables more accurate interpretation of the data when combined with pathway analysis. We applied our methods on a real dataset for the analysis of rice (Oryza sativa L. Japonica nipponbare) upon drought stress. The result showed that TRAP was able to detect pathways more accurately than several existing methods. TRAP is available at http://biohealth.snu.ac.kr/software/TRAP/. Copyright © 2014 Elsevier Inc. All rights reserved.

Ground testing of Arabidopsis preservation protocol for the microarray analysis to be used in the ISS EMCS Multigen-2 experiment

NASA Astrophysics Data System (ADS)

Kittang, Ann-Iren; Kvaløy, Brita; Winge, Per; Iversen, Tor-Henning

2010-11-01

Gene expression analysis using microarrays has proved to be an important method in life science. The opportunity to grow higher plants on the International Space Station (ISS) opens up the possibility for gene expression profiling of plants grown in microgravity. The work presented focuses on how to meet the scientific requirements of plant growth and the sample preservation, given the technical and operational constraints associated with space research. The growth chamber (Multigen-2 Science Testing Unit) and a protocol suggested to be used in the European Modular Cultivation System (EMCS) Multigen-2 experiment on the ISS to grow and later preserve Arabidopsis seedlings, were tested on ground. The results showed that most of the plants developed normally. In order to avoid high population stress the number of seedlings per growth area should be reduced. The RNAlater preservation method to be used in the space experiment was compared with a quick freeze in Liquid Nitrogen (LN2). The RNA from samples preserved in RNAlater at room temperature for 24 h was slightly more degraded than the RNA from the LN2 preserved samples (RNA integrity number, RIN: 7.7 and 8.6, respectively). However, the RNA quality and quantity was satisfactory for microarray analysis. Of the genes analysed, 74 genes (0.28%) were significantly differentially expressed, most of them showing moderate to low regulation. Among the genes induced in the RNAlater preserved samples, three salt inducible transcription factors (ZAT10, SZF1 and SZF2) were identified, suggesting that the high salt concentration in RNAlater causes salt stress before the transcription stopped. In conclusion, the Multigen-2 preservation protocol tested here will allow for the genes regulated by microgravity in the space experiment to be revealed. The results do indicate that not all the biological processes are stopped instantly by the RNAlater. The limited diffusion indirectly caused by the microgravity may potentially result in a different degree of salt stress in the test compared to the 1 × g control during the space experiment. This has to be accounted for during the evaluation of the results. Since slightly degraded RNA was observed, further optimalisation of the preservation protocol will be performed.

A perspective on microarrays: current applications, pitfalls, and potential uses

PubMed Central

Jaluria, Pratik; Konstantopoulos, Konstantinos; Betenbaugh, Michael; Shiloach, Joseph

2007-01-01

With advances in robotics, computational capabilities, and the fabrication of high quality glass slides coinciding with increased genomic information being available on public databases, microarray technology is increasingly being used in laboratories around the world. In fact, fields as varied as: toxicology, evolutionary biology, drug development and production, disease characterization, diagnostics development, cellular physiology and stress responses, and forensics have benefiting from its use. However, for many researchers not familiar with microarrays, current articles and reviews often address neither the fundamental principles behind the technology nor the proper designing of experiments. Although, microarray technology is relatively simple, conceptually, its practice does require careful planning and detailed understanding of the limitations inherently present. Without these considerations, it can be exceedingly difficult to ascertain valuable information from microarray data. Therefore, this text aims to outline key features in microarray technology, paying particular attention to current applications as outlined in recent publications, experimental design, statistical methods, and potential uses. Furthermore, this review is not meant to be comprehensive, but rather substantive; highlighting important concepts and detailing steps necessary to conduct and interpret microarray experiments. Collectively, the information included in this text will highlight the versatility of microarray technology and provide a glimpse of what the future may hold. PMID:17254338

Heterologous oligonucleotide microarrays for transcriptomics in a non-model species; a proof-of-concept study of drought stress in Musa

PubMed Central

Davey, Mark W; Graham, Neil S; Vanholme, Bartel; Swennen, Rony; May, Sean T; Keulemans, Johan

2009-01-01

Background 'Systems-wide' approaches such as microarray RNA-profiling are ideally suited to the study of the complex overlapping responses of plants to biotic and abiotic stresses. However, commercial microarrays are only available for a limited number of plant species and development costs are so substantial as to be prohibitive for most research groups. Here we evaluate the use of cross-hybridisation to Affymetrix oligonucleotide GeneChip® microarrays to profile the response of the banana (Musa spp.) leaf transcriptome to drought stress using a genomic DNA (gDNA)-based probe-selection strategy to improve the efficiency of detection of differentially expressed Musa transcripts. Results Following cross-hybridisation of Musa gDNA to the Rice GeneChip® Genome Array, ~33,700 gene-specific probe-sets had a sufficiently high degree of homology to be retained for transcriptomic analyses. In a proof-of-concept approach, pooled RNA representing a single biological replicate of control and drought stressed leaves of the Musa cultivar 'Cachaco' were hybridised to the Affymetrix Rice Genome Array. A total of 2,910 Musa gene homologues with a >2-fold difference in expression levels were subsequently identified. These drought-responsive transcripts included many functional classes associated with plant biotic and abiotic stress responses, as well as a range of regulatory genes known to be involved in coordinating abiotic stress responses. This latter group included members of the ERF, DREB, MYB, bZIP and bHLH transcription factor families. Fifty-two of these drought-sensitive Musa transcripts were homologous to genes underlying QTLs for drought and cold tolerance in rice, including in 2 instances QTLs associated with a single underlying gene. The list of drought-responsive transcripts also included genes identified in publicly-available comparative transcriptomics experiments. Conclusion Our results demonstrate that despite the general paucity of nucleotide sequence data in Musa and only distant phylogenetic relations to rice, gDNA probe-based cross-hybridisation to the Rice GeneChip® is a highly promising strategy to study complex biological responses and illustrates the potential of such strategies for gene discovery in non-model species. PMID:19758430

The global response of Nostoc punctiforme ATCC 29133 to UVA stress, assessed in a temporal DNA microarray study.

PubMed

Soule, Tanya; Gao, Qunjie; Stout, Valerie; Garcia-Pichel, Ferran

2013-01-01

Cyanobacteria in nature are exposed not only to the visible spectrum of sunlight but also to its harmful ultraviolet components (UVA and UVB). We used Nostoc punctiforme ATCC 29133 as a model to study the UVA response by analyzing global gene expression patterns using genomic microarrays. UVA exposure resulted in the statistically detectable differential expression of 573 genes of the 6903 that were probed, compared with that of the control cultures. Of those genes, 473 were up-regulated, while only 100 were down-regulated. Many of the down-regulated genes were involved in photosynthetic pigment biosynthesis, indicating a significant shift in this metabolism. As expected, we detected the up-regulation of genes encoding antioxidant enzymes and the sunscreen, scytonemin. However, a majority of the up-regulated genes, 47%, were unassignable bioinformatically to known functional categories, suggesting that the UVA stress response is not well understood. Interestingly, the most dramatic up-regulation involved several contiguous genes of unassigned metabolism on plasmid A. This is the first global UVA stress response analysis of any phototrophic microorganism and the differential expression of 8% of the genes of the Nostoc genome indicates that adaptation to UVA in Nostoc has been an evolutionary force of significance. © 2012 Wiley Periodicals, Inc. Photochemistry and Photobiology © 2012 The American Society of Photobiology.

Whole-genome transcriptional analysis of heavy metal stresses inCaulobacter crescentus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Ping; Brodie, Eoin L.; Suzuki, Yohey

2005-09-21

The bacterium Caulobacter crescentus and related stalkbacterial species are known for their distinctive ability to live in lownutrient environments, a characteristic of most heavy metal contaminatedsites. Caulobacter crescentus is a model organism for studying cell cycleregulation with well developed genetics. We have identified the pathwaysresponding to heavy metal toxicity in C. crescentus to provide insightsfor possible application of Caulobacter to environmental restoration. Weexposed C. crescentus cells to four heavy metals (chromium, cadmium,selenium and uranium) and analyzed genome wide transcriptional activitiespost exposure using a Affymetrix GeneChip microarray. C. crescentusshowed surprisingly high tolerance to uranium, a possible mechanism forwhich may be formationmore » of extracellular calcium-uranium-phosphateprecipitates. The principal response to these metals was protectionagainst oxidative stress (up-regulation of manganese-dependent superoxidedismutase, sodA). Glutathione S-transferase, thioredoxin, glutaredoxinsand DNA repair enzymes responded most strongly to cadmium and chromate.The cadmium and chromium stress response also focused on reducing theintracellular metal concentration, with multiple efflux pumps employed toremove cadmium while a sulfate transporter was down-regulated to reducenon-specific uptake of chromium. Membrane proteins were also up-regulatedin response to most of the metals tested. A two-component signaltransduction system involved in the uranium response was identified.Several differentially regulated transcripts from regions previously notknown to encode proteins were identified, demonstrating the advantage ofevaluating the transcriptome using whole genome microarrays.« less

Mitochondrial dysfunction, oxidative stress and apoptosis revealed by proteomic and transcriptomic analyses of the striata in two mouse models of Parkinson’s disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chin, Mark H.; Qian, Weijun; Wang, Haixing

2008-02-10

The molecular mechanisms underlying the changes in the nigrostriatal pathway in Parkinson disease (PD) are not completely understood. Here we use mass spectrometry and microarrays to study the proteomic and transcriptomic changes in the striatum of two mouse models of PD, induced by the distinct neurotoxins 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and methamphetamine (METH). Proteomic analyses resulted in the identification and relative quantification of 912 proteins with two or more unique peptides and 85 proteins with significant abundance changes following neurotoxin treatment. Similarly, microarray analyses revealed 181 genes with significant changes in mRNA following neurotoxin treatment. The combined protein and gene list providesmore » a clearer picture of the potential mechanisms underlying neurodegeneration observed in PD. Functional analysis of this combined list revealed a number of significant categories, including mitochondrial dysfunction, oxidative stress response and apoptosis. Additionally, codon usage and miRNAs may play an important role in translational control in the striatum. These results constitute one of the largest datasets integrating protein and transcript changes for these neurotoxin models with many similar endpoint phenotypes but distinct mechanisms.« less

Assessing differential expression in two-color microarrays: a resampling-based empirical Bayes approach.

PubMed

Li, Dongmei; Le Pape, Marc A; Parikh, Nisha I; Chen, Will X; Dye, Timothy D

2013-01-01

Microarrays are widely used for examining differential gene expression, identifying single nucleotide polymorphisms, and detecting methylation loci. Multiple testing methods in microarray data analysis aim at controlling both Type I and Type II error rates; however, real microarray data do not always fit their distribution assumptions. Smyth's ubiquitous parametric method, for example, inadequately accommodates violations of normality assumptions, resulting in inflated Type I error rates. The Significance Analysis of Microarrays, another widely used microarray data analysis method, is based on a permutation test and is robust to non-normally distributed data; however, the Significance Analysis of Microarrays method fold change criteria are problematic, and can critically alter the conclusion of a study, as a result of compositional changes of the control data set in the analysis. We propose a novel approach, combining resampling with empirical Bayes methods: the Resampling-based empirical Bayes Methods. This approach not only reduces false discovery rates for non-normally distributed microarray data, but it is also impervious to fold change threshold since no control data set selection is needed. Through simulation studies, sensitivities, specificities, total rejections, and false discovery rates are compared across the Smyth's parametric method, the Significance Analysis of Microarrays, and the Resampling-based empirical Bayes Methods. Differences in false discovery rates controls between each approach are illustrated through a preterm delivery methylation study. The results show that the Resampling-based empirical Bayes Methods offer significantly higher specificity and lower false discovery rates compared to Smyth's parametric method when data are not normally distributed. The Resampling-based empirical Bayes Methods also offers higher statistical power than the Significance Analysis of Microarrays method when the proportion of significantly differentially expressed genes is large for both normally and non-normally distributed data. Finally, the Resampling-based empirical Bayes Methods are generalizable to next generation sequencing RNA-seq data analysis.

Transfection microarray and the applications.

PubMed

Miyake, Masato; Yoshikawa, Tomohiro; Fujita, Satoshi; Miyake, Jun

2009-05-01

Microarray transfection has been extensively studied for high-throughput functional analysis of mammalian cells. However, control of efficiency and reproducibility are the critical issues for practical use. By using solid-phase transfection accelerators and nano-scaffold, we provide a highly efficient and reproducible microarray-transfection device, "transfection microarray". The device would be applied to the limited number of available primary cells and stem cells not only for large-scale functional analysis but also reporter-based time-lapse cellular event analysis.

Gene Expression Profiling of the Hypoxia Signaling Pathway in Hypoxia-Inducible Factor 1α Null Mouse Embryonic Fibroblasts

PubMed Central

Vengellur, Ajith; Woods, Barbara G.; Ryan, Heather E.; Johnson, Randall S.; Lapres, John J.

2003-01-01

Hypoxia is defined as a deficiency of oxygen reaching the tissues of the body, and it plays a critical role in development and pathological conditions, such as cancer. Once tumors outgrow their blood supply, their central portion becomes hypoxic and the tumor stimulates angiogenesis through the activation of the hypoxia-inducible factors (HIFs). HIFs are transcription factors that are regulated in an oxygen-dependent manner by a group of prolyl hydroxylases (known as PHDs or HPHs). Our understanding of hypoxia signaling is limited by our incomplete knowledge of HIF target genes. cDNA microarrays and a cell line lacking a principal HIF protein, HIF1α, were used to identify a more complete set of hypoxia-regulated genes. The microarrays identified a group of 286 clones that were significantly influenced by hypoxia and 54 of these were coordinately regulated by cobalt chloride. The expression profile of HIF1α −/− cells also identified a group of downregulated genes encoding enzymes involved in protecting cells from oxidative stress, offering an explanation for the increased sensitivity of HIF1α −/− cells to agents that promote this type of response. The microarray studies confirmed the hypoxia-induced expression of the HIF regulating prolyl hydroxylase, PHD2. An analysis of the members of the PHD family revealed that they are differentially regulated by cobalt chloride and hypoxia. These results suggest that HIF1α is the predominant isoform in fibroblasts and that it regulates a wide battery of genes critical for normal cellular function and survival under various stresses. PMID:14686790

Analysis of ripening-related gene expression in papaya using an Arabidopsis-based microarray

PubMed Central

2012-01-01

Background Papaya (Carica papaya L.) is a commercially important crop that produces climacteric fruits with a soft and sweet pulp that contain a wide range of health promoting phytochemicals. Despite its importance, little is known about transcriptional modifications during papaya fruit ripening and their control. In this study we report the analysis of ripe papaya transcriptome by using a cross-species (XSpecies) microarray technique based on the phylogenetic proximity between papaya and Arabidopsis thaliana. Results Papaya transcriptome analyses resulted in the identification of 414 ripening-related genes with some having their expression validated by qPCR. The transcription profile was compared with that from ripening tomato and grape. There were many similarities between papaya and tomato especially with respect to the expression of genes encoding proteins involved in primary metabolism, regulation of transcription, biotic and abiotic stress and cell wall metabolism. XSpecies microarray data indicated that transcription factors (TFs) of the MADS-box, NAC and AP2/ERF gene families were involved in the control of papaya ripening and revealed that cell wall-related gene expression in papaya had similarities to the expression profiles seen in Arabidopsis during hypocotyl development. Conclusion The cross-species array experiment identified a ripening-related set of genes in papaya allowing the comparison of transcription control between papaya and other fruit bearing taxa during the ripening process. PMID:23256600

Microarray technology for major chemical contaminants analysis in food: current status and prospects.

PubMed

Zhang, Zhaowei; Li, Peiwu; Hu, Xiaofeng; Zhang, Qi; Ding, Xiaoxia; Zhang, Wen

2012-01-01

Chemical contaminants in food have caused serious health issues in both humans and animals. Microarray technology is an advanced technique suitable for the analysis of chemical contaminates. In particular, immuno-microarray approach is one of the most promising methods for chemical contaminants analysis. The use of microarrays for the analysis of chemical contaminants is the subject of this review. Fabrication strategies and detection methods for chemical contaminants are discussed in detail. Application to the analysis of mycotoxins, biotoxins, pesticide residues, and pharmaceutical residues is also described. Finally, future challenges and opportunities are discussed.

Transcriptome profiling of low temperature-treated cassava apical shoots showed dynamic responses of tropical plant to cold stress

PubMed Central

2012-01-01

Background Cassava is an important tropical root crop adapted to a wide range of environmental stimuli such as drought and acid soils. Nevertheless, it is an extremely cold-sensitive tropical species. Thus far, there is limited information about gene regulation and signalling pathways related to the cold stress response in cassava. The development of microarray technology has accelerated the study of global transcription profiling under certain conditions. Results A 60-mer oligonucleotide microarray representing 20,840 genes was used to perform transcriptome profiling in apical shoots of cassava subjected to cold at 7°C for 0, 4 and 9 h. A total of 508 transcripts were identified as early cold-responsive genes in which 319 sequences had functional descriptions when aligned with Arabidopsis proteins. Gene ontology annotation analysis identified many cold-relevant categories, including 'Response to abiotic and biotic stimulus', 'Response to stress', 'Transcription factor activity', and 'Chloroplast'. Various stress-associated genes with a wide range of biological functions were found, such as signal transduction components (e.g., MAP kinase 4), transcription factors (TFs, e.g., RAP2.11), and reactive oxygen species (ROS) scavenging enzymes (e.g., catalase 2), as well as photosynthesis-related genes (e.g., PsaL). Seventeen major TF families including many well-studied members (e.g., AP2-EREBP) were also involved in the early response to cold stress. Meanwhile, KEGG pathway analysis uncovered many important pathways, such as 'Plant hormone signal transduction' and 'Starch and sucrose metabolism'. Furthermore, the expression changes of 32 genes under cold and other abiotic stress conditions were validated by real-time RT-PCR. Importantly, most of the tested stress-responsive genes were primarily expressed in mature leaves, stem cambia, and fibrous roots rather than apical buds and young leaves. As a response to cold stress in cassava, an increase in transcripts and enzyme activities of ROS scavenging genes and the accumulation of total soluble sugars (including sucrose and glucose) were also detected. Conclusions The dynamic expression changes reflect the integrative controlling and transcriptome regulation of the networks in the cold stress response of cassava. The biological processes involved in the signal perception and physiological response might shed light on the molecular mechanisms related to cold tolerance in tropical plants and provide useful candidate genes for genetic improvement. PMID:22321773

DNA microarray analyses reveal a post-irradiation differential time-dependent gene expression profile in yeast cells exposed to X-rays and gamma-rays.

PubMed

Kimura, Shinzo; Ishidou, Emi; Kurita, Sakiko; Suzuki, Yoshiteru; Shibato, Junko; Rakwal, Randeep; Iwahashi, Hitoshi

2006-07-21

Ionizing radiation (IR) is the most enigmatic of genotoxic stress inducers in our environment that has been around from the eons of time. IR is generally considered harmful, and has been the subject of numerous studies, mostly looking at the DNA damaging effects in cells and the repair mechanisms therein. Moreover, few studies have focused on large-scale identification of cellular responses to IR, and to this end, we describe here an initial study on the transcriptional responses of the unicellular genome model, yeast (Saccharomyces cerevisiae strain S288C), by cDNA microarray. The effect of two different IR, X-rays, and gamma (gamma)-rays, was investigated by irradiating the yeast cells cultured in YPD medium with 50 Gy doses of X- and gamma-rays, followed by resuspension of the cells in YPD for time-course experiments. The samples were collected for microarray analysis at 20, 40, and 80 min after irradiation. Microarray analysis revealed a time-course transcriptional profile of changed gene expressions. Up-regulated genes belonged to the functional categories mainly related to cell cycle and DNA processing, cell rescue defense and virulence, protein and cell fate, and metabolism (X- and gamma-rays). Similarly, for X- and gamma-rays, the down-regulated genes belonged to mostly transcription and protein synthesis, cell cycle and DNA processing, control of cellular organization, cell fate, and C-compound and carbohydrate metabolism categories, respectively. This study provides for the first time a snapshot of the genome-wide mRNA expression profiles in X- and gamma-ray post-irradiated yeast cells and comparatively interprets/discusses the changed gene functional categories as effects of these two radiations vis-à-vis their energy levels.

Differential gene expression profiles of peripheral blood mononuclear cells in childhood asthma.

PubMed

Kong, Qian; Li, Wen-Jing; Huang, Hua-Rong; Zhong, Ying-Qiang; Fang, Jian-Pei

2015-05-01

Asthma is a common childhood disease with strong genetic components. This study compared whole-genome expression differences between asthmatic young children and healthy controls to identify gene signatures of childhood asthma. Total RNA extracted from peripheral blood mononuclear cells (PBMC) was subjected to microarray analysis. QRT-PCR was performed to verify the microarray results. Classification and functional characterization of differential genes were illustrated by hierarchical clustering and gene ontology analysis. Multiple logistic regression (MLR) analysis, receiver operating characteristic (ROC) curve analysis, and discriminate power were used to scan asthma-specific diagnostic markers. For fold-change>2 and p < 0.05, there were 758 named differential genes. The results of QRT-PCR confirmed successfully the array data. Hierarchical clustering divided 29 highly possible genes into seven categories and the genes in the same cluster were likely to possess similar expression patterns or functions. Gene ontology analysis presented that differential genes primarily enriched in immune response, response to stress or stimulus, and regulation of apoptosis in biological process. MLR and ROC curve analysis revealed that the combination of ADAM33, Smad7, and LIGHT possessed excellent discriminating power. The combination of ADAM33, Smad7, and LIGHT would be a reliable and useful childhood asthma model for prediction and diagnosis.

Contributions to Statistical Problems Related to Microarray Data

ERIC Educational Resources Information Center

Hong, Feng

2009-01-01

Microarray is a high throughput technology to measure the gene expression. Analysis of microarray data brings many interesting and challenging problems. This thesis consists three studies related to microarray data. First, we propose a Bayesian model for microarray data and use Bayes Factors to identify differentially expressed genes. Second, we…

Overexpression of Grain Amaranth (Amaranthus hypochondriacus) AhERF or AhDOF Transcription Factors in Arabidopsis thaliana Increases Water Deficit- and Salt-Stress Tolerance, Respectively, via Contrasting Stress-Amelioration Mechanisms

PubMed Central

Massange-Sánchez, Julio A.; Palmeros-Suárez, Paola A.; Espitia-Rangel, Eduardo; Rodríguez-Arévalo, Isaac; Sánchez-Segura, Lino; Martínez-Gallardo, Norma A.; Alatorre-Cobos, Fulgencio; Tiessen, Axel; Délano-Frier, John P.

2016-01-01

Two grain amaranth transcription factor (TF) genes were overexpressed in Arabidopsis plants. The first, coding for a group VII ethylene response factor TF (i.e., AhERF-VII) conferred tolerance to water-deficit stress (WS) in transgenic Arabidopsis without affecting vegetative or reproductive growth. A significantly lower water-loss rate in detached leaves coupled to a reduced stomatal opening in leaves of plants subjected to WS was associated with this trait. WS tolerance was also associated with an increased antioxidant enzyme activity and the accumulation of putative stress-related secondary metabolites. However, microarray and GO data did not indicate an obvious correlation between WS tolerance, stomatal closure, and abscisic acid (ABA)-related signaling. This scenario suggested that stomatal closure during WS in these plants involved ABA-independent mechanisms, possibly involving reactive oxygen species (ROS). WS tolerance may have also involved other protective processes, such as those employed for methyl glyoxal detoxification. The second, coding for a class A and cluster I DNA binding with one finger TF (i.e., AhDof-AI) provided salt-stress (SS) tolerance with no evident fitness penalties. The lack of an obvious development-related phenotype contrasted with microarray and GO data showing an enrichment of categories and genes related to developmental processes, particularly flowering. SS tolerance also correlated with increased superoxide dismutase activity but not with augmented stomatal closure. Additionally, microarray and GO data indicated that, contrary to AhERF-VII, SS tolerance conferred by AhDof-AI in Arabidopsis involved ABA-dependent and ABA-independent stress amelioration mechanisms. PMID:27749893

Gene expression profiling in respond to TBT exposure in small abalone Haliotis diversicolor.

PubMed

Jia, Xiwei; Zou, Zhihua; Wang, Guodong; Wang, Shuhong; Wang, Yilei; Zhang, Ziping

2011-10-01

In this study, we investigated the gene expression profiling of small abalone, Haliotis diversicolor by tributyltin (TBT) exposure using a cDNA microarray containing 2473 unique transcripts. Totally, 107 up-regulated genes and 41 down-regulated genes were found. For further investigation of candidate genes from microarray data and EST analysis, quantitative real-time PCR was performed at 6 h, 24 h, 48 h, 96 h and 192 h TBT exposure. 26 genes were found to be significantly differentially expressed in different time course, 3 of them were unknown. Some gene homologues like cellulose, endo-beta-1,4-glucanase, ferritin subunit 1 and thiolester containing protein II CG7052-PB might be the good biomarker candidate for TBT monitor. The identification of stress response genes and their expression profiles will permit detailed investigation of the defense responses of small abalone genes. Published by Elsevier Ltd.

Microarrays in brain research: the good, the bad and the ugly.

PubMed

Mirnics, K

2001-06-01

Making sense of microarray data is a complex process, in which the interpretation of findings will depend on the overall experimental design and judgement of the investigator performing the analysis. As a result, differences in tissue harvesting, microarray types, sample labelling and data analysis procedures make post hoc sharing of microarray data a great challenge. To ensure rapid and meaningful data exchange, we need to create some order out of the existing chaos. In these ground-breaking microarray standardization and data sharing efforts, NIH agencies should take a leading role

HDBStat!: a platform-independent software suite for statistical analysis of high dimensional biology data.

PubMed

Trivedi, Prinal; Edwards, Jode W; Wang, Jelai; Gadbury, Gary L; Srinivasasainagendra, Vinodh; Zakharkin, Stanislav O; Kim, Kyoungmi; Mehta, Tapan; Brand, Jacob P L; Patki, Amit; Page, Grier P; Allison, David B

2005-04-06

Many efforts in microarray data analysis are focused on providing tools and methods for the qualitative analysis of microarray data. HDBStat! (High-Dimensional Biology-Statistics) is a software package designed for analysis of high dimensional biology data such as microarray data. It was initially developed for the analysis of microarray gene expression data, but it can also be used for some applications in proteomics and other aspects of genomics. HDBStat! provides statisticians and biologists a flexible and easy-to-use interface to analyze complex microarray data using a variety of methods for data preprocessing, quality control analysis and hypothesis testing. Results generated from data preprocessing methods, quality control analysis and hypothesis testing methods are output in the form of Excel CSV tables, graphs and an Html report summarizing data analysis. HDBStat! is a platform-independent software that is freely available to academic institutions and non-profit organizations. It can be downloaded from our website http://www.soph.uab.edu/ssg_content.asp?id=1164.

Combining suppressive subtractive hybridization and cDNA microarrays to identify dietary phosphorus-responsive genes of the rainbow trout (Oncorhynchus mykiss) kidney.

PubMed

Lake, Jennifer; Gravel, Catherine; Koko, Gabriel Koffi D; Robert, Claude; Vandenberg, Grant W

2010-03-01

Phosphorus (P)-responsive genes and how they regulate renal adaptation to phosphorous-deficient diets in animals, including fish, are not well understood. RNA abundance profiling using cDNA microarrays is an efficient approach to study nutrient-gene interactions and identify these dietary P-responsive genes. To test the hypothesis that dietary P-responsive genes are differentially expressed in fish fed varying P levels, rainbow trout were fed a practical high-P diet (R20: 0.96% P) or a low-P diet (R0: 0.38% P) for 7 weeks. The differentially-expressed genes between dietary groups were identified and compared from the kidney by combining suppressive subtractive hybridization (SSH) with cDNA microarray analysis. A number of genes were confirmed by real-time PCR, and correlated with plasma and bone P concentrations. Approximately 54 genes were identified as potential dietary P-responsive after 7 weeks on a diet deficient in P according to cDNA microarray analysis. Of 18 selected genes, 13 genes were confirmed to be P-responsive at 7 weeks by real-time PCR analysis, including: iNOS, cytochrome b, cytochrome c oxidase subunit II , alpha-globin I, beta-globin, ATP synthase, hyperosmotic protein 21, COL1A3, Nkef, NDPK, glucose phosphate isomerase 1, Na+/H+ exchange protein and GDP dissociation inhibitor 2. Many of these dietary P-responsive genes responded in a moderate way (R0/R20 ratio: <2-3 or >0.5) and in a transient manner to dietary P limitation. In summary, renal adaptation to dietary P deficiency in trout involves changes in the expression of several genes, suggesting a profile of metabolic stress, since many of these differentially-expressed candidates are associated with the cellular adaptative responses. Crown Copyright 2009. Published by Elsevier Inc. All rights reserved.

Rice tolerance to suboptimal low temperatures relies on the maintenance of the photosynthetic capacity.

PubMed

Gazquez, Ayelén; Vilas, Juan Manuel; Colman Lerner, Jorge Esteban; Maiale, Santiago Javier; Calzadilla, Pablo Ignacio; Menéndez, Ana Bernardina; Rodríguez, Andrés Alberto

2018-06-01

The purpose of this research was to identify differences between two contrasting rice cultivars in their response to suboptimal low temperatures stress. A transcriptomic analysis of the seedlings was performed and results were complemented with biochemical and physiological analyses. The microarray analysis showed downregulation of many genes related with PSII and particularly with the oxygen evolving complex in the sensitive cultivar IR50. Complementary studies indicated that the PSII performance, the degree of oxygen evolving complex coupling with the PSII core and net photosynthetic rate diminished in this cultivar in response to the stress. However, the tolerant cultivar Koshihikari was able to maintain its energy equilibrium by sustaining the photosynthetic capacity. The increase of oleic acid in Koshihikari could be related with membrane remodelling of the chloroplasts and hence contribute to tolerance. Overall, these results work as a ground for future analyses that look forward to characterize possible mechanisms to tolerate this stress. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Transcriptome Analysis of Escherichia coli O157:H7 Exposed to Lysates of Lettuce Leaves ▿

PubMed Central

Kyle, Jennifer L.; Parker, Craig T.; Goudeau, Danielle; Brandl, Maria T.

2010-01-01

Harvesting and processing of leafy greens inherently cause plant tissue damage, creating niches on leaves that human pathogens can exploit. We previously demonstrated that Escherichia coli O157:H7 (EcO157) multiplies more rapidly on shredded leaves than on intact leaves (M. T. Brandl, Appl. Environ. Microbiol. 74:5285-5289, 2008). To investigate how EcO157 cells adapt to physicochemical conditions in injured lettuce tissue, we used microarray-based whole-genome transcriptional profiling to characterize gene expression patterns in EcO157 after 15- and 30-min exposures to romaine lettuce lysates. Multiple carbohydrate transport systems that have a role in the utilization of substrates known to be prevalent in plant cells were activated in EcO157. This indicates the availability to the human pathogen of a variety of carbohydrates released from injured plant cells that may promote its extensive growth in leaf lysates and, thus, in wounded leaf tissue. In addition, microarray analysis revealed the upregulation of numerous genes associated with EcO157 attachment and virulence, with oxidative stress and antimicrobial resistance (including the OxyR and Mar regulons), with detoxification of noxious compounds, and with DNA repair. Upregulation of oxidative stress and antimicrobial resistance genes in EcO157 was confirmed on shredded lettuce by quantitative reverse transcription-PCR. We further demonstrate that this adaptation to stress conditions imparts the pathogen with increased resistance to hydrogen peroxide and calcium hypochlorite. This enhanced resistance to chlorinated sanitizers combined with increased expression of virulence determinants and multiplication at sites of injury on the leaves may help explain the association of processed leafy greens with outbreaks of EcO157. PMID:20061451

Transcriptome response to elevated CO2, water deficit, and thermal stress in peanut

USDA-ARS?s Scientific Manuscript database

Previously, our laboratories have performed gene expression studies using EST sequencing and spotted microarrays to investigate tissue-specific gene expression and response to abiotic stress. While these studies have provided valuable insight into these processes, they are constrained by sequencer t...

A close connection between the PERK and IRE arms of the UPR and the transcriptional regulation of autophagy.

PubMed

Deegan, Shane; Koryga, Izabela; Glynn, Sharon A; Gupta, Sanjeev; Gorman, Adrienne M; Samali, Afshin

2015-01-02

Endoplasmic reticulum (ER) stress is known to lead to activation of both the unfolded protein response (UPR) and autophagy. Although regulatory connections have been identified between the UPR and autophagy, it is still unclear to what extent the UPR regulates the genes involved at the different stages of the autophagy pathway. Here, we carried out a microarray analysis of HCT116 cells subjected to ER stress and observed the transcriptional upregulation of a large cohort of autophagy-related genes. Of particular interest, we identified the transcriptional upregulation of the autophagy receptor genes SQSTM1/p62, NBR1 and BNIP3L/NIX in response to ER stress and show that the inhibition of the UPR transmembrane receptors, PERK and IRE1, abrogates this upregulation. Copyright © 2014 Elsevier Inc. All rights reserved.

ArrayNinja: An Open Source Platform for Unified Planning and Analysis of Microarray Experiments.

PubMed

Dickson, B M; Cornett, E M; Ramjan, Z; Rothbart, S B

2016-01-01

Microarray-based proteomic platforms have emerged as valuable tools for studying various aspects of protein function, particularly in the field of chromatin biochemistry. Microarray technology itself is largely unrestricted in regard to printable material and platform design, and efficient multidimensional optimization of assay parameters requires fluidity in the design and analysis of custom print layouts. This motivates the need for streamlined software infrastructure that facilitates the combined planning and analysis of custom microarray experiments. To this end, we have developed ArrayNinja as a portable, open source, and interactive application that unifies the planning and visualization of microarray experiments and provides maximum flexibility to end users. Array experiments can be planned, stored to a private database, and merged with the imaged results for a level of data interaction and centralization that is not currently attainable with available microarray informatics tools. © 2016 Elsevier Inc. All rights reserved.

Genome-Wide Identification, Evolutionary Expansion, and Expression Profile of Homeodomain-Leucine Zipper Gene Family in Poplar (Populus trichocarpa)

PubMed Central

Hu, Ruibo; Chi, Xiaoyuan; Chai, Guohua; Kong, Yingzhen; He, Guo; Wang, Xiaoyu; Shi, Dachuan; Zhang, Dongyuan; Zhou, Gongke

2012-01-01

Background Homeodomain-leucine zipper (HD-ZIP) proteins are plant-specific transcriptional factors known to play crucial roles in plant development. Although sequence phylogeny analysis of Populus HD-ZIPs was carried out in a previous study, no systematic analysis incorporating genome organization, gene structure, and expression compendium has been conducted in model tree species Populus thus far. Principal Findings In this study, a comprehensive analysis of Populus HD-ZIP gene family was performed. Sixty-three full-length HD-ZIP genes were found in Populus genome. These Populus HD-ZIP genes were phylogenetically clustered into four distinct subfamilies (HD-ZIP I–IV) and predominately distributed across 17 linkage groups (LG). Fifty genes from 25 Populus paralogous pairs were located in the duplicated blocks of Populus genome and then preferentially retained during the sequential evolutionary courses. Genomic organization analyses indicated that purifying selection has played a pivotal role in the retention and maintenance of Populus HD-ZIP gene family. Microarray analysis has shown that 21 Populus paralogous pairs have been differentially expressed across different tissues and under various stresses, with five paralogous pairs showing nearly identical expression patterns, 13 paralogous pairs being partially redundant and three paralogous pairs diversifying significantly. Quantitative real-time RT-PCR (qRT-PCR) analysis performed on 16 selected Populus HD-ZIP genes in different tissues and under both drought and salinity stresses confirms their tissue-specific and stress-inducible expression patterns. Conclusions Genomic organizations indicated that segmental duplications contributed significantly to the expansion of Populus HD-ZIP gene family. Exon/intron organization and conserved motif composition of Populus HD-ZIPs are highly conservative in the same subfamily, suggesting the members in the same subfamilies may also have conservative functionalities. Microarray and qRT-PCR analyses showed that 89% (56 out of 63) of Populus HD-ZIPs were duplicate genes that might have been retained by substantial subfunctionalization. Taken together, these observations may lay the foundation for future functional analysis of Populus HD-ZIP genes to unravel their biological roles. PMID:22359569

Microarray analysis identifies candidate genes for key roles in coral development

PubMed Central

Grasso, Lauretta C; Maindonald, John; Rudd, Stephen; Hayward, David C; Saint, Robert; Miller, David J; Ball, Eldon E

2008-01-01

Background Anthozoan cnidarians are amongst the simplest animals at the tissue level of organization, but are surprisingly complex and vertebrate-like in terms of gene repertoire. As major components of tropical reef ecosystems, the stony corals are anthozoans of particular ecological significance. To better understand the molecular bases of both cnidarian development in general and coral-specific processes such as skeletogenesis and symbiont acquisition, microarray analysis was carried out through the period of early development – when skeletogenesis is initiated, and symbionts are first acquired. Results Of 5081 unique peptide coding genes, 1084 were differentially expressed (P ≤ 0.05) in comparisons between four different stages of coral development, spanning key developmental transitions. Genes of likely relevance to the processes of settlement, metamorphosis, calcification and interaction with symbionts were characterised further and their spatial expression patterns investigated using whole-mount in situ hybridization. Conclusion This study is the first large-scale investigation of developmental gene expression for any cnidarian, and has provided candidate genes for key roles in many aspects of coral biology, including calcification, metamorphosis and symbiont uptake. One surprising finding is that some of these genes have clear counterparts in higher animals but are not present in the closely-related sea anemone Nematostella. Secondly, coral-specific processes (i.e. traits which distinguish corals from their close relatives) may be analogous to similar processes in distantly related organisms. This first large-scale application of microarray analysis demonstrates the potential of this approach for investigating many aspects of coral biology, including the effects of stress and disease. PMID:19014561

Gene expression profile in cardiovascular disease and preeclampsia: a meta-analysis of the transcriptome based on raw data from human studies deposited in Gene Expression Omnibus.

PubMed

Sitras, V; Fenton, C; Acharya, G

2015-02-01

Cardiovascular disease (CVD) and preeclampsia (PE) share common clinical features. We aimed to identify common transcriptomic signatures involved in CVD and PE in humans. Meta-analysis of individual raw microarray data deposited in GEO, obtained from blood samples of patients with CVD versus controls and placental samples from women with PE versus healthy women with uncomplicated pregnancies. Annotation of cases versus control samples was taken directly from the microarray documentation. Genes that showed a significant differential expression in the majority of experiments were selected for subsequent analysis. Hypergeometric gene list analysis was performed using Bioconductor GOstats package. Bioinformatic analysis was performed in PANTHER. Seven studies in CVD and 5 studies in PE were eligible for meta-analysis. A total of 181 genes were found to be differentially expressed in microarray studies investigating gene expression in blood samples obtained from patients with CVD compared to controls and 925 genes were differentially expressed between preeclamptic and healthy placentas. Among these differentially expressed genes, 22 were common between CVD and PE. Bioinformatic analysis of these genes revealed oxidative stress, p-53 pathway feedback, inflammation mediated by chemokines and cytokines, interleukin signaling, B-cell activation, PDGF signaling, Wnt signaling, integrin signaling and Alzheimer disease pathways to be involved in the pathophysiology of both CVD and PE. Metabolism, development, response to stimulus, immune response and cell communication were the associated biologic processes in both conditions. Gene set enrichment analysis showed the following overlapping pathways between CVD and PE: TGF-β-signaling, apoptosis, graft-versus-host disease, allograft rejection, chemokine signaling, steroid hormone synthesis, type I and II diabetes mellitus, VEGF signaling, pathways in cancer, GNRH signaling, Huntingtons disease and Notch signaling. CVD and PE share same common traits in their gene expression profile indicating common pathways in their pathophysiology. Copyright © 2014 Elsevier Ltd. All rights reserved.

WebArray: an online platform for microarray data analysis

PubMed Central

Xia, Xiaoqin; McClelland, Michael; Wang, Yipeng

2005-01-01

Background Many cutting-edge microarray analysis tools and algorithms, including commonly used limma and affy packages in Bioconductor, need sophisticated knowledge of mathematics, statistics and computer skills for implementation. Commercially available software can provide a user-friendly interface at considerable cost. To facilitate the use of these tools for microarray data analysis on an open platform we developed an online microarray data analysis platform, WebArray, for bench biologists to utilize these tools to explore data from single/dual color microarray experiments. Results The currently implemented functions were based on limma and affy package from Bioconductor, the spacings LOESS histogram (SPLOSH) method, PCA-assisted normalization method and genome mapping method. WebArray incorporates these packages and provides a user-friendly interface for accessing a wide range of key functions of limma and others, such as spot quality weight, background correction, graphical plotting, normalization, linear modeling, empirical bayes statistical analysis, false discovery rate (FDR) estimation, chromosomal mapping for genome comparison. Conclusion WebArray offers a convenient platform for bench biologists to access several cutting-edge microarray data analysis tools. The website is freely available at . It runs on a Linux server with Apache and MySQL. PMID:16371165

Transcript Profiling Reveals the Presence of Abiotic Stress and Developmental Stage Specific Ascorbate Oxidase Genes in Plants

PubMed Central

Batth, Rituraj; Singh, Kapil; Kumari, Sumita; Mustafiz, Ananda

2017-01-01

Abiotic stress and climate change is the major concern for plant growth and crop yield. Abiotic stresses lead to enhanced accumulation of reactive oxygen species (ROS) consequently resulting in cellular damage and major losses in crop yield. One of the major scavengers of ROS is ascorbate (AA) which acts as first line of defense against external oxidants. An enzyme named ascorbate oxidase (AAO) is known to oxidize AA and deleteriously affect the plant system in response to stress. Genome-wide analysis of AAO gene family has led to the identification of five, three, seven, four, and six AAO genes in Oryza sativa, Arabidopsis, Glycine max, Zea mays, and Sorghum bicolor genomes, respectively. Expression profiling of these genes was carried out in response to various abiotic stresses and during various stages of vegetative and reproductive development using publicly available microarray database. Expression analysis in Oryza sativa revealed tissue specific expression of AAO genes wherein few members were exclusively expressed in either root or shoot. These genes were found to be regulated by both developmental cues as well as diverse stress conditions. The qRT-PCR analysis in response to salinity and drought stress in rice shoots revealed OsAAO2 to be the most stress responsive gene. On the other hand, OsAAO3 and OsAAO4 genes showed enhanced expression in roots under salinity/drought stresses. This study provides lead about important stress responsive AAO genes in various crop plants, which could be used to engineer climate resilient crop plants. PMID:28261251

Tissue specific and abiotic stress regulated transcription of histidine kinases in plants is also influenced by diurnal rhythm

PubMed Central

Singh, Anupama; Kushwaha, Hemant R.; Soni, Praveen; Gupta, Himanshu; Singla-Pareek, Sneh L.; Pareek, Ashwani

2015-01-01

Two-component system (TCS) is one of the key signal sensing machinery which enables species to sense environmental stimuli. It essentially comprises of three major components, sensory histidine kinase proteins (HKs), histidine phosphotransfer proteins (Hpts), and response regulator proteins (RRs). The members of the TCS family have already been identified in Arabidopsis and rice but the knowledge about their functional indulgence during various abiotic stress conditions remains meager. Current study is an attempt to carry out comprehensive analysis of the expression of TCS members in response to various abiotic stress conditions and in various plant tissues in Arabidopsis and rice using MPSS and publicly available microarray data. The analysis suggests that despite having almost similar number of genes, rice expresses higher number of TCS members during various abiotic stress conditions than Arabidopsis. We found that the TCS machinery is regulated by not only various abiotic stresses, but also by the tissue specificity. Analysis of expression of some representative members of TCS gene family showed their regulation by the diurnal cycle in rice seedlings, thus bringing-in another level of their transcriptional control. Thus, we report a highly complex and tight regulatory network of TCS members, as influenced by the tissue, abiotic stress signal, and diurnal rhythm. The insights on the comparative expression analysis presented in this study may provide crucial leads toward dissection of diverse role(s) of the various TCS family members in Arabidopsis and rice. PMID:26442025

Evolution of the MIDTAL microarray: the adaption and testing of oligonucleotide 18S and 28S rDNA probes and evaluation of subsequent microarray generations with Prymnesium spp. cultures and field samples.

PubMed

McCoy, Gary R; Touzet, Nicolas; Fleming, Gerard T A; Raine, Robin

2015-07-01

The toxic microalgal species Prymnesium parvum and Prymnesium polylepis are responsible for numerous fish kills causing economic stress on the aquaculture industry and, through the consumption of contaminated shellfish, can potentially impact on human health. Monitoring of toxic phytoplankton is traditionally carried out by light microscopy. However, molecular methods of identification and quantification are becoming more common place. This study documents the optimisation of the novel Microarrays for the Detection of Toxic Algae (MIDTAL) microarray from its initial stages to the final commercial version now available from Microbia Environnement (France). Existing oligonucleotide probes used in whole-cell fluorescent in situ hybridisation (FISH) for Prymnesium species from higher group probes to species-level probes were adapted and tested on the first-generation microarray. The combination and interaction of numerous other probes specific for a whole range of phytoplankton taxa also spotted on the chip surface caused high cross reactivity, resulting in false-positive results on the microarray. The probe sequences were extended for the subsequent second-generation microarray, and further adaptations of the hybridisation protocol and incubation temperatures significantly reduced false-positive readings from the first to the second-generation chip, thereby increasing the specificity of the MIDTAL microarray. Additional refinement of the subsequent third-generation microarray protocols with the addition of a poly-T amino linker to the 5' end of each probe further enhanced the microarray performance but also highlighted the importance of optimising RNA labelling efficiency when testing with natural seawater samples from Killary Harbour, Ireland.

Characterization of barley Prp1 gene and its expression during seed development and under abiotic stress.

PubMed

Jiang, Qian-Tao; Liu, Tao; Ma, Jian; Wei, Yu-Ming; Lu, Zhen-Xiang; Lan, Xiu-Jin; Dai, Shou-Fen; Zheng, You-Liang

2011-10-01

The pre-mRNA processing (Prp1) gene encodes a spliceosomal protein. It was firstly identified in fission yeast and plays a regular role during spliceosome activation and cell cycle. Plant Prp1 genes have only been identified from rice, Sorghum and Arabidopsis thaliana. In this study, we reported the identification and isolation of a novel Prp1 gene from barley, and further explored its expressional pattern by using real-time quantitative RTPCR, promoter prediction and analysis of microarray data. The putative barley Prp1 protein has a similar primary structure features to those of other known Prp1 protein in this family. The results of amino acid comparison indicated that Prp1 protein of barley and other plant species has a highly conserved 30 termnal region while their 50 sequences greatly varied. The results of expressional analysis revealed that the expression level of barley Prp1 gene is always stable in different vegetative tissues, except it is up-regulated at the mid- and late stages of seed development or under the condition of cold stress. This kind of expressional pattern for barley Prp1 is also supported by our results of comparison of microarray data from barley, rice and Arabidopsis. For the molecular mechanism of its expressional pattern, we conclude that the expression of Prp1 gene may be up-regulated by the increase of pre-mRNAs and not be constitutive or ubiquitous.

Reverse-engineering the Arabidopsis thaliana transcriptional network under changing environmental conditions

PubMed Central

Carrera, Javier; Rodrigo, Guillermo; Jaramillo, Alfonso; Elena, Santiago F

2009-01-01

Background Understanding the molecular mechanisms plants have evolved to adapt their biological activities to a constantly changing environment is an intriguing question and one that requires a systems biology approach. Here we present a network analysis of genome-wide expression data combined with reverse-engineering network modeling to dissect the transcriptional control of Arabidopsis thaliana. The regulatory network is inferred by using an assembly of microarray data containing steady-state RNA expression levels from several growth conditions, developmental stages, biotic and abiotic stresses, and a variety of mutant genotypes. Results We show that the A. thaliana regulatory network has the characteristic properties of hierarchical networks. We successfully applied our quantitative network model to predict the full transcriptome of the plant for a set of microarray experiments not included in the training dataset. We also used our model to analyze the robustness in expression levels conferred by network motifs such as the coherent feed-forward loop. In addition, the meta-analysis presented here has allowed us to identify regulatory and robust genetic structures. Conclusions These data suggest that A. thaliana has evolved high connectivity in terms of transcriptional regulation among cellular functions involved in response and adaptation to changing environments, while gene networks constitutively expressed or less related to stress response are characterized by a lower connectivity. Taken together, these findings suggest conserved regulatory strategies that have been selected during the evolutionary history of this eukaryote. PMID:19754933

Issues in the analysis of oligonucleotide tiling microarrays for transcript mapping

NASA Technical Reports Server (NTRS)

Royce, Thomas E.; Rozowsky, Joel S.; Bertone, Paul; Samanta, Manoj; Stolc, Viktor; Weissman, Sherman; Snyder, Michael; Gerstein, Mark

2005-01-01

Traditional microarrays use probes complementary to known genes to quantitate the differential gene expression between two or more conditions. Genomic tiling microarray experiments differ in that probes that span a genomic region at regular intervals are used to detect the presence or absence of transcription. This difference means the same sets of biases and the methods for addressing them are unlikely to be relevant to both types of experiment. We introduce the informatics challenges arising in the analysis of tiling microarray experiments as open problems to the scientific community and present initial approaches for the analysis of this nascent technology.

BRIC-17 Mapping Spaceflight-Induced Hypoxic Signaling and Response in Plants

NASA Technical Reports Server (NTRS)

Gilroy, Simon; Choi, Won-Gyu; Swanson, Sarah

2012-01-01

Goals of this work are: (1) Define global changes in gene expression patterns in Arabidopsis plants grown in microgravity using whole genome microarrays (2) Compare to mutants resistant to low oxygen challenge using whole genome microarrays Also measuring root and shoot size Outcomes from this research are: (1) Provide fundamental information on plant responses to the stresses inherent in spaceflight (2) Potential for informing on genetic strategies to engineer plants for optimal growth in space

Comparison of gene expression responses to hypoxia in viviparous (Xiphophorus) and oviparous (Oryzias) fishes using a medaka microarray.

PubMed

Boswell, Mikki G; Wells, Melissa C; Kirk, Lyndsey M; Ju, Zhenlin; Zhang, Ziping; Booth, Rachell E; Walter, Ronald B

2009-03-01

Gene expression profiling using DNA microarray technology is a useful tool for assessing gene transcript level responses after an organism is exposed to environmental stress. Herein, we detail results from studies using an 8 k medaka (Oryzias latipes) microarray to assess modulated gene expression patterns upon hypoxia exposure of the live-bearing aquaria fish, Xiphophorus maculatus. To assess the reproducibility and reliability of using the medaka array in cross-genus hybridization, a two-factor ANOVA analysis of gene expression was employed. The data show the tissue source of the RNA used for array hybridization contributed more to the observed response of modulated gene targets than did the species source of the RNA. In addition, hierarchical clustering via heat map analyses of groupings of tissues and species (Xiphophorus and medaka) suggests that hypoxia induced similar responses in the same tissues from these two diverse aquatic model organisms. Our Xiphophorus results indicate 206 brain, 37 liver, and 925 gill gene targets exhibit hypoxia induced expression changes. Analysis of the Xiphophorus data to determine those features exhibiting a significant (p<0.05)+/-3 fold change produced only two gene targets within brain tissue and 80 features within gill tissue. Of these 82 characterized features, 39 were identified via homology searching (cut-off E-value of 1 x 10(-5)) and placed into one or more biological process gene ontology groups. Among these 39 genes, metabolic energy changes and manipulation was the most affected biological pathway (13 genes).

Hyperosmotic Stress Reduces Melanin Production by Altering Melanosome Formation

PubMed Central

Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Choi, Dong-Hwa; Park, Kyuhee; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

2014-01-01

Many tissues of the human body encounter hyperosmotic stress. The effect of extracellular osmotic changes on melanin production has not yet been elucidated. In this study, we determined that hyperosmotic stress induced by organic osmolytes results in reduced melanin production in human melanoma MNT-1 cells. Under hyperosmotic stress, few pigmented mature melanosomes were detected, but there was an increase in swollen vacuoles. These vacuoles were stained with an anti-M6PR antibody that recognizes late endosomal components and with anti-TA99 and anti-HMB45 antibodies, implying that melanosome formation was affected by hyperosmotic stress. Electron microscopic analysis revealed that the M6PR-positive swollen vacuoles were multi-layered and contained melanized granules, and they produced melanin when L-DOPA was applied, indicating that these vacuoles were still capable of producing melanin, but the inner conditions were not compatible with melanin production. The vacuolation phenomenon induced by hyperosmotic conditions disappeared with treatment with the PI3K activator 740 Y-P, indicating that the PI3K pathway is affected by hyperosmotic conditions and is responsible for the proper formation and maturation of melanosomes. The microarray analysis showed alterations of the vesicle organization and transport under hyperosmotic stress. Our findings suggest that melanogenesis could be regulated by physiological conditions, such as osmotic pressure. PMID:25170965

Hsf and Hsp gene families in Populus: genome-wide identification, organization and correlated expression during development and in stress responses.

PubMed

Zhang, Jin; Liu, Bobin; Li, Jianbo; Zhang, Li; Wang, Yan; Zheng, Huanquan; Lu, Mengzhu; Chen, Jun

2015-03-14

Heat shock proteins (Hsps) are molecular chaperones that are involved in many normal cellular processes and stress responses, and heat shock factors (Hsfs) are the transcriptional activators of Hsps. Hsfs and Hsps are widely coordinated in various biological processes. Although the roles of Hsfs and Hsps in stress responses have been well characterized in Arabidopsis, their roles in perennial woody species undergoing various environmental stresses remain unclear. Here, a comprehensive identification and analysis of Hsf and Hsp families in poplars is presented. In Populus trichocarpa, we identified 42 paralogous pairs, 66.7% resulting from a whole genome duplication. The gene structure and motif composition are relatively conserved in each subfamily. Microarray and quantitative real-time RT-PCR analyses showed that most of the Populus Hsf and Hsp genes are differentially expressed upon exposure to various stresses. A coexpression network between Populus Hsf and Hsp genes was generated based on their expression. Coordinated relationships were validated by transient overexpression and subsequent qPCR analyses. The comprehensive analysis indicates that different sets of PtHsps are downstream of particular PtHsfs and provides a basis for functional studies aimed at revealing the roles of these families in poplar development and stress responses.

Hyperosmotic stress reduces melanin production by altering melanosome formation.

PubMed

Bin, Bum-Ho; Bhin, Jinhyuk; Yang, Seung Ha; Choi, Dong-Hwa; Park, Kyuhee; Shin, Dong Wook; Lee, Ai-Young; Hwang, Daehee; Cho, Eun-Gyung; Lee, Tae Ryong

2014-01-01

Many tissues of the human body encounter hyperosmotic stress. The effect of extracellular osmotic changes on melanin production has not yet been elucidated. In this study, we determined that hyperosmotic stress induced by organic osmolytes results in reduced melanin production in human melanoma MNT-1 cells. Under hyperosmotic stress, few pigmented mature melanosomes were detected, but there was an increase in swollen vacuoles. These vacuoles were stained with an anti-M6PR antibody that recognizes late endosomal components and with anti-TA99 and anti-HMB45 antibodies, implying that melanosome formation was affected by hyperosmotic stress. Electron microscopic analysis revealed that the M6PR-positive swollen vacuoles were multi-layered and contained melanized granules, and they produced melanin when L-DOPA was applied, indicating that these vacuoles were still capable of producing melanin, but the inner conditions were not compatible with melanin production. The vacuolation phenomenon induced by hyperosmotic conditions disappeared with treatment with the PI3K activator 740 Y-P, indicating that the PI3K pathway is affected by hyperosmotic conditions and is responsible for the proper formation and maturation of melanosomes. The microarray analysis showed alterations of the vesicle organization and transport under hyperosmotic stress. Our findings suggest that melanogenesis could be regulated by physiological conditions, such as osmotic pressure.

The Development of Protein Microarrays and Their Applications in DNA-Protein and Protein-Protein Interaction Analyses of Arabidopsis Transcription Factors

PubMed Central

Gong, Wei; He, Kun; Covington, Mike; Dinesh-Kumar, S. P.; Snyder, Michael; Harmer, Stacey L.; Zhu, Yu-Xian; Deng, Xing Wang

2009-01-01

We used our collection of Arabidopsis transcription factor (TF) ORFeome clones to construct protein microarrays containing as many as 802 TF proteins. These protein microarrays were used for both protein-DNA and protein-protein interaction analyses. For protein-DNA interaction studies, we examined AP2/ERF family TFs and their cognate cis-elements. By careful comparison of the DNA-binding specificity of 13 TFs on the protein microarray with previous non-microarray data, we showed that protein microarrays provide an efficient and high throughput tool for genome-wide analysis of TF-DNA interactions. This microarray protein-DNA interaction analysis allowed us to derive a comprehensive view of DNA-binding profiles of AP2/ERF family proteins in Arabidopsis. It also revealed four TFs that bound the EE (evening element) and had the expected phased gene expression under clock-regulation, thus providing a basis for further functional analysis of their roles in clock regulation of gene expression. We also developed procedures for detecting protein interactions using this TF protein microarray and discovered four novel partners that interact with HY5, which can be validated by yeast two-hybrid assays. Thus, plant TF protein microarrays offer an attractive high-throughput alternative to traditional techniques for TF functional characterization on a global scale. PMID:19802365

Karyotype versus microarray testing for genetic abnormalities after stillbirth.

PubMed

Reddy, Uma M; Page, Grier P; Saade, George R; Silver, Robert M; Thorsten, Vanessa R; Parker, Corette B; Pinar, Halit; Willinger, Marian; Stoll, Barbara J; Heim-Hall, Josefine; Varner, Michael W; Goldenberg, Robert L; Bukowski, Radek; Wapner, Ronald J; Drews-Botsch, Carolyn D; O'Brien, Barbara M; Dudley, Donald J; Levy, Brynn

2012-12-06

Genetic abnormalities have been associated with 6 to 13% of stillbirths, but the true prevalence may be higher. Unlike karyotype analysis, microarray analysis does not require live cells, and it detects small deletions and duplications called copy-number variants. The Stillbirth Collaborative Research Network conducted a population-based study of stillbirth in five geographic catchment areas. Standardized postmortem examinations and karyotype analyses were performed. A single-nucleotide polymorphism array was used to detect copy-number variants of at least 500 kb in placental or fetal tissue. Variants that were not identified in any of three databases of apparently unaffected persons were then classified into three groups: probably benign, clinical significance unknown, or pathogenic. We compared the results of karyotype and microarray analyses of samples obtained after delivery. In our analysis of samples from 532 stillbirths, microarray analysis yielded results more often than did karyotype analysis (87.4% vs. 70.5%, P<0.001) and provided better detection of genetic abnormalities (aneuploidy or pathogenic copy-number variants, 8.3% vs. 5.8%; P=0.007). Microarray analysis also identified more genetic abnormalities among 443 antepartum stillbirths (8.8% vs. 6.5%, P=0.02) and 67 stillbirths with congenital anomalies (29.9% vs. 19.4%, P=0.008). As compared with karyotype analysis, microarray analysis provided a relative increase in the diagnosis of genetic abnormalities of 41.9% in all stillbirths, 34.5% in antepartum stillbirths, and 53.8% in stillbirths with anomalies. Microarray analysis is more likely than karyotype analysis to provide a genetic diagnosis, primarily because of its success with nonviable tissue, and is especially valuable in analyses of stillbirths with congenital anomalies or in cases in which karyotype results cannot be obtained. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development.).

Emerging Use of Gene Expression Microarrays in Plant Physiology

DOE PAGES

Wullschleger, Stan D.; Difazio, Stephen P.

2003-01-01

Microarrays have become an important technology for the global analysis of gene expression in humans, animals, plants, and microbes. Implemented in the context of a well-designed experiment, cDNA and oligonucleotide arrays can provide highthroughput, simultaneous analysis of transcript abundance for hundreds, if not thousands, of genes. However, despite widespread acceptance, the use of microarrays as a tool to better understand processes of interest to the plant physiologist is still being explored. To help illustrate current uses of microarrays in the plant sciences, several case studies that we believe demonstrate the emerging application of gene expression arrays in plant physiology weremore » selected from among the many posters and presentations at the 2003 Plant and Animal Genome XI Conference. Based on this survey, microarrays are being used to assess gene expression in plants exposed to the experimental manipulation of air temperature, soil water content and aluminium concentration in the root zone. Analysis often includes characterizing transcript profiles for multiple post-treatment sampling periods and categorizing genes with common patterns of response using hierarchical clustering techniques. In addition, microarrays are also providing insights into developmental changes in gene expression associated with fibre and root elongation in cotton and maize, respectively. Technical and analytical limitations of microarrays are discussed and projects attempting to advance areas of microarray design and data analysis are highlighted. Finally, although much work remains, we conclude that microarrays are a valuable tool for the plant physiologist interested in the characterization and identification of individual genes and gene families with potential application in the fields of agriculture, horticulture and forestry.« less

A genome-wide 20 K citrus microarray for gene expression analysis

PubMed Central

Martinez-Godoy, M Angeles; Mauri, Nuria; Juarez, Jose; Marques, M Carmen; Santiago, Julia; Forment, Javier; Gadea, Jose

2008-01-01

Background Understanding of genetic elements that contribute to key aspects of citrus biology will impact future improvements in this economically important crop. Global gene expression analysis demands microarray platforms with a high genome coverage. In the last years, genome-wide EST collections have been generated in citrus, opening the possibility to create new tools for functional genomics in this crop plant. Results We have designed and constructed a publicly available genome-wide cDNA microarray that include 21,081 putative unigenes of citrus. As a functional companion to the microarray, a web-browsable database [1] was created and populated with information about the unigenes represented in the microarray, including cDNA libraries, isolated clones, raw and processed nucleotide and protein sequences, and results of all the structural and functional annotation of the unigenes, like general description, BLAST hits, putative Arabidopsis orthologs, microsatellites, putative SNPs, GO classification and PFAM domains. We have performed a Gene Ontology comparison with the full set of Arabidopsis proteins to estimate the genome coverage of the microarray. We have also performed microarray hybridizations to check its usability. Conclusion This new cDNA microarray replaces the first 7K microarray generated two years ago and allows gene expression analysis at a more global scale. We have followed a rational design to minimize cross-hybridization while maintaining its utility for different citrus species. Furthermore, we also provide access to a website with full structural and functional annotation of the unigenes represented in the microarray, along with the ability to use this site to directly perform gene expression analysis using standard tools at different publicly available servers. Furthermore, we show how this microarray offers a good representation of the citrus genome and present the usefulness of this genomic tool for global studies in citrus by using it to catalogue genes expressed in citrus globular embryos. PMID:18598343

A Java-based tool for the design of classification microarrays.

PubMed

Meng, Da; Broschat, Shira L; Call, Douglas R

2008-08-04

Classification microarrays are used for purposes such as identifying strains of bacteria and determining genetic relationships to understand the epidemiology of an infectious disease. For these cases, mixed microarrays, which are composed of DNA from more than one organism, are more effective than conventional microarrays composed of DNA from a single organism. Selection of probes is a key factor in designing successful mixed microarrays because redundant sequences are inefficient and limited representation of diversity can restrict application of the microarray. We have developed a Java-based software tool, called PLASMID, for use in selecting the minimum set of probe sequences needed to classify different groups of plasmids or bacteria. The software program was successfully applied to several different sets of data. The utility of PLASMID was illustrated using existing mixed-plasmid microarray data as well as data from a virtual mixed-genome microarray constructed from different strains of Streptococcus. Moreover, use of data from expression microarray experiments demonstrated the generality of PLASMID. In this paper we describe a new software tool for selecting a set of probes for a classification microarray. While the tool was developed for the design of mixed microarrays-and mixed-plasmid microarrays in particular-it can also be used to design expression arrays. The user can choose from several clustering methods (including hierarchical, non-hierarchical, and a model-based genetic algorithm), several probe ranking methods, and several different display methods. A novel approach is used for probe redundancy reduction, and probe selection is accomplished via stepwise discriminant analysis. Data can be entered in different formats (including Excel and comma-delimited text), and dendrogram, heat map, and scatter plot images can be saved in several different formats (including jpeg and tiff). Weights generated using stepwise discriminant analysis can be stored for analysis of subsequent experimental data. Additionally, PLASMID can be used to construct virtual microarrays with genomes from public databases, which can then be used to identify an optimal set of probes.

Enhancing interdisciplinary mathematics and biology education: a microarray data analysis course bridging these disciplines.

PubMed

Tra, Yolande V; Evans, Irene M

2010-01-01

BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course.

Enhancing Interdisciplinary Mathematics and Biology Education: A Microarray Data Analysis Course Bridging These Disciplines

PubMed Central

Evans, Irene M.

2010-01-01

BIO2010 put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on microarray data analysis. We started using Genome Consortium for Active Teaching (GCAT) materials and Microarray Genome and Clustering Tool software and added R statistical software along with Bioconductor packages. In response to student feedback, one microarray data set was fully analyzed in class, starting from preprocessing to gene discovery to pathway analysis using the latter software. A class project was to conduct a similar analysis where students analyzed their own data or data from a published journal paper. This exercise showed the impact that filtering, preprocessing, and different normalization methods had on gene inclusion in the final data set. We conclude that this course achieved its goals to equip students with skills to analyze data from a microarray experiment. We offer our insight about collaborative teaching as well as how other faculty might design and implement a similar interdisciplinary course. PMID:20810954

Chromosomal Microarray versus Karyotyping for Prenatal Diagnosis

PubMed Central

Wapner, Ronald J.; Martin, Christa Lese; Levy, Brynn; Ballif, Blake C.; Eng, Christine M.; Zachary, Julia M.; Savage, Melissa; Platt, Lawrence D.; Saltzman, Daniel; Grobman, William A.; Klugman, Susan; Scholl, Thomas; Simpson, Joe Leigh; McCall, Kimberly; Aggarwal, Vimla S.; Bunke, Brian; Nahum, Odelia; Patel, Ankita; Lamb, Allen N.; Thom, Elizabeth A.; Beaudet, Arthur L.; Ledbetter, David H.; Shaffer, Lisa G.; Jackson, Laird

2013-01-01

Background Chromosomal microarray analysis has emerged as a primary diagnostic tool for the evaluation of developmental delay and structural malformations in children. We aimed to evaluate the accuracy, efficacy, and incremental yield of chromosomal microarray analysis as compared with karyotyping for routine prenatal diagnosis. Methods Samples from women undergoing prenatal diagnosis at 29 centers were sent to a central karyotyping laboratory. Each sample was split in two; standard karyotyping was performed on one portion and the other was sent to one of four laboratories for chromosomal microarray. Results We enrolled a total of 4406 women. Indications for prenatal diagnosis were advanced maternal age (46.6%), abnormal result on Down’s syndrome screening (18.8%), structural anomalies on ultrasonography (25.2%), and other indications (9.4%). In 4340 (98.8%) of the fetal samples, microarray analysis was successful; 87.9% of samples could be used without tissue culture. Microarray analysis of the 4282 nonmosaic samples identified all the aneuploidies and unbalanced rearrangements identified on karyotyping but did not identify balanced translocations and fetal triploidy. In samples with a normal karyotype, microarray analysis revealed clinically relevant deletions or duplications in 6.0% with a structural anomaly and in 1.7% of those whose indications were advanced maternal age or positive screening results. Conclusions In the context of prenatal diagnostic testing, chromosomal microarray analysis identified additional, clinically significant cytogenetic information as compared with karyotyping and was equally efficacious in identifying aneuploidies and unbalanced rearrangements but did not identify balanced translocations and triploidies. (Funded by the Eunice Kennedy Shriver National Institute of Child Health and Human Development and others; ClinicalTrials.gov number, NCT01279733.) PMID:23215555

Analysis of microarray leukemia data using an efficient MapReduce-based K-nearest-neighbor classifier.

PubMed

Kumar, Mukesh; Rath, Nitish Kumar; Rath, Santanu Kumar

2016-04-01

Microarray-based gene expression profiling has emerged as an efficient technique for classification, prognosis, diagnosis, and treatment of cancer. Frequent changes in the behavior of this disease generates an enormous volume of data. Microarray data satisfies both the veracity and velocity properties of big data, as it keeps changing with time. Therefore, the analysis of microarray datasets in a small amount of time is essential. They often contain a large amount of expression, but only a fraction of it comprises genes that are significantly expressed. The precise identification of genes of interest that are responsible for causing cancer are imperative in microarray data analysis. Most existing schemes employ a two-phase process such as feature selection/extraction followed by classification. In this paper, various statistical methods (tests) based on MapReduce are proposed for selecting relevant features. After feature selection, a MapReduce-based K-nearest neighbor (mrKNN) classifier is also employed to classify microarray data. These algorithms are successfully implemented in a Hadoop framework. A comparative analysis is done on these MapReduce-based models using microarray datasets of various dimensions. From the obtained results, it is observed that these models consume much less execution time than conventional models in processing big data. Copyright © 2016 Elsevier Inc. All rights reserved.

High hydrostatic pressure leads to free radicals accumulation in yeast cells triggering oxidative stress.

PubMed

Bravim, Fernanda; Mota, Mainã M; Fernandes, A Alberto R; Fernandes, Patricia M B

2016-08-01

Saccharomyces cerevisiae is a unicellular organism that during the fermentative process is exposed to a variable environment; hence, resistance to multiple stress conditions is a desirable trait. The stress caused by high hydrostatic pressure (HHP) in S. cerevisiae resembles the injuries generated by other industrial stresses. In this study, it was confirmed that gene expression pattern in response to HHP displays an oxidative stress response profile which is expanded upon hydrostatic pressure release. Actually, reactive oxygen species (ROS) concentration level increased in yeast cells exposed to HHP treatment and an incubation period at room pressure led to a decrease in intracellular ROS concentration. On the other hand, ethylic, thermic and osmotic stresses did not result in any ROS accumulation in yeast cells. Microarray analysis revealed an upregulation of genes related to methionine metabolism, appearing to be a specific cellular response to HHP, and not related to other stresses, such as heat and osmotic stresses. Next, we investigated whether enhanced oxidative stress tolerance leads to enhanced tolerance to HHP stress. Overexpression of STF2 is known to enhance tolerance to oxidative stress and we show that it also leads to enhanced tolerance to HHP stress. © FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Mapping of oxidative stress responses of human tumor cells following photodynamic therapy using hexaminolevulinate

PubMed Central

Cekaite, Lina; Peng, Qian; Reiner, Andrew; Shahzidi, Susan; Tveito, Siri; Furre, Ingegerd E; Hovig, Eivind

2007-01-01

Background Photodynamic therapy (PDT) involves systemic or topical administration of a lesion-localizing photosensitizer or its precursor, followed by irradiation of visible light to cause singlet oxygen-induced damage to the affected tissue. A number of mechanisms seem to be involved in the protective responses to PDT, including activation of transcription factors, heat shock proteins, antioxidant enzymes and apoptotic pathways. Results In this study, we address the effects of a destructive/lethal hexaminolevulinate (HAL) mediated PDT dose on the transcriptome by using transcriptional exon evidence oligo microarrays. Here, we confirm deviations in the steady state expression levels of previously identified early defence response genes and extend this to include unreported PDT inducible gene groups, most notably the metallothioneins and histones. HAL-PDT mediated stress also altered expression of genes encoded by mitochondrial DNA (mtDNA). Further, we report PDT stress induced alternative splicing. Specifically, the ATF3 alternative isoform (deltaZip2) was up-regulated, while the full-length variant was not changed by the treatment. Results were independently verified by two different technological microarray platforms. Good microarray, RT-PCR and Western immunoblotting correlation for selected genes support these findings. Conclusion Here, we report new insights into how destructive/lethal PDT alters the transcriptome not only at the transcriptional level but also at post-transcriptional level via alternative splicing. PMID:17692132

Microarray and growth analyses identify differences and similarities of early corn response to weeds, shade, and nitrogen stress

USDA-ARS?s Scientific Manuscript database

Weed interference with crop growth is often attributed to water, nutrient, or light competition; however, specific physiological responses to these stresses are not well described. This study’s objective was to compare growth, yield, and gene expression responses of corn to nitrogen (N), low light (...

Direct labeling of serum proteins by fluorescent dye for antibody microarray.

PubMed

Klimushina, M V; Gumanova, N G; Metelskaya, V A

2017-05-06

Analysis of serum proteome by antibody microarray is used to identify novel biomarkers and to study signaling pathways including protein phosphorylation and protein-protein interactions. Labeling of serum proteins is important for optimal performance of the antibody microarray. Proper choice of fluorescent label and optimal concentration of protein loaded on the microarray ensure good quality of imaging that can be reliably scanned and processed by the software. We have optimized direct serum protein labeling using fluorescent dye Arrayit Green 540 (Arrayit Corporation, USA) for antibody microarray. Optimized procedure produces high quality images that can be readily scanned and used for statistical analysis of protein composition of the serum. Copyright © 2017 Elsevier Inc. All rights reserved.

High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces cerevisiae distillery strain

PubMed Central

Bravim, Fernanda; Lippman, Soyeon I.; da Silva, Lucas F.; Souza, Diego T.; Fernandes, A. Alberto R.; Masuda, Claudio A.; Broach, James R.

2016-01-01

High hydrostatic pressure (HHP) is a stress that exerts broad effects on microorganisms with characteristics similar to those of common environmental stresses. In this study, we aimed to identify genetic mechanisms that can enhance alcoholic fermentation of wild Saccharomyces cerevisiae isolated from Brazilian spirit fermentation vats. Accordingly, we performed a time course microarray analysis on a S. cerevisiae strain submitted to mild sublethal pressure treatment of 50 MPa for 30 min at room temperature, followed by incubation for 5, 10 and 15 min without pressure treatment. The obtained transcriptional profiles demonstrate the importance of post-pressurisation period on the activation of several genes related to cell recovery and stress tolerance. Based on these results, we over-expressed genes strongly induced by HHP in the same wild yeast strain and identified genes, particularly SYM1, whose over-expression results in enhanced ethanol production and stress tolerance upon fermentation. The present study validates the use of HHP as a biotechnological tool for the fermentative industries. PMID:22915193

Isolation of Polysomal RNA for Analyzing Stress-Responsive Genes Regulated at the Translational Level in Plants.

PubMed

Li, Yong-Fang; Mahalingam, Ramamurthy; Sunkar, Ramanjulu

2017-01-01

Alteration of gene expression is an essential mechanism, which allows plants to respond and adapt to adverse environmental conditions. Transcriptome and proteome analyses in plants exposed to abiotic stresses revealed that protein levels are not correlated with the changes in corresponding mRNAs, indicating regulation at translational level is another major regulator for gene expression. Analysis of translatome, which refers to all mRNAs associated with ribosomes, thus has the potential to bridge the gap between transcriptome and proteome. Polysomal RNA profiling and recently developed ribosome profiling (Ribo-seq) are two main methods for translatome analysis at global level. Here, we describe the classical procedure for polysomal RNA isolation by sucrose gradient ultracentrifugation followed by highthroughput RNA-seq to identify genes regulated at translational level. Polysomal RNA can be further used for a variety of downstream applications including Northern blot analysis, qRT-PCR, RNase protection assay, and microarray-based gene expression profiling.

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Daly, Don S.; Willse, Alan R.

The Automated Microarray Image Analysis (AMIA) Toolbox for MATLAB is a flexible, open-source microarray image analysis tool that allows the user to customize analysis of sets of microarray images. This tool provides several methods of identifying and quantify spot statistics, as well as extensive diagnostic statistics and images to identify poor data quality or processing. The open nature of this software allows researchers to understand the algorithms used to provide intensity estimates and to modify them easily if desired.

ELISA-BASE: An Integrated Bioinformatics Tool for Analyzing and Tracking ELISA Microarray Data

DOE Office of Scientific and Technical Information (OSTI.GOV)

White, Amanda M.; Collett, James L.; Seurynck-Servoss, Shannon L.

ELISA-BASE is an open-source database for capturing, organizing and analyzing protein enzyme-linked immunosorbent assay (ELISA) microarray data. ELISA-BASE is an extension of the BioArray Soft-ware Environment (BASE) database system, which was developed for DNA microarrays. In order to make BASE suitable for protein microarray experiments, we developed several plugins for importing and analyzing quantitative ELISA microarray data. Most notably, our Protein Microarray Analysis Tool (ProMAT) for processing quantita-tive ELISA data is now available as a plugin to the database.

Resveratrol supplementation confers neuroprotection in cortical brain tissue of nonhuman primates fed a high-fat/sucrose diet

PubMed Central

Bernier, Michel; Wahl, Devin; Ali, Ahmed; Allard, Joanne; Faulkner, Shakeela; Wnorowski, Artur; Sanghvi, Mitesh; Moaddel, Ruin; Alfaras, Irene; Mattison, Julie A.; Tarantini, Stefano; Tucsek, Zsuzsanna; Ungvari, Zoltan; Csiszar, Anna; Pearson, Kevin J.; de Cabo, Rafael

2016-01-01

Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in preventing pancreatic beta cell dysfunction, arterial stiffening and metabolic decline induced by high-fat/high-sugar (HFS) diet in nonhuman primates. Here, the analysis was extended to examine whether RSV may reduce dietary stress toxicity in the cerebral cortex of the same cohort of treated animals. Middle-aged male rhesus monkeys were fed for 2 years with HFS alone or combined with RSV, after which whole-genome microarray analysis of cerebral cortex tissue was carried out along with ELISA, immunofluorescence, and biochemical analyses to examine markers of vascular health and inflammation in the cerebral cortices. A number of genes and pathways that were differentially modulated in these dietary interventions indicated an exacerbation of neuroinflammation (e.g., oxidative stress markers, apoptosis, NF-κB activation) in HFS-fed animals and protection by RSV treatment. The decreased expression of mitochondrial aldehyde dehydrogenase 2, dysregulation in endothelial nitric oxide synthase, and reduced capillary density induced by HFS stress were rescued by RSV supplementation. Our results suggest that long-term RSV treatment confers neuroprotection against cerebral vascular dysfunction during nutrient stress. PMID:27070252

Resveratrol supplementation confers neuroprotection in cortical brain tissue of nonhuman primates fed a high-fat/sucrose diet.

PubMed

Bernier, Michel; Wahl, Devin; Ali, Ahmed; Allard, Joanne; Faulkner, Shakeela; Wnorowski, Artur; Sanghvi, Mitesh; Moaddel, Ruin; Alfaras, Irene; Mattison, Julie A; Tarantini, Stefano; Tucsek, Zsuzsanna; Ungvari, Zoltan; Csiszar, Anna; Pearson, Kevin J; de Cabo, Rafael

2016-05-01

Previous studies have shown positive effects of long-term resveratrol (RSV) supplementation in preventing pancreatic beta cell dysfunction, arterial stiffening and metabolic decline induced by high-fat/high-sugar (HFS) diet in nonhuman primates. Here, the analysis was extended to examine whether RSV may reduce dietary stress toxicity in the cerebral cortex of the same cohort of treated animals. Middle-aged male rhesus monkeys were fed for 2 years with HFS alone or combined with RSV, after which whole-genome microarray analysis of cerebral cortex tissue was carried out along with ELISA, immunofluorescence, and biochemical analyses to examine markers of vascular health and inflammation in the cerebral cortices. A number of genes and pathways that were differentially modulated in these dietary interventions indicated an exacerbation of neuroinflammation (e.g., oxidative stress markers, apoptosis, NF-κB activation) in HFS-fed animals and protection by RSV treatment. The decreased expression of mitochondrial aldehyde dehydrogenase 2, dysregulation in endothelial nitric oxide synthase, and reduced capillary density induced by HFS stress were rescued by RSV supplementation. Our results suggest that long-term RSV treatment confers neuroprotection against cerebral vascular dysfunction during nutrient stress.

Transcriptome analysis in non-model species: a new method for the analysis of heterologous hybridization on microarrays

PubMed Central

2010-01-01

Background Recent developments in high-throughput methods of analyzing transcriptomic profiles are promising for many areas of biology, including ecophysiology. However, although commercial microarrays are available for most common laboratory models, transcriptome analysis in non-traditional model species still remains a challenge. Indeed, the signal resulting from heterologous hybridization is low and difficult to interpret because of the weak complementarity between probe and target sequences, especially when no microarray dedicated to a genetically close species is available. Results We show here that transcriptome analysis in a species genetically distant from laboratory models is made possible by using MAXRS, a new method of analyzing heterologous hybridization on microarrays. This method takes advantage of the design of several commercial microarrays, with different probes targeting the same transcript. To illustrate and test this method, we analyzed the transcriptome of king penguin pectoralis muscle hybridized to Affymetrix chicken microarrays, two organisms separated by an evolutionary distance of approximately 100 million years. The differential gene expression observed between different physiological situations computed by MAXRS was confirmed by real-time PCR on 10 genes out of 11 tested. Conclusions MAXRS appears to be an appropriate method for gene expression analysis under heterologous hybridization conditions. PMID:20509979

Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses

PubMed Central

2011-01-01

Background Salt stress hinders the growth of plants and reduces crop production worldwide. However, different plant species might possess different adaptive mechanisms to mitigate salt stress. We conducted a detailed pathway analysis of transcriptional dynamics in the roots of Medicago truncatula seedlings under salt stress and selected a transcription factor gene, MtCBF4, for experimental validation. Results A microarray experiment was conducted using root samples collected 6, 24, and 48 h after application of 180 mM NaCl. Analysis of 11 statistically significant expression profiles revealed different behaviors between primary and secondary metabolism pathways in response to external stress. Secondary metabolism that helps to maintain osmotic balance was induced. One of the highly induced transcription factor genes was successfully cloned, and was named MtCBF4. Phylogenetic analysis revealed that MtCBF4, which belongs to the AP2-EREBP transcription factor family, is a novel member of the CBF transcription factor in M. truncatula. MtCBF4 is shown to be a nuclear-localized protein. Expression of MtCBF4 in M. truncatula was induced by most of the abiotic stresses, including salt, drought, cold, and abscisic acid, suggesting crosstalk between these abiotic stresses. Transgenic Arabidopsis over-expressing MtCBF4 enhanced tolerance to drought and salt stress, and activated expression of downstream genes that contain DRE elements. Over-expression of MtCBF4 in M. truncatula also enhanced salt tolerance and induced expression level of corresponding downstream genes. Conclusion Comprehensive transcriptomic analysis revealed complex mechanisms exist in plants in response to salt stress. The novel transcription factor gene MtCBF4 identified here played an important role in response to abiotic stresses, indicating that it might be a good candidate gene for genetic improvement to produce stress-tolerant plants. PMID:21718548

A Human Lectin Microarray for Sperm Surface Glycosylation Analysis *

PubMed Central

Sun, Yangyang; Cheng, Li; Gu, Yihua; Xin, Aijie; Wu, Bin; Zhou, Shumin; Guo, Shujuan; Liu, Yin; Diao, Hua; Shi, Huijuan; Wang, Guangyu; Tao, Sheng-ce

2016-01-01

Glycosylation is one of the most abundant and functionally important protein post-translational modifications. As such, technology for efficient glycosylation analysis is in high demand. Lectin microarrays are a powerful tool for such investigations and have been successfully applied for a variety of glycobiological studies. However, most of the current lectin microarrays are primarily constructed from plant lectins, which are not well suited for studies of human glycosylation because of the extreme complexity of human glycans. Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane-associated proteins including heat shock protein 90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays. PMID:27364157

Rice Phospholipase A Superfamily: Organization, Phylogenetic and Expression Analysis during Abiotic Stresses and Development

PubMed Central

Singh, Amarjeet; Baranwal, Vinay; Shankar, Alka; Kanwar, Poonam; Ranjan, Rajeev; Yadav, Sandeep; Pandey, Amita; Kapoor, Sanjay; Pandey, Girdhar K.

2012-01-01

Background Phospholipase A (PLA) is an important group of enzymes responsible for phospholipid hydrolysis in lipid signaling. PLAs have been implicated in abiotic stress signaling and developmental events in various plants species. Genome-wide analysis of PLA superfamily has been carried out in dicot plant Arabidopsis. A comprehensive genome-wide analysis of PLAs has not been presented yet in crop plant rice. Methodology/Principal Findings A comprehensive bioinformatics analysis identified a total of 31 PLA encoding genes in the rice genome, which are divided into three classes; phospholipase A1 (PLA1), patatin like phospholipases (pPLA) and low molecular weight secretory phospholipase A2 (sPLA2) based on their sequences and phylogeny. A subset of 10 rice PLAs exhibited chromosomal duplication, emphasizing the role of duplication in the expansion of this gene family in rice. Microarray expression profiling revealed a number of PLA members expressing differentially and significantly under abiotic stresses and reproductive development. Comparative expression analysis with Arabidopsis PLAs revealed a high degree of functional conservation between the orthologs in two plant species, which also indicated the vital role of PLAs in stress signaling and plant development across different plant species. Moreover, sub-cellular localization of a few candidates suggests their differential localization and functional role in the lipid signaling. Conclusion/Significance The comprehensive analysis and expression profiling would provide a critical platform for the functional characterization of the candidate PLA genes in crop plants. PMID:22363522

Monitoring Transcriptomic Changes in Soil-Grown Roots and Shoots of Arabidopsis thaliana Subjected to a Progressive Drought Stress.

PubMed

Bashir, Khurram; Rasheed, Sultana; Matsui, Akihiro; Iida, Kei; Tanaka, Maho; Seki, Motoaki

2018-01-01

Numerous experiments have been performed in Arabidopsis to monitor changes in gene expression that occur in response to a variety of abiotic and biotic stresses, different growth conditions, and at various developmental stages. In addition, gene expression patterns have also been characterized among wild-type and mutant genotypes. Despite these numerous reports, transcriptional changes occurring in roots of soil-grown plants subjected to a progressive drought stress have remained undocumented. To fill this gap, we established a system that allows one to establish water-deficit conditions and to collect root and shoot samples with minimal damage to the root system. Arabidopsis plants are grown in a ceramic-based granular soil and subjected to progressive drought stress by withholding water. Root and shoot samples were collected separately, RNA was purified, and a microarray analysis of drought-stressed roots and shoots was performed at 0, 1, 3, 5, 7, and 9 days after the onset of drought stress treatment. Here, we describe the detailed protocol used to analyze the transcriptomic changes occurring in roots and shoots of soil-grown Arabidopsis subjected to a progressive drought stress.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gentry, T.; Schadt, C.; Zhou, J.

Microarray technology has the unparalleled potential tosimultaneously determine the dynamics and/or activities of most, if notall, of the microbial populations in complex environments such as soilsand sediments. Researchers have developed several types of arrays thatcharacterize the microbial populations in these samples based on theirphylogenetic relatedness or functional genomic content. Several recentstudies have used these microarrays to investigate ecological issues;however, most have only analyzed a limited number of samples withrelatively few experiments utilizing the full high-throughput potentialof microarray analysis. This is due in part to the unique analyticalchallenges that these samples present with regard to sensitivity,specificity, quantitation, and data analysis. Thismore » review discussesspecific applications of microarrays to microbial ecology research alongwith some of the latest studies addressing the difficulties encounteredduring analysis of complex microbial communities within environmentalsamples. With continued development, microarray technology may ultimatelyachieve its potential for comprehensive, high-throughput characterizationof microbial populations in near real-time.« less

Linking the salt transcriptome with physiological responses of a salt-resistant Populus species as a strategy to identify genes important for stress acclimation.

PubMed

Brinker, Monika; Brosché, Mikael; Vinocur, Basia; Abo-Ogiala, Atef; Fayyaz, Payam; Janz, Dennis; Ottow, Eric A; Cullmann, Andreas D; Saborowski, Joachim; Kangasjärvi, Jaakko; Altman, Arie; Polle, Andrea

2010-12-01

To investigate early salt acclimation mechanisms in a salt-tolerant poplar species (Populus euphratica), the kinetics of molecular, metabolic, and physiological changes during a 24-h salt exposure were measured. Three distinct phases of salt stress were identified by analyses of the osmotic pressure and the shoot water potential: dehydration, salt accumulation, and osmotic restoration associated with ionic stress. The duration and intensity of these phases differed between leaves and roots. Transcriptome analysis using P. euphratica-specific microarrays revealed clusters of coexpressed genes in these phases, with only 3% overlapping salt-responsive genes in leaves and roots. Acclimation of cellular metabolism to high salt concentrations involved remodeling of amino acid and protein biosynthesis and increased expression of molecular chaperones (dehydrins, osmotin). Leaves suffered initially from dehydration, which resulted in changes in transcript levels of mitochondrial and photosynthetic genes, indicating adjustment of energy metabolism. Initially, decreases in stress-related genes were found, whereas increases occurred only when leaves had restored the osmotic balance by salt accumulation. Comparative in silico analysis of the poplar stress regulon with Arabidopsis (Arabidopsis thaliana) orthologs was used as a strategy to reduce the number of candidate genes for functional analysis. Analysis of Arabidopsis knockout lines identified a lipocalin-like gene (AtTIL) and a gene encoding a protein with previously unknown functions (AtSIS) to play roles in salt tolerance. In conclusion, by dissecting the stress transcriptome of tolerant species, novel genes important for salt endurance can be identified.

Linking the Salt Transcriptome with Physiological Responses of a Salt-Resistant Populus Species as a Strategy to Identify Genes Important for Stress Acclimation1[W][OA

PubMed Central

Brinker, Monika; Brosché, Mikael; Vinocur, Basia; Abo-Ogiala, Atef; Fayyaz, Payam; Janz, Dennis; Ottow, Eric A.; Cullmann, Andreas D.; Saborowski, Joachim; Kangasjärvi, Jaakko; Altman, Arie; Polle, Andrea

2010-01-01

To investigate early salt acclimation mechanisms in a salt-tolerant poplar species (Populus euphratica), the kinetics of molecular, metabolic, and physiological changes during a 24-h salt exposure were measured. Three distinct phases of salt stress were identified by analyses of the osmotic pressure and the shoot water potential: dehydration, salt accumulation, and osmotic restoration associated with ionic stress. The duration and intensity of these phases differed between leaves and roots. Transcriptome analysis using P. euphratica-specific microarrays revealed clusters of coexpressed genes in these phases, with only 3% overlapping salt-responsive genes in leaves and roots. Acclimation of cellular metabolism to high salt concentrations involved remodeling of amino acid and protein biosynthesis and increased expression of molecular chaperones (dehydrins, osmotin). Leaves suffered initially from dehydration, which resulted in changes in transcript levels of mitochondrial and photosynthetic genes, indicating adjustment of energy metabolism. Initially, decreases in stress-related genes were found, whereas increases occurred only when leaves had restored the osmotic balance by salt accumulation. Comparative in silico analysis of the poplar stress regulon with Arabidopsis (Arabidopsis thaliana) orthologs was used as a strategy to reduce the number of candidate genes for functional analysis. Analysis of Arabidopsis knockout lines identified a lipocalin-like gene (AtTIL) and a gene encoding a protein with previously unknown functions (AtSIS) to play roles in salt tolerance. In conclusion, by dissecting the stress transcriptome of tolerant species, novel genes important for salt endurance can be identified. PMID:20959419

Fully automated analysis of multi-resolution four-channel micro-array genotyping data

NASA Astrophysics Data System (ADS)

Abbaspour, Mohsen; Abugharbieh, Rafeef; Podder, Mohua; Tebbutt, Scott J.

2006-03-01

We present a fully-automated and robust microarray image analysis system for handling multi-resolution images (down to 3-micron with sizes up to 80 MBs per channel). The system is developed to provide rapid and accurate data extraction for our recently developed microarray analysis and quality control tool (SNP Chart). Currently available commercial microarray image analysis applications are inefficient, due to the considerable user interaction typically required. Four-channel DNA microarray technology is a robust and accurate tool for determining genotypes of multiple genetic markers in individuals. It plays an important role in the state of the art trend where traditional medical treatments are to be replaced by personalized genetic medicine, i.e. individualized therapy based on the patient's genetic heritage. However, fast, robust, and precise image processing tools are required for the prospective practical use of microarray-based genetic testing for predicting disease susceptibilities and drug effects in clinical practice, which require a turn-around timeline compatible with clinical decision-making. In this paper we have developed a fully-automated image analysis platform for the rapid investigation of hundreds of genetic variations across multiple genes. Validation tests indicate very high accuracy levels for genotyping results. Our method achieves a significant reduction in analysis time, from several hours to just a few minutes, and is completely automated requiring no manual interaction or guidance.

Superoxide Dismutases, SOD1 and SOD2, Play a Distinct Role in the Fat Body during Pupation in Silkworm Bombyx mori

PubMed Central

Nojima, Yosui; Ito, Katsuhiko; Ono, Hiromasa; Nakazato, Takeru; Bono, Hidemasa; Yokoyama, Takeshi; Sato, Ryoichi; Suetsugu, Yoshitaka; Nakamura, Yuki; Yamamoto, Kimiko; Satoh, Jun-ichi; Tabunoki, Hiroko; Fugo, Hajime

2015-01-01

One way that aerobic biological systems counteract the generation of reactive oxygen species (ROS) is with superoxide dismutase proteins SOD1 and SOD2 that metabolize superoxide radicals to molecular oxygen and hydrogen peroxide or scavenge oxygen radicals produced by the extensive oxidation-reduction and electron-transport reactions that occur in mitochondria. We characterized SOD1 and SOD2 of Bombyx mori isolated from the fat body of larvae. Immunological analysis demonstrated the presence of BmSOD1 and BmSOD2 in the silk gland, midgut, fat body, Malpighian tubules, testis and ovary from larvae to adults. We found that BmSOD2 had a unique expression pattern in the fat body through the fifth instar larval developmental stage. The anti-oxidative functions of BmSOD1 and BmSOD2 were assessed by exposing larvae to insecticide rotenone or vasodilator isosorbide dinitrate, which is an ROS generator in BmN4 cells; however, exposure to these compounds had no effect on the expression levels of either BmSOD protein. Next, we investigated the physiological role of BmSOD1 and BmSOD2 under environmental oxidative stress, applied through whole-body UV irradiation and assayed using quantitative RT-PCR, immunoblotting and microarray analysis. The mRNA expression level of both BmSOD1 and BmSOD2 was markedly increased but protein expression level was increased only slightly. To examine the differences in mRNA and protein level due to UV irradiation intensity, we performed microarray analysis. Gene set enrichment analysis revealed that genes in the insulin signaling pathway and PPAR signaling pathway were significantly up-regulated after 6 and 12 hours of UV irradiation. Taken together, the activities of BmSOD1 and BmSOD2 may be related to the response to UV irradiation stress in B. mori. These results suggest that BmSOD1 and BmSOD2 modulate environmental oxidative stress in the cell and have a specific role in fat body of B. mori during pupation. PMID:25714339

Transcriptome Analysis of ABA/JA-Dual Responsive Genes in Rice Shoot and Root.

PubMed

Kim, Jin-Ae; Bhatnagar, Nikita; Kwon, Soon Jae; Min, Myung Ki; Moon, Seok-Jun; Yoon, In Sun; Kwon, Taek-Ryoun; Kim, Sun Tae; Kim, Beom-Gi

2018-01-01

The phytohormone abscisic acid (ABA) enables plants to adapt to adverse environmental conditions through the modulation of metabolic pathways and of growth and developmental programs. We used comparative microarray analysis to identify genes exhibiting ABA-dependent expression and other hormone-dependent expression among them in Oryza sativa shoot and root. We identified 854 genes as significantly up- or down-regulated in root or shoot under ABA treatment condition. Most of these genes had similar expression profiles in root and shoot under ABA treatment condition, whereas 86 genes displayed opposite expression responses in root and shoot. To examine the crosstalk between ABA and other hormones, we compared the expression profiles of the ABA-dependently regulated genes under several different hormone treatment conditions. Interestingly, around half of the ABA-dependently expressed genes were also regulated by jasmonic acid based on microarray data analysis. We searched the promoter regions of these genes for cis-elements that could be responsible for their responsiveness to both hormones, and found that ABRE and MYC2 elements, among others, were common to the promoters of genes that were regulated by both ABA and JA. These results show that ABA and JA might have common gene expression regulation system and might explain why the JA could function for both abiotic and biotic stress tolerance.

Salmonella Enteritidis Strains from Poultry Exhibit Differential Responses to Acid Stress, Oxidative Stress, and Survival in the Egg Albumen

PubMed Central

Casavant, Carol; Hawley, Quincy; Addwebi, Tarek; Call, Douglas R.; Guard, Jean

2012-01-01

Abstract Salmonella Enteritidis is the major foodborne pathogen that is primarily transmitted by contaminated chicken meat and eggs. We recently demonstrated that Salmonella Enteritidis strains from poultry differ in their ability to invade human intestinal cells and cause disease in orally challenged mice. Here we hypothesized that the differential virulence of Salmonella Enteritidis strains is due to the differential fitness in the adverse environments that may be encountered during infection in the host. The responses of a panel of six Salmonella Enteritidis strains to acid stress, oxidative stress, survival in egg albumen, and the ability to cause infection in chickens were analyzed. This analysis allowed classification of strains into two categories, stress-sensitive and stress-resistant, with the former showing significantly (p<0.05) reduced survival in acidic (gastric phase of infection) and oxidative (intestinal and systemic phase of infection) stress. Stress-sensitive strains also showed impaired intestinal colonization and systemic dissemination in orally inoculated chickens and failed to survive/grow in egg albumen. Comparative genomic hybridization microarray analysis revealed no differences at the discriminatory level of the whole gene content between stress-sensitive and stress-resistant strains. However, sequencing of rpoS, a stress-regulatory gene, revealed that one of the three stress-sensitive strains carried an insertion mutation in the rpoS resulting in truncation of σS. Finding that one of the stress-sensitive strains carried an easily identifiable small polymorphism within a stress-response gene suggests that the other strains may also have small polymorphisms elsewhere in the genome, which likely impact regulation of stress or virulence associated genes in some manner. PMID:22304629

Using co-expression analysis and stress-based screens to uncover Arabidopsis peroxisomal proteins involved in drought response

DOE PAGES

Li, Jiying; Hu, Jianping; Bassham, Diane

2015-09-14

Peroxisomes are essential organelles that house a wide array of metabolic reactions important for plant growth and development. However, our knowledge regarding the role of peroxisomal proteins in various biological processes, including plant stress response, is still incomplete. Recent proteomic studies of plant peroxisomes significantly increased the number of known peroxisomal proteins and greatly facilitated the study of peroxisomes at the systems level. The objectives of this study were to determine whether genes that encode peroxisomal proteins with related functions are co-expressed in Arabidopsis and identify peroxisomal proteins involved in stress response using in silico analysis and mutant screens. Usingmore » microarray data from online databases, we performed hierarchical clustering analysis to generate a comprehensive view of transcript level changes for Arabidopsis peroxisomal genes during development and under abiotic and biotic stress conditions. Many genes involved in the same metabolic pathways exhibited co-expression, some genes known to be involved in stress response are regulated by the corresponding stress conditions, and function of some peroxisomal proteins could be predicted based on their coexpression pattern. Since drought caused expression changes to the highest number of genes that encode peroxisomal proteins, we subjected a subset of Arabidopsis peroxisomal mutants to a drought stress assay. Mutants of the LON2 protease and the photorespiratory enzyme hydroxypyruvate reductase 1 (HPR1) showed enhanced susceptibility to drought, suggesting the involvement of peroxisomal quality control and photorespiration in drought resistance. Lastly, our study provided a global view of how genes that encode peroxisomal proteins respond to developmental and environmental cues and began to reveal additional peroxisomal proteins involved in stress response, thus opening up new avenues to investigate the role of peroxisomes in plant adaptation to environmental stresses.« less

Overexpression of pigeonpea stress-induced cold and drought regulatory gene (CcCDR) confers drought, salt, and cold tolerance in Arabidopsis

PubMed Central

Tamirisa, Srinath; Vudem, Dashavantha Reddy; Khareedu, Venkateswara Rao

2014-01-01

A potent cold and drought regulatory protein-encoding gene (CcCDR) was isolated from the subtractive cDNA library of pigeonpea plants subjected to drought stress. CcCDR was induced by different abiotic stress conditions in pigeonpea. Overexpression of CcCDR in Arabidopsis thaliana imparted enhanced tolerance against major abiotic stresses, namely drought, salinity, and low temperature, as evidenced by increased biomass, root length, and chlorophyll content. Transgenic plants also showed increased levels of antioxidant enzymes, proline, and reducing sugars under stress conditions. Furthermore, CcCDR-transgenic plants showed enhanced relative water content, osmotic potential, and cell membrane stability, as well as hypersensitivity to abscisic acid (ABA) as compared with control plants. Localization studies confirmed that CcCDR could enter the nucleus, as revealed by intense fluorescence, indicating its possible interaction with various nuclear proteins. Microarray analysis revealed that 1780 genes were up-regulated in CcCDR-transgenics compared with wild-type plants. Real-time PCR analysis on selected stress-responsive genes, involved in ABA-dependent and -independent signalling networks, revealed higher expression levels in transgenic plants, suggesting that CcCDR acts upstream of these genes. The overall results demonstrate the explicit role of CcCDR in conferring multiple abiotic stress tolerance at the whole-plant level. The multifunctional CcCDR seems promising as a prime candidate gene for enhancing abiotic stress tolerance in diverse plants. PMID:24868035

Identification and characterization of wheat long non-protein coding RNAs responsive to powdery mildew infection and heat stress by using microarray analysis and SBS sequencing

PubMed Central

2011-01-01

Background Biotic and abiotic stresses, such as powdery mildew infection and high temperature, are important limiting factors for yield and grain quality in wheat production. Emerging evidences suggest that long non-protein coding RNAs (npcRNAs) are developmentally regulated and play roles in development and stress responses of plants. However, identification of long npcRNAs is limited to a few plant species, such as Arabidopsis, rice and maize, no systematic identification of long npcRNAs and their responses to abiotic and biotic stresses is reported in wheat. Results In this study, by using computational analysis and experimental approach we identified 125 putative wheat stress responsive long npcRNAs, which are not conserved among plant species. Among them, some were precursors of small RNAs such as microRNAs and siRNAs, two long npcRNAs were identified as signal recognition particle (SRP) 7S RNA variants, and three were characterized as U3 snoRNAs. We found that wheat long npcRNAs showed tissue dependent expression patterns and were responsive to powdery mildew infection and heat stress. Conclusion Our results indicated that diverse sets of wheat long npcRNAs were responsive to powdery mildew infection and heat stress, and could function in wheat responses to both biotic and abiotic stresses, which provided a starting point to understand their functions and regulatory mechanisms in the future. PMID:21473757

PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

EPA Science Inventory

Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

S-Adenosylmethionine Prevents Mallory Denk Body Formation in Drug-Primed Mice by Inhibiting the Epigenetic Memory

PubMed Central

Li, Jun; Bardag-Gorce, Fawzia; Dedes, Jennifer; French, Barbara Alan; Amidi, Fataneh; Oliva, Joan; French, Samuel William

2010-01-01

In previous studies, microarray analysis of livers from mice fed diethyl-1,4-dihydro-2,4,6-trimethyl-3,5-pyridine decarboxylate (DDC) for 10 weeks followed by 1 month of drug withdrawal (drug-primed mice) and then 7 days of drug refeeding showed an increase in the expression of numerous genes referred to here as the molecular cellular memory. This memory predisposes the liver to Mallory Denk body formation in response to drug refeeding. In the current study, drug-primed mice were refed DDC with or without a daily dose of S-adenosylmethionine (SAMe; 4 g/kg of body weight). The livers were studied for evidence of oxidative stress and changes in gene expression with microarray analysis. SAMe prevented Mallory Denk body formation in vivo. The molecular cellular memory induced by DDC refeeding lasted for 4 months after drug withdrawal and was not manifest when SAMe was added to the diet in the in vivo experiment. Liver cells from drug-primed mice spontaneously formed Mallory Denk bodies in primary tissue cultures. SAMe prevented Mallory Denk bodies when it was added to the culture medium. Conclusion SAMe treatment prevented Mallory Denk body formation in vivo and in vitro by preventing the expression of a molecular cellular memory induced by prior DDC feeding. No evidence for the involvement of oxidative stress in induction of the memory was found. The molecular memory included the up-regulation of the expression of genes associated with the development of liver cell preneoplasia. PMID:18098314

Identification of rice genes associated with cosmic-ray response via co-expression gene network analysis.

PubMed

Hwang, Sun-Goo; Kim, Dong Sub; Hwang, Jung Eun; Han, A-Reum; Jang, Cheol Seong

2014-05-15

In order to better understand the biological systems that are affected in response to cosmic ray (CR), we conducted weighted gene co-expression network analysis using the module detection method. By using the Pearson's correlation coefficient (PCC) value, we evaluated complex gene-gene functional interactions between 680 CR-responsive probes from integrated microarray data sets, which included large-scale transcriptional profiling of 1000 microarray samples. These probes were divided into 6 distinct modules that contained 20 enriched gene ontology (GO) functions, such as oxidoreductase activity, hydrolase activity, and response to stimulus and stress. In particular, modules 1 and 2 commonly showed enriched annotation categories such as oxidoreductase activity, including enriched cis-regulatory elements known as ROS-specific regulators. These results suggest that the ROS-mediated irradiation response pathway is affected by CR in modules 1 and 2. We found 243 ionizing radiation (IR)-responsive probes that exhibited similarities in expression patterns in various irradiation microarray data sets. The expression patterns of 6 randomly selected IR-responsive genes were evaluated by quantitative reverse transcription polymerase chain reaction following treatment with CR, gamma rays (GR), and ion beam (IB); similar patterns were observed among these genes under these 3 treatments. Moreover, we constructed subnetworks of IR-responsive genes and evaluated the expression levels of their neighboring genes following GR treatment; similar patterns were observed among them. These results of network-based analyses might provide a clue to understanding the complex biological system related to the CR response in plants. Copyright © 2014 Elsevier B.V. All rights reserved.

Transcriptome-Wide Identification of Reference Genes for Expression Analysis of Soybean Responses to Drought Stress along the Day.

PubMed

Marcolino-Gomes, Juliana; Rodrigues, Fabiana Aparecida; Fuganti-Pagliarini, Renata; Nakayama, Thiago Jonas; Ribeiro Reis, Rafaela; Bouças Farias, Jose Renato; Harmon, Frank G; Correa Molinari, Hugo Bruno; Correa Molinari, Mayla Daiane; Nepomuceno, Alexandre

2015-01-01

The soybean transcriptome displays strong variation along the day in optimal growth conditions and also in response to adverse circumstances, like drought stress. However, no study conducted to date has presented suitable reference genes, with stable expression along the day, for relative gene expression quantification in combined studies on drought stress and diurnal oscillations. Recently, water deficit responses have been associated with circadian clock oscillations at the transcription level, revealing the existence of hitherto unknown processes and increasing the demand for studies on plant responses to drought stress and its oscillation during the day. We performed data mining from a transcriptome-wide background using microarrays and RNA-seq databases to select an unpublished set of candidate reference genes, specifically chosen for the normalization of gene expression in studies on soybean under both drought stress and diurnal oscillations. Experimental validation and stability analysis in soybean plants submitted to drought stress and sampled during a 24 h timecourse showed that four of these newer reference genes (FYVE, NUDIX, Golgin-84 and CYST) indeed exhibited greater expression stability than the conventionally used housekeeping genes (ELF1-β and β-actin) under these conditions. We also demonstrated the effect of using reference candidate genes with different stability values to normalize the relative expression data from a drought-inducible soybean gene (DREB5) evaluated in different periods of the day.

GeneXplorer: an interactive web application for microarray data visualization and analysis.

PubMed

Rees, Christian A; Demeter, Janos; Matese, John C; Botstein, David; Sherlock, Gavin

2004-10-01

When publishing large-scale microarray datasets, it is of great value to create supplemental websites where either the full data, or selected subsets corresponding to figures within the paper, can be browsed. We set out to create a CGI application containing many of the features of some of the existing standalone software for the visualization of clustered microarray data. We present GeneXplorer, a web application for interactive microarray data visualization and analysis in a web environment. GeneXplorer allows users to browse a microarray dataset in an intuitive fashion. It provides simple access to microarray data over the Internet and uses only HTML and JavaScript to display graphic and annotation information. It provides radar and zoom views of the data, allows display of the nearest neighbors to a gene expression vector based on their Pearson correlations and provides the ability to search gene annotation fields. The software is released under the permissive MIT Open Source license, and the complete documentation and the entire source code are freely available for download from CPAN http://search.cpan.org/dist/Microarray-GeneXplorer/.

Transcriptome-wide analysis of WRKY transcription factors in wheat and their leaf rust responsive expression profiling.

PubMed

Satapathy, Lopamudra; Singh, Dharmendra; Ranjan, Prashant; Kumar, Dhananjay; Kumar, Manish; Prabhu, Kumble Vinod; Mukhopadhyay, Kunal

2014-12-01

WRKY, a plant-specific transcription factor family, has important roles in pathogen defense, abiotic cues and phytohormone signaling, yet little is known about their roles and molecular mechanism of function in response to rust diseases in wheat. We identified 100 TaWRKY sequences using wheat Expressed Sequence Tag database of which 22 WRKY sequences were novel. Identified proteins were characterized based on their zinc finger motifs and phylogenetic analysis clustered them into six clades consisting of class IIc and class III WRKY proteins. Functional annotation revealed major functions in metabolic and cellular processes in control plants; whereas response to stimuli, signaling and defense in pathogen inoculated plants, their major molecular function being binding to DNA. Tag-based expression analysis of the identified genes revealed differential expression between mock and Puccinia triticina inoculated wheat near isogenic lines. Gene expression was also performed with six rust-related microarray experiments at Gene Expression Omnibus database. TaWRKY10, 15, 17 and 56 were common in both tag-based and microarray-based differential expression analysis and could be representing rust specific WRKY genes. The obtained results will bestow insight into the functional characterization of WRKY transcription factors responsive to leaf rust pathogenesis that can be used as candidate genes in molecular breeding programs to improve biotic stress tolerance in wheat.

Nanotechnology: moving from microarrays toward nanoarrays.

PubMed

Chen, Hua; Li, Jun

2007-01-01

Microarrays are important tools for high-throughput analysis of biomolecules. The use of microarrays for parallel screening of nucleic acid and protein profiles has become an industry standard. A few limitations of microarrays are the requirement for relatively large sample volumes and elongated incubation time, as well as the limit of detection. In addition, traditional microarrays make use of bulky instrumentation for the detection, and sample amplification and labeling are quite laborious, which increase analysis cost and delays the time for obtaining results. These problems limit microarray techniques from point-of-care and field applications. One strategy for overcoming these problems is to develop nanoarrays, particularly electronics-based nanoarrays. With further miniaturization, higher sensitivity, and simplified sample preparation, nanoarrays could potentially be employed for biomolecular analysis in personal healthcare and monitoring of trace pathogens. In this chapter, it is intended to introduce the concept and advantage of nanotechnology and then describe current methods and protocols for novel nanoarrays in three aspects: (1) label-free nucleic acids analysis using nanoarrays, (2) nanoarrays for protein detection by conventional optical fluorescence microscopy as well as by novel label-free methods such as atomic force microscopy, and (3) nanoarray for enzymatic-based assay. These nanoarrays will have significant applications in drug discovery, medical diagnosis, genetic testing, environmental monitoring, and food safety inspection.

A meta-data based method for DNA microarray imputation.

PubMed

Jörnsten, Rebecka; Ouyang, Ming; Wang, Hui-Yu

2007-03-29

DNA microarray experiments are conducted in logical sets, such as time course profiling after a treatment is applied to the samples, or comparisons of the samples under two or more conditions. Due to cost and design constraints of spotted cDNA microarray experiments, each logical set commonly includes only a small number of replicates per condition. Despite the vast improvement of the microarray technology in recent years, missing values are prevalent. Intuitively, imputation of missing values is best done using many replicates within the same logical set. In practice, there are few replicates and thus reliable imputation within logical sets is difficult. However, it is in the case of few replicates that the presence of missing values, and how they are imputed, can have the most profound impact on the outcome of downstream analyses (e.g. significance analysis and clustering). This study explores the feasibility of imputation across logical sets, using the vast amount of publicly available microarray data to improve imputation reliability in the small sample size setting. We download all cDNA microarray data of Saccharomyces cerevisiae, Arabidopsis thaliana, and Caenorhabditis elegans from the Stanford Microarray Database. Through cross-validation and simulation, we find that, for all three species, our proposed imputation using data from public databases is far superior to imputation within a logical set, sometimes to an astonishing degree. Furthermore, the imputation root mean square error for significant genes is generally a lot less than that of non-significant ones. Since downstream analysis of significant genes, such as clustering and network analysis, can be very sensitive to small perturbations of estimated gene effects, it is highly recommended that researchers apply reliable data imputation prior to further analysis. Our method can also be applied to cDNA microarray experiments from other species, provided good reference data are available.

Expanding frontiers in plant transcriptomics in aid of functional genomics and molecular breeding.

PubMed

Agarwal, Pinky; Parida, Swarup K; Mahto, Arunima; Das, Sweta; Mathew, Iny Elizebeth; Malik, Naveen; Tyagi, Akhilesh K

2014-12-01

The transcript pool of a plant part, under any given condition, is a collection of mRNAs that will pave the way for a biochemical reaction of the plant to stimuli. Over the past decades, transcriptome study has advanced from Northern blotting to RNA sequencing (RNA-seq), through other techniques, of which real-time quantitative polymerase chain reaction (PCR) and microarray are the most significant ones. The questions being addressed by such studies have also matured from a solitary process to expression atlas and marker-assisted genetic enhancement. Not only genes and their networks involved in various developmental processes of plant parts have been elucidated, but also stress tolerant genes have been highlighted. The transcriptome of a plant with altered expression of a target gene has given information about the downstream genes. Marker information has been used for breeding improved varieties. Fortunately, the data generated by transcriptome analysis has been made freely available for ample utilization and comparison. The review discusses this wide variety of transcriptome data being generated in plants, which includes developmental stages, abiotic and biotic stress, effect of altered gene expression, as well as comparative transcriptomics, with a special emphasis on microarray and RNA-seq. Such data can be used to determine the regulatory gene networks, which can subsequently be utilized for generating improved plant varieties. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification, occurrence, and validation of DRE and ABRE Cis-regulatory motifs in the promoter regions of genes of Arabidopsis thaliana.

PubMed

Mishra, Sonal; Shukla, Aparna; Upadhyay, Swati; Sanchita; Sharma, Pooja; Singh, Seema; Phukan, Ujjal J; Meena, Abha; Khan, Feroz; Tripathi, Vineeta; Shukla, Rakesh Kumar; Shrama, Ashok

2014-04-01

Plants posses a complex co-regulatory network which helps them to elicit a response under diverse adverse conditions. We used an in silico approach to identify the genes with both DRE and ABRE motifs in their promoter regions in Arabidopsis thaliana. Our results showed that Arabidopsis contains a set of 2,052 genes with ABRE and DRE motifs in their promoter regions. Approximately 72% or more of the total predicted 2,052 genes had a gap distance of less than 400 bp between DRE and ABRE motifs. For positional orientation of the DRE and ABRE motifs, we found that the DR form (one in direct and the other one in reverse orientation) was more prevalent than other forms. These predicted 2,052 genes include 155 transcription factors. Using microarray data from The Arabidopsis Information Resource (TAIR) database, we present 44 transcription factors out of 155 which are upregulated by more than twofold in response to osmotic stress and ABA treatment. Fifty-one transcripts from the one predicted above were validated using semiquantitative expression analysis to support the microarray data in TAIR. Taken together, we report a set of genes containing both DRE and ABRE motifs in their promoter regions in A. thaliana, which can be useful to understand the role of ABA under osmotic stress condition. © 2013 Institute of Botany, Chinese Academy of Sciences.

Insights into the Sigma-1 receptor chaperone’s cellular functions: a microarray report

PubMed Central

Tsai, Shang-Yi; Rothman, Richard Kyle; Su, Tsung-Ping

2013-01-01

We previously demonstrated that Sig-1Rs are critical regulators in neuronal morphogenesis and development via the regulation of oxidative stress and mitochondrial functions. In the present study, we sought to identify pathways and genes that are affected by Sig-1R. Gene expression profiles were examined in rat hippocampal neurons that had been cultured for18 days in vitro (DIV). The cells were transduced with AAV siRNA targeting Sig-1R on DIV 10 for 7 days, followed by gene expression analysis using a rat genome cDNA array. The gene array results indicated that Sig-1R knockdown hampered cellular functions including steroid biogenesis, protein ubiquitination, actin cytoskeleton network, and Nrf-2 mediated oxidative stress. Many of the cellular components important for actin polymerization and synapse plasticity, including F-actin capping protein and neurofilaments, were significantly changed in AAV-siSig-1R neurons. Further, cytochrome c was reduced in AAV-Sig-1R neurons whereas free-radical generating enzymes including cytochrome p450 and cytochrome b-245 were increased. The microarray results also suggest that Sig-1Rs may regulate genes that are involved in the pathogenesis of many CNS diseases including Alzheimer’s disease and Parkinson’s disease. These data further confirmed that Sig-1Rs play critical roles in the CNS and thus these findings may aid in future development of therapeutic treatments targeting neurodegenerative disorders. PMID:21905129

Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes.

PubMed

Dong, Xiangshu; Kim, Wan Kyu; Lim, Yong-Pyo; Kim, Yeon-Ki; Hur, Yoonkang

2013-02-01

We investigated the mechanism regulating cytoplasmic male sterility (CMS) in Brassica rapa ssp. pekinensis using floral bud transcriptome analyses of Ogura-CMS Chinese cabbage and its maintainer line in B. rapa 300-K oligomeric probe (Br300K) microarrays. Ogura-CMS Chinese cabbage produced few and infertile pollen grains on indehiscent anthers. Compared to the maintainer line, CMS plants had shorter filaments and plant growth, and delayed flowering and pollen development. In microarray analysis, 4646 genes showed different expression, depending on floral bud size, between Ogura-CMS and its maintainer line. We found 108 and 62 genes specifically expressed in Ogura-CMS and its maintainer line, respectively. Ogura-CMS line-specific genes included stress-related, redox-related, and B. rapa novel genes. In the maintainer line, genes related to pollen coat and germination were specifically expressed in floral buds longer than 3mm, suggesting insufficient expression of these genes in Ogura-CMS is directly related to dysfunctional pollen. In addition, many nuclear genes associated with auxin response, ATP synthesis, pollen development and stress response had delayed expression in Ogura-CMS plants compared to the maintainer line, which is consistent with the delay in growth and development of Ogura-CMS plants. Delayed expression may reduce pollen grain production and/or cause sterility, implying that mitochondrial, retrograde signaling delays nuclear gene expression. Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.

Transcriptome analysis supports viral infection and fluoride toxicity as contributors to chronic kidney disease of unknown etiology (CKDu) in Sri Lanka.

PubMed

Sayanthooran, Saravanabavan; Gunerathne, Lishanthe; Abeysekera, Tilak D J; Magana-Arachchi, Dhammika N

2018-05-28

Chronic kidney disease of unknown etiology (CKDu), having epidemic characteristics, is being diagnosed increasingly in certain tropical regions of the world, mainly Latin America and Sri Lanka. They have been observed primarily in farming communities and current hypotheses point toward many environmental and occupational triggers. CKDu does not have common etiologies of chronic kidney disease (CKD) such as hypertension, diabetes, or autoimmune disease. We aimed to understand the molecular processes underlying CKDu in Sri Lanka using transcriptome analysis. RNA extracted from whole blood was reverse transcribed and used for microarray analysis using the Human HT-12 v.4 array (Illumina). Pathway analysis was carried out using ingenuity pathway analysis (IPA-Qiagen). Microarray results were validated using real-time PCR of five selected genes. Pathways related to innate immune response, including interferon signaling, inflammasome signaling and TREM1 signaling had the most significant positive activation z scores, where as EIF2 signaling and mTOR signaling had the most significant negative activation z scores. Pathways previously linked to fluoride toxicity; G-protein activation, Cdc42 signaling, Rac signaling and RhoA signaling were activated in CKDu patients. The most significantly activated biological functions were cell death, cell movement and antimicrobial response. Significant toxicological functions were mitochondrial dysfunction, oxidative stress and apoptosis. Based on the molecular pathway analysis in CKDu patients and review of literature, viral infections and fluoride toxicity appear to be contributing to the molecular mechanisms underlying CKDu.

BABAR: an R package to simplify the normalisation of common reference design microarray-based transcriptomic datasets

PubMed Central

2010-01-01

Background The development of DNA microarrays has facilitated the generation of hundreds of thousands of transcriptomic datasets. The use of a common reference microarray design allows existing transcriptomic data to be readily compared and re-analysed in the light of new data, and the combination of this design with large datasets is ideal for 'systems'-level analyses. One issue is that these datasets are typically collected over many years and may be heterogeneous in nature, containing different microarray file formats and gene array layouts, dye-swaps, and showing varying scales of log2- ratios of expression between microarrays. Excellent software exists for the normalisation and analysis of microarray data but many data have yet to be analysed as existing methods struggle with heterogeneous datasets; options include normalising microarrays on an individual or experimental group basis. Our solution was to develop the Batch Anti-Banana Algorithm in R (BABAR) algorithm and software package which uses cyclic loess to normalise across the complete dataset. We have already used BABAR to analyse the function of Salmonella genes involved in the process of infection of mammalian cells. Results The only input required by BABAR is unprocessed GenePix or BlueFuse microarray data files. BABAR provides a combination of 'within' and 'between' microarray normalisation steps and diagnostic boxplots. When applied to a real heterogeneous dataset, BABAR normalised the dataset to produce a comparable scaling between the microarrays, with the microarray data in excellent agreement with RT-PCR analysis. When applied to a real non-heterogeneous dataset and a simulated dataset, BABAR's performance in identifying differentially expressed genes showed some benefits over standard techniques. Conclusions BABAR is an easy-to-use software tool, simplifying the simultaneous normalisation of heterogeneous two-colour common reference design cDNA microarray-based transcriptomic datasets. We show BABAR transforms real and simulated datasets to allow for the correct interpretation of these data, and is the ideal tool to facilitate the identification of differentially expressed genes or network inference analysis from transcriptomic datasets. PMID:20128918

Melatonin attenuates dextran sodium sulfate induced colitis with sleep deprivation: possible mechanism by microarray analysis.

PubMed

Chung, Sook Hee; Park, Young Sook; Kim, Ok Soon; Kim, Ja Hyun; Baik, Haing Woon; Hong, Young Ok; Kim, Sang Su; Shin, Jae-Ho; Jun, Jin-Hyun; Jo, Yunju; Ahn, Sang Bong; Jo, Young Kwan; Son, Byoung Kwan; Kim, Seong Hwan

2014-06-01

Inflammatory bowel disease is a chronic inflammatory condition of the gastrointestinal tract. It can be aggravated by stress, like sleep deprivation, and improved by anti-inflammatory agents, like melatonin. We aimed to investigate the effects of sleep deprivation and melatonin on inflammation. We also investigated genes regulated by sleep deprivation and melatonin. In the 2% DSS induced colitis mice model, sleep deprivation was induced using modified multiple platform water bath. Melatonin was injected after induction of colitis and colitis with sleep deprivation. Also mRNA was isolated from the colon of mice and analyzed via microarray and real-time PCR. Sleep deprivation induced reduction of body weight, and it was difficult for half of the mice to survive. Sleep deprivation aggravated, and melatonin attenuated the severity of colitis. In microarrays and real-time PCR of mice colon tissues, mRNA of adiponectin and aquaporin 8 were downregulated by sleep deprivation and upregulated by melatonin. However, mRNA of E2F transcription factor (E2F2) and histocompatibility class II antigen A, beta 1 (H2-Ab1) were upregulated by sleep deprivation and downregulated by melatonin. Melatonin improves and sleep deprivation aggravates inflammation of colitis in mice. Adiponectin, aquaporin 8, E2F2 and H2-Ab1 may be involved in the inflammatory change aggravated by sleep deprivation and attenuated by melatonin.

The efficacy of microarray screening for autosomal recessive retinitis pigmentosa in routine clinical practice

PubMed Central

van Huet, Ramon A. C.; Pierrache, Laurence H.M.; Meester-Smoor, Magda A.; Klaver, Caroline C.W.; van den Born, L. Ingeborgh; Hoyng, Carel B.; de Wijs, Ilse J.; Collin, Rob W. J.; Hoefsloot, Lies H.

2015-01-01

Purpose To determine the efficacy of multiple versions of a commercially available arrayed primer extension (APEX) microarray chip for autosomal recessive retinitis pigmentosa (arRP). Methods We included 250 probands suspected of arRP who were genetically analyzed with the APEX microarray between January 2008 and November 2013. The mode of inheritance had to be autosomal recessive according to the pedigree (including isolated cases). If the microarray identified a heterozygous mutation, we performed Sanger sequencing of exons and exon–intron boundaries of that specific gene. The efficacy of this microarray chip with the additional Sanger sequencing approach was determined by the percentage of patients that received a molecular diagnosis. We also collected data from genetic tests other than the APEX analysis for arRP to provide a detailed description of the molecular diagnoses in our study cohort. Results The APEX microarray chip for arRP identified the molecular diagnosis in 21 (8.5%) of the patients in our cohort. Additional Sanger sequencing yielded a second mutation in 17 patients (6.8%), thereby establishing the molecular diagnosis. In total, 38 patients (15.2%) received a molecular diagnosis after analysis using the microarray and additional Sanger sequencing approach. Further genetic analyses after a negative result of the arRP microarray (n = 107) resulted in a molecular diagnosis of arRP (n = 23), autosomal dominant RP (n = 5), X-linked RP (n = 2), and choroideremia (n = 1). Conclusions The efficacy of the commercially available APEX microarray chips for arRP appears to be low, most likely caused by the limitations of this technique and the genetic and allelic heterogeneity of RP. Diagnostic yields up to 40% have been reported for next-generation sequencing (NGS) techniques that, as expected, thereby outperform targeted APEX analysis. PMID:25999674

The search for the neurobiological basis of vulnerability to drug abuse: using microarrays to investigate the role of stress and individual differences.

PubMed

Kabbaj, M; Evans, S; Watson, S J; Akil, H

2004-01-01

Basic neurobiological studies have led to great progress in our understanding of the mechanisms of action of drugs of abuse. Much has been learned about the brain response from the moment a psychoactive drug enters the organism onwards, including the psychological, neurobiological and peripheral effects of repeated drug administration, withdrawal and re-exposure. However, to relate this knowledge to the human experience requires further research on the antecedents of drug-taking behavior and the factors that predispose particular individuals to drug seeking and drug abuse. Thus, it is important to address several issues at the fundamental level: (1) Why are some individuals more vulnerable to drugs of abuse more than others? Is there a broader dimension or dimensions of emotional reactivity that contribute to this difference in vulnerability? (2) What is the effect of psychosocial stress on drug-seeking and drug-taking behavior, and are the effects distinct across individuals? (3) Since both drug-taking behavior and stress have sustained and pervasive effects on the brain, can we use microarrays to discern the "neural signature" or "neural phenotype" associated with these processes, and can we distinguish this signature across individuals with differing propensities to taking drugs? In the present paper, we summarize some of our early attempts at addressing these questions. We rely on animal studies aimed at characterizing the emotional and stress reactivity of rats with different propensities to self-administer drugs (high responders and low responders); we briefly describe the effect of a psychosocial stressor on these animals; we then detail a study using microarray technology aimed at investigating the "neural phenotype" associated with social defeat stress in the high vs. low responder animals. This "discovery" approach is used as a starting place for identifying novel mechanisms that might alter the vulnerability of different individuals to drug-seeking behavior. The power and limits of this approach, and its future directions, are discussed within this general framework.

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factors under Multiple Stresses in Brassica napus

PubMed Central

He, Yajun; Mao, Shaoshuai; Gao, Yulong; Zhu, Liying; Wu, Daoming; Cui, Yixin; Li, Jiana; Qian, Wei

2016-01-01

WRKY transcription factors play important roles in responses to environmental stress stimuli. Using a genome-wide domain analysis, we identified 287 WRKY genes with 343 WRKY domains in the sequenced genome of Brassica napus, 139 in the A sub-genome and 148 in the C sub-genome. These genes were classified into eight groups based on phylogenetic analysis. In the 343 WRKY domains, a total of 26 members showed divergence in the WRKY domain, and 21 belonged to group I. This finding suggested that WRKY genes in group I are more active and variable compared with genes in other groups. Using genome-wide identification and analysis of the WRKY gene family in Brassica napus, we observed genome duplication, chromosomal/segmental duplications and tandem duplication. All of these duplications contributed to the expansion of the WRKY gene family. The duplicate segments that were detected indicated that genome duplication events occurred in the two diploid progenitors B. rapa and B. olearecea before they combined to form B. napus. Analysis of the public microarray database and EST database for B. napus indicated that 74 WRKY genes were induced or preferentially expressed under stress conditions. According to the public QTL data, we identified 77 WRKY genes in 31 QTL regions related to various stress tolerance. We further evaluated the expression of 26 BnaWRKY genes under multiple stresses by qRT-PCR. Most of the genes were induced by low temperature, salinity and drought stress, indicating that the WRKYs play important roles in B. napus stress responses. Further, three BnaWRKY genes were strongly responsive to the three multiple stresses simultaneously, which suggests that these 3 WRKY may have multi-functional roles in stress tolerance and can potentially be used in breeding new rapeseed cultivars. We also found six tandem repeat pairs exhibiting similar expression profiles under the various stress conditions, and three pairs were mapped in the stress related QTL regions, indicating tandem duplicate WRKYs in the adaptive responses to environmental stimuli during the evolution process. Our results provide a framework for future studies regarding the function of WRKY genes in response to stress in B. napus. PMID:27322342

Genome-Wide Identification and Expression Analysis of WRKY Transcription Factors under Multiple Stresses in Brassica napus.

PubMed

He, Yajun; Mao, Shaoshuai; Gao, Yulong; Zhu, Liying; Wu, Daoming; Cui, Yixin; Li, Jiana; Qian, Wei

2016-01-01

WRKY transcription factors play important roles in responses to environmental stress stimuli. Using a genome-wide domain analysis, we identified 287 WRKY genes with 343 WRKY domains in the sequenced genome of Brassica napus, 139 in the A sub-genome and 148 in the C sub-genome. These genes were classified into eight groups based on phylogenetic analysis. In the 343 WRKY domains, a total of 26 members showed divergence in the WRKY domain, and 21 belonged to group I. This finding suggested that WRKY genes in group I are more active and variable compared with genes in other groups. Using genome-wide identification and analysis of the WRKY gene family in Brassica napus, we observed genome duplication, chromosomal/segmental duplications and tandem duplication. All of these duplications contributed to the expansion of the WRKY gene family. The duplicate segments that were detected indicated that genome duplication events occurred in the two diploid progenitors B. rapa and B. olearecea before they combined to form B. napus. Analysis of the public microarray database and EST database for B. napus indicated that 74 WRKY genes were induced or preferentially expressed under stress conditions. According to the public QTL data, we identified 77 WRKY genes in 31 QTL regions related to various stress tolerance. We further evaluated the expression of 26 BnaWRKY genes under multiple stresses by qRT-PCR. Most of the genes were induced by low temperature, salinity and drought stress, indicating that the WRKYs play important roles in B. napus stress responses. Further, three BnaWRKY genes were strongly responsive to the three multiple stresses simultaneously, which suggests that these 3 WRKY may have multi-functional roles in stress tolerance and can potentially be used in breeding new rapeseed cultivars. We also found six tandem repeat pairs exhibiting similar expression profiles under the various stress conditions, and three pairs were mapped in the stress related QTL regions, indicating tandem duplicate WRKYs in the adaptive responses to environmental stimuli during the evolution process. Our results provide a framework for future studies regarding the function of WRKY genes in response to stress in B. napus.

Genome-wide identification and characterisation of F-box family in maize.

PubMed

Jia, Fengjuan; Wu, Bingjiang; Li, Hui; Huang, Jinguang; Zheng, Chengchao

2013-11-01

F-box-containing proteins, as the key components of the protein degradation machinery, are widely distributed in higher plants and are considered as one of the largest known families of regulatory proteins. The F-box protein family plays a crucial role in plant growth and development and in response to biotic and abiotic stresses. However, systematic analysis of the F-box family in maize (Zea mays) has not been reported yet. In this paper, we identified and characterised the maize F-box genes in a genome-wide scale, including phylogenetic analysis, chromosome distribution, gene structure, promoter analysis and gene expression profiles. A total of 359 F-box genes were identified and divided into 15 subgroups by phylogenetic analysis. The F-box domain was relatively conserved, whereas additional motifs outside the F-box domain may indicate the functional diversification of maize F-box genes. These genes were unevenly distributed in ten maize chromosomes, suggesting that they expanded in the maize genome because of tandem and segmental duplication events. The expression profiles suggested that the maize F-box genes had temporal and spatial expression patterns. Putative cis-acting regulatory DNA elements involved in abiotic stresses were observed in maize F-box gene promoters. The gene expression profiles under abiotic stresses also suggested that some genes participated in stress responsive pathways. Furthermore, ten genes were chosen for quantitative real-time PCR analysis under drought stress and the results were consistent with the microarray data. This study has produced a comparative genomics analysis of the maize ZmFBX gene family that can be used in further studies to uncover their roles in maize growth and development.

The dehydration stress of couch grass is associated with its lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA.

PubMed

Janská, Anna; Svoboda, Pavel; Spiwok, Vojtěch; Kučera, Ladislav; Ovesná, Jaroslava

2018-05-02

The wild relatives of crop species represent a potentially valuable source of novel genetic variation, particularly in the context of improving the crop's level of tolerance to abiotic stress. The mechanistic basis of these tolerances remains largely unexplored. Here, the focus was to characterize the transcriptomic response of the nodes (meristematic tissue) of couch grass (a relative of barley) to dehydration stress, and to compare it to that of the barley crown formed by both a drought tolerant and a drought sensitive barley cultivar. Many of the genes up-regulated in the nodes by the stress were homologs of genes known to be mediated by abscisic acid during the response to drought, or were linked to either development or lipid metabolism. Transporters also featured prominently, as did genes acting on root architecture. The resilience of the couch grass node arise from both their capacity to develop an altered, more effective root architecture, but also from their formation of a lipid barrier on their outer surface and their ability to modify both their lipid metabolism and transporter activity when challenged by dehydration stress. Our analysis revealed the nature of dehydration stress response in couch grass. We suggested the tolerance is associated with lipid metabolism, the induction of transporters and the re-programming of development coordinated by ABA. We also proved the applicability of barley microarray for couch grass stress-response analysis.

Principles of gene microarray data analysis.

PubMed

Mocellin, Simone; Rossi, Carlo Riccardo

2007-01-01

The development of several gene expression profiling methods, such as comparative genomic hybridization (CGH), differential display, serial analysis of gene expression (SAGE), and gene microarray, together with the sequencing of the human genome, has provided an opportunity to monitor and investigate the complex cascade of molecular events leading to tumor development and progression. The availability of such large amounts of information has shifted the attention of scientists towards a nonreductionist approach to biological phenomena. High throughput technologies can be used to follow changing patterns of gene expression over time. Among them, gene microarray has become prominent because it is easier to use, does not require large-scale DNA sequencing, and allows for the parallel quantification of thousands of genes from multiple samples. Gene microarray technology is rapidly spreading worldwide and has the potential to drastically change the therapeutic approach to patients affected with tumor. Therefore, it is of paramount importance for both researchers and clinicians to know the principles underlying the analysis of the huge amount of data generated with microarray technology.

16S rRNA gene-based phylogenetic microarray for simultaneous identification of members of the genus Burkholderia.

PubMed

Schönmann, Susan; Loy, Alexander; Wimmersberger, Céline; Sobek, Jens; Aquino, Catharine; Vandamme, Peter; Frey, Beat; Rehrauer, Hubert; Eberl, Leo

2009-04-01

For cultivation-independent and highly parallel analysis of members of the genus Burkholderia, an oligonucleotide microarray (phylochip) consisting of 131 hierarchically nested 16S rRNA gene-targeted oligonucleotide probes was developed. A novel primer pair was designed for selective amplification of a 1.3 kb 16S rRNA gene fragment of Burkholderia species prior to microarray analysis. The diagnostic performance of the microarray for identification and differentiation of Burkholderia species was tested with 44 reference strains of the genera Burkholderia, Pandoraea, Ralstonia and Limnobacter. Hybridization patterns based on presence/absence of probe signals were interpreted semi-automatically using the novel likelihood-based strategy of the web-tool Phylo- Detect. Eighty-eight per cent of the reference strains were correctly identified at the species level. The evaluated microarray was applied to investigate shifts in the Burkholderia community structure in acidic forest soil upon addition of cadmium, a condition that selected for Burkholderia species. The microarray results were in agreement with those obtained from phylogenetic analysis of Burkholderia 16S rRNA gene sequences recovered from the same cadmiumcontaminated soil, demonstrating the value of the Burkholderia phylochip for determinative and environmental studies.

Support vector machine and principal component analysis for microarray data classification

NASA Astrophysics Data System (ADS)

Astuti, Widi; Adiwijaya

2018-03-01

Cancer is a leading cause of death worldwide although a significant proportion of it can be cured if it is detected early. In recent decades, technology called microarray takes an important role in the diagnosis of cancer. By using data mining technique, microarray data classification can be performed to improve the accuracy of cancer diagnosis compared to traditional techniques. The characteristic of microarray data is small sample but it has huge dimension. Since that, there is a challenge for researcher to provide solutions for microarray data classification with high performance in both accuracy and running time. This research proposed the usage of Principal Component Analysis (PCA) as a dimension reduction method along with Support Vector Method (SVM) optimized by kernel functions as a classifier for microarray data classification. The proposed scheme was applied on seven data sets using 5-fold cross validation and then evaluation and analysis conducted on term of both accuracy and running time. The result showed that the scheme can obtained 100% accuracy for Ovarian and Lung Cancer data when Linear and Cubic kernel functions are used. In term of running time, PCA greatly reduced the running time for every data sets.

Experimental Approaches to Microarray Analysis of Tumor Samples

ERIC Educational Resources Information Center

Furge, Laura Lowe; Winter, Michael B.; Meyers, Jacob I.; Furge, Kyle A.

2008-01-01

Comprehensive measurement of gene expression using high-density nucleic acid arrays (i.e. microarrays) has become an important tool for investigating the molecular differences in clinical and research samples. Consequently, inclusion of discussion in biochemistry, molecular biology, or other appropriate courses of microarray technologies has…

Multiplex cDNA quantification method that facilitates the standardization of gene expression data

PubMed Central

Gotoh, Osamu; Murakami, Yasufumi; Suyama, Akira

2011-01-01

Microarray-based gene expression measurement is one of the major methods for transcriptome analysis. However, current microarray data are substantially affected by microarray platforms and RNA references because of the microarray method can provide merely the relative amounts of gene expression levels. Therefore, valid comparisons of the microarray data require standardized platforms, internal and/or external controls and complicated normalizations. These requirements impose limitations on the extensive comparison of gene expression data. Here, we report an effective approach to removing the unfavorable limitations by measuring the absolute amounts of gene expression levels on common DNA microarrays. We have developed a multiplex cDNA quantification method called GEP-DEAN (Gene expression profiling by DCN-encoding-based analysis). The method was validated by using chemically synthesized DNA strands of known quantities and cDNA samples prepared from mouse liver, demonstrating that the absolute amounts of cDNA strands were successfully measured with a sensitivity of 18 zmol in a highly multiplexed manner in 7 h. PMID:21415008

Spot detection and image segmentation in DNA microarray data.

PubMed

Qin, Li; Rueda, Luis; Ali, Adnan; Ngom, Alioune

2005-01-01

Following the invention of microarrays in 1994, the development and applications of this technology have grown exponentially. The numerous applications of microarray technology include clinical diagnosis and treatment, drug design and discovery, tumour detection, and environmental health research. One of the key issues in the experimental approaches utilising microarrays is to extract quantitative information from the spots, which represent genes in a given experiment. For this process, the initial stages are important and they influence future steps in the analysis. Identifying the spots and separating the background from the foreground is a fundamental problem in DNA microarray data analysis. In this review, we present an overview of state-of-the-art methods for microarray image segmentation. We discuss the foundations of the circle-shaped approach, adaptive shape segmentation, histogram-based methods and the recently introduced clustering-based techniques. We analytically show that clustering-based techniques are equivalent to the one-dimensional, standard k-means clustering algorithm that utilises the Euclidean distance.

Split-plot microarray experiments: issues of design, power and sample size.

PubMed

Tsai, Pi-Wen; Lee, Mei-Ling Ting

2005-01-01

This article focuses on microarray experiments with two or more factors in which treatment combinations of the factors corresponding to the samples paired together onto arrays are not completely random. A main effect of one (or more) factor(s) is confounded with arrays (the experimental blocks). This is called a split-plot microarray experiment. We utilise an analysis of variance (ANOVA) model to assess differentially expressed genes for between-array and within-array comparisons that are generic under a split-plot microarray experiment. Instead of standard t- or F-test statistics that rely on mean square errors of the ANOVA model, we use a robust method, referred to as 'a pooled percentile estimator', to identify genes that are differentially expressed across different treatment conditions. We illustrate the design and analysis of split-plot microarray experiments based on a case application described by Jin et al. A brief discussion of power and sample size for split-plot microarray experiments is also presented.

Genome-wide analysis of drought induced gene expression changes in flax (Linum usitatissimum).

PubMed

Dash, Prasanta K; Cao, Yongguo; Jailani, Abdul K; Gupta, Payal; Venglat, Prakash; Xiang, Daoquan; Rai, Rhitu; Sharma, Rinku; Thirunavukkarasu, Nepolean; Abdin, Malik Z; Yadava, Devendra K; Singh, Nagendra K; Singh, Jas; Selvaraj, Gopalan; Deyholos, Mike; Kumar, Polumetla Ananda; Datla, Raju

2014-01-01

A robust phenotypic plasticity to ward off adverse environmental conditions determines performance and productivity in crop plants. Flax (linseed), is an important cash crop produced for natural textile fiber (linen) or oilseed with many health promoting products. This crop is prone to drought stress and yield losses in many parts of the world. Despite recent advances in drought research in a number of important crops, related progress in flax is very limited. Since, response of this plant to drought stress has not been addressed at the molecular level; we conducted microarray analysis to capture transcriptome associated with induced drought in flax. This study identified 183 differentially expressed genes (DEGs) associated with diverse cellular, biophysical and metabolic programs in flax. The analysis also revealed especially the altered regulation of cellular and metabolic pathways governing photosynthesis. Additionally, comparative transcriptome analysis identified a plethora of genes that displayed differential regulation both spatially and temporally. These results revealed co-regulated expression of 26 genes in both shoot and root tissues with implications for drought stress response. Furthermore, the data also showed that more genes are upregulated in roots compared to shoots, suggesting that roots may play important and additional roles in response to drought in flax. With prolonged drought treatment, the number of DEGs increased in both tissue types. Differential expression of selected genes was confirmed by qRT-PCR, thus supporting the suggested functional association of these intrinsic genes in maintaining growth and homeostasis in response to imminent drought stress in flax. Together the present study has developed foundational and new transcriptome data sets for drought stress in flax.

Identification of genes involved in the ACC-mediated control of root cell elongation in Arabidopsis thaliana

PubMed Central

2012-01-01

Background Along the root axis of Arabidopsis thaliana, cells pass through different developmental stages. In the apical meristem repeated cycles of division increase the numbers of cells. Upon leaving the meristem, these cells pass the transition zone where they are physiologically and mechanically prepared to undergo subsequent rapid elongation. During the process of elongation epidermal cells increase their length by 300% in a couple of hours. When elongation ceases, the cells acquire their final size, shape and functions (in the differentiation zone). Ethylene administered as its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) is capable of inhibiting elongation in a concentration-dependent way. Using a microarray analysis, genes and/or processes involved in this elongation arrest are identified. Results Using a CATMA-microarray analysis performed on control and 3h ACC-treated roots, 240 differentially expressed genes were identified. Quantitative Real-Time RT-PCR analysis of the 10 most up and down regulated genes combined with literature search confirmed the accurateness of the analysis. This revealed that inhibition of cell elongation is, at least partly, caused by restricting the events that under normal growth conditions initiate elongation and by increasing the processes that normally stop cellular elongation at the end of the elongation/onset of differentiation zone. Conclusions ACC interferes with cell elongation in the Arabidopsis thaliana roots by inhibiting cells from entering the elongation process and by immediately stimulating the formation of cross-links in cell wall components, diminishing the remaining elongation capacity. From the analysis of the differentially expressed genes, it becomes clear that many genes identified in this response, are also involved in several other kind of stress responses. This suggests that many responses originate from individual elicitors, but that somewhere in the downstream signaling cascade, these are converged to a ’common pathway’. Furthermore, several potential keyplayers, such as transcription factors and auxin-responsive genes, were identified by the microarray analysis. They await further analysis to reveal their exact role in the control of cell elongation. PMID:23134674

Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni

PubMed Central

Lockyer, Anne E; Spinks, Jenny; Kane, Richard A; Hoffmann, Karl F; Fitzpatrick, Jennifer M; Rollinson, David; Noble, Leslie R; Jones, Catherine S

2008-01-01

Background Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. Results We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1α and EF-2. Conclusion Production of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination. PMID:19114004

Biomphalaria glabrata transcriptome: cDNA microarray profiling identifies resistant- and susceptible-specific gene expression in haemocytes from snail strains exposed to Schistosoma mansoni.

PubMed

Lockyer, Anne E; Spinks, Jenny; Kane, Richard A; Hoffmann, Karl F; Fitzpatrick, Jennifer M; Rollinson, David; Noble, Leslie R; Jones, Catherine S

2008-12-29

Biomphalaria glabrata is an intermediate snail host for Schistosoma mansoni, one of the important schistosomes infecting man. B. glabrata/S. mansoni provides a useful model system for investigating the intimate interactions between host and parasite. Examining differential gene expression between S. mansoni-exposed schistosome-resistant and susceptible snail lines will identify genes and pathways that may be involved in snail defences. We have developed a 2053 element cDNA microarray for B. glabrata containing clones from ORESTES (Open Reading frame ESTs) libraries, suppression subtractive hybridization (SSH) libraries and clones identified in previous expression studies. Snail haemocyte RNA, extracted from parasite-challenged resistant and susceptible snails, 2 to 24 h post-exposure to S. mansoni, was hybridized to the custom made cDNA microarray and 98 differentially expressed genes or gene clusters were identified, 94 resistant-associated and 4 susceptible-associated. Quantitative PCR analysis verified the cDNA microarray results for representative transcripts. Differentially expressed genes were annotated and clustered using gene ontology (GO) terminology and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis. 61% of the identified differentially expressed genes have no known function including the 4 susceptible strain-specific transcripts. Resistant strain-specific expression of genes implicated in innate immunity of invertebrates was identified, including hydrolytic enzymes such as cathepsin L, a cysteine proteinase involved in lysis of phagocytosed particles; metabolic enzymes such as ornithine decarboxylase, the rate-limiting enzyme in the production of polyamines, important in inflammation and infection processes, as well as scavenging damaging free radicals produced during production of reactive oxygen species; stress response genes such as HSP70; proteins involved in signalling, such as importin 7 and copine 1, cytoplasmic intermediate filament (IF) protein and transcription enzymes such as elongation factor 1alpha and EF-2. Production of the first cDNA microarray for profiling gene expression in B. glabrata provides a foundation for expanding our understanding of pathways and genes involved in the snail internal defence system (IDS). We demonstrate resistant strain-specific expression of genes potentially associated with the snail IDS, ranging from signalling and inflammation responses through to lysis of proteinacous products (encapsulated sporocysts or phagocytosed parasite components) and processing/degradation of these targeted products by ubiquitination.

Detection of cryptic pathogenic copy number variations and constitutional loss of heterozygosity using high resolution SNP microarray analysis in 117 patients referred for cytogenetic analysis and impact on clinical practice.

PubMed

Bruno, D L; Ganesamoorthy, D; Schoumans, J; Bankier, A; Coman, D; Delatycki, M; Gardner, R J M; Hunter, M; James, P A; Kannu, P; McGillivray, G; Pachter, N; Peters, H; Rieubland, C; Savarirayan, R; Scheffer, I E; Sheffield, L; Tan, T; White, S M; Yeung, A; Bowman, Z; Ngo, C; Choy, K W; Cacheux, V; Wong, L; Amor, D J; Slater, H R

2009-02-01

Microarray genome analysis is realising its promise for improving detection of genetic abnormalities in individuals with mental retardation and congenital abnormality. Copy number variations (CNVs) are now readily detectable using a variety of platforms and a major challenge is the distinction of pathogenic from ubiquitous, benign polymorphic CNVs. The aim of this study was to investigate replacement of time consuming, locus specific testing for specific microdeletion and microduplication syndromes with microarray analysis, which theoretically should detect all known syndromes with CNV aetiologies as well as new ones. Genome wide copy number analysis was performed on 117 patients using Affymetrix 250K microarrays. 434 CNVs (195 losses and 239 gains) were found, including 18 pathogenic CNVs and 9 identified as "potentially pathogenic". Almost all pathogenic CNVs were larger than 500 kb, significantly larger than the median size of all CNVs detected. Segmental regions of loss of heterozygosity larger than 5 Mb were found in 5 patients. Genome microarray analysis has improved diagnostic success in this group of patients. Several examples of recently discovered "new syndromes" were found suggesting they are more common than previously suspected and collectively are likely to be a major cause of mental retardation. The findings have several implications for clinical practice. The study revealed the potential to make genetic diagnoses that were not evident in the clinical presentation, with implications for pretest counselling and the consent process. The importance of contributing novel CNVs to high quality databases for genotype-phenotype analysis and review of guidelines for selection of individuals for microarray analysis is emphasised.

Genome-wide analysis of the WRKY gene family in cotton.

PubMed

Dou, Lingling; Zhang, Xiaohong; Pang, Chaoyou; Song, Meizhen; Wei, Hengling; Fan, Shuli; Yu, Shuxun

2014-12-01

WRKY proteins are major transcription factors involved in regulating plant growth and development. Although many studies have focused on the functional identification of WRKY genes, our knowledge concerning many areas of WRKY gene biology is limited. For example, in cotton, the phylogenetic characteristics, global expression patterns, molecular mechanisms regulating expression, and target genes/pathways of WRKY genes are poorly characterized. Therefore, in this study, we present a genome-wide analysis of the WRKY gene family in cotton (Gossypium raimondii and Gossypium hirsutum). We identified 116 WRKY genes in G. raimondii from the completed genome sequence, and we cloned 102 WRKY genes in G. hirsutum. Chromosomal location analysis indicated that WRKY genes in G. raimondii evolved mainly from segmental duplication followed by tandem amplifications. Phylogenetic analysis of alga, bryophyte, lycophyta, monocot and eudicot WRKY domains revealed family member expansion with increasing complexity of the plant body. Microarray, expression profiling and qRT-PCR data revealed that WRKY genes in G. hirsutum may regulate the development of fibers, anthers, tissues (roots, stems, leaves and embryos), and are involved in the response to stresses. Expression analysis showed that most group II and III GhWRKY genes are highly expressed under diverse stresses. Group I members, representing the ancestral form, seem to be insensitive to abiotic stress, with low expression divergence. Our results indicate that cotton WRKY genes might have evolved by adaptive duplication, leading to sensitivity to diverse stresses. This study provides fundamental information to inform further analysis and understanding of WRKY gene functions in cotton species.

Microarray and Pathway Analysis Reveal Distinct Mechanisms Underlying Cannabinoid-Mediated Modulation of LPS-Induced Activation of BV-2 Microglial Cells

PubMed Central

Juknat, Ana; Kozela, Ewa; Rimmerman, Neta; Levy, Rivka; Gao, Fuying; Coppola, Giovanni; Geschwind, Daniel; Vogel, Zvi

2013-01-01

Cannabinoids are known to exert immunosuppressive activities. However, the mechanisms which contribute to these effects are unknown. Using lipopolysaccharide (LPS) to activate BV-2 microglial cells, we examined how Δ9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and cannabidiol (CBD) the non-psychoactive component, modulate the inflammatory response. Microarray analysis of genome-wide mRNA levels was performed using Illumina platform and the resulting expression patterns analyzed using the Ingenuity Pathway Analysis to identify functional subsets of genes, and the Ingenuity System Database to denote the gene networks regulated by CBD and THC. From the 5338 transcripts that were differentially expressed across treatments, 400 transcripts were found to be upregulated by LPS, 502 by CBD+LPS and 424 by THC+LPS, while 145 were downregulated by LPS, 297 by CBD+LPS and 149 by THC+LPS, by 2-fold or more (p≤0.005). Results clearly link the effects of CBD and THC to inflammatory signaling pathways and identify new cannabinoid targets in the MAPK pathway (Dusp1, Dusp8, Dusp2), cell cycle related (Cdkn2b, Gadd45a) as well as JAK/STAT regulatory molecules (Socs3, Cish, Stat1). The impact of CBD on LPS-stimulated gene expression was greater than that of THC. We attribute this difference to the fact that CBD highly upregulated several genes encoding negative regulators of both NFκB and AP-1 transcriptional activities, such as Trib3 and Dusp1 known to be modulated through Nrf2 activation. The CBD-specific expression profile reflected changes associated with oxidative stress and glutathione depletion via Trib3 and expression of ATF4 target genes. Furthermore, the CBD affected genes were shown to be controlled by nuclear factors usually involved in regulation of stress response and inflammation, mainly via Nrf2/Hmox1 axis and the Nrf2/ATF4-Trib3 pathway. These observations indicate that CBD, and less so THC, induce a cellular stress response and that this response underlies their high immunosuppressant activities. PMID:23637839

The F-box family genes as key elements in response to salt, heavy mental, and drought stresses in Medicago truncatula.

PubMed

Song, Jian Bo; Wang, Yan Xiang; Li, Hai Bo; Li, Bo Wen; Zhou, Zhao Sheng; Gao, Shuai; Yang, Zhi Min

2015-07-01

F-box protein is a subunit of Skp1-Rbx1-Cul1-F-box protein (SCF) complex with typically conserved F-box motifs of approximately 40 amino acids and is one of the largest protein families in eukaryotes. F-box proteins play critical roles in selective and specific protein degradation through the 26S proteasome. In this study, we bioinformatically identified 972 putative F-box proteins from Medicago truncatula genome. Our analysis showed that in addition to the conserved motif, the F-box proteins have several other functional domains in their C-terminal regions (e.g., LRRs, Kelch, FBA, and PP2), some of which were found to be M. truncatula species-specific. By phylogenetic analysis of the F-box motifs, these proteins can be classified into three major families, and each family can be further grouped into more subgroups. Analysis of the genomic distribution of F-box genes on M. truncatula chromosomes revealed that the evolutional expansion of F-box genes in M. truncatula was probably due to localized gene duplications. To investigate the possible response of the F-box genes to abiotic stresses, both publicly available and customer-prepared microarrays were analyzed. Most of the F-box protein genes can be responding to salt and heavy metal stresses. Real-time PCR analysis confirmed that some of the F-box protein genes containing heat, drought, salicylic acid, and abscisic acid responsive cis-elements were able to respond to the abiotic stresses.

A genome-wide expression profile of salt-responsive genes in the apple rootstock Malus zumi.

PubMed

Li, Qingtian; Liu, Jia; Tan, Dunxian; Allan, Andrew C; Jiang, Yuzhuang; Xu, Xuefeng; Han, Zhenhai; Kong, Jin

2013-10-18

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees.

A Genome-Wide Expression Profile of Salt-Responsive Genes in the Apple Rootstock Malus zumi

PubMed Central

Li, Qingtian; Liu, Jia; Tan, Dunxian; Allan, Andrew C.; Jiang, Yuzhuang; Xu, Xuefeng; Han, Zhenhai; Kong, Jin

2013-01-01

In some areas of cultivation, a lack of salt tolerance severely affects plant productivity. Apple, Malus x domestica Borkh., is sensitive to salt, and, as a perennial woody plant the mechanism of salt stress adaption will be different from that of annual herbal model plants, such as Arabidopsis. Malus zumi is a salt tolerant apple rootstock, which survives high salinity (up to 0.6% NaCl). To examine the mechanism underlying this tolerance, a genome-wide expression analysis was performed, using a cDNA library constructed from salt-treated seedlings of Malus zumi. A total of 15,000 cDNA clones were selected for microarray analysis. In total a group of 576 cDNAs, of which expression changed more than four-fold, were sequenced and 18 genes were selected to verify their expression pattern under salt stress by semi-quantitative RT-PCR. Our genome-wide expression analysis resulted in the isolation of 50 novel Malus genes and the elucidation of a new apple-specific mechanism of salt tolerance, including the stabilization of photosynthesis under stress, involvement of phenolic compounds, and sorbitol in ROS scavenging and osmoprotection. The promoter regions of 111 genes were analyzed by PlantCARE, suggesting an intensive cross-talking of abiotic stress in Malus zumi. An interaction network of salt responsive genes was constructed and molecular regulatory pathways of apple were deduced. Our research will contribute to gene function analysis and further the understanding of salt-tolerance mechanisms in fruit trees. PMID:24145753

Microarray analysis of potential genes in the pathogenesis of recurrent oral ulcer.

PubMed

Han, Jingying; He, Zhiwei; Li, Kun; Hou, Lu

2015-01-01

Recurrent oral ulcer seriously threatens patients' daily life and health. This study investigated potential genes and pathways that participate in the pathogenesis of recurrent oral ulcer by high throughput bioinformatic analysis. RT-PCR and Western blot were applied to further verify screened interleukins effect. Recurrent oral ulcer related genes were collected from websites and papers, and further found out from Human Genome 280 6.0 microarray data. Each pathway of recurrent oral ulcer related genes were got through chip hybridization. RT-PCR was applied to test four recurrent oral ulcer related genes to verify the microarray data. Data transformation, scatter plot, clustering analysis, and expression pattern analysis were used to analyze recurrent oral ulcer related gene expression changes. Recurrent oral ulcer gene microarray was successfully established. Microarray showed that 551 genes involved in recurrent oral ulcer activity and 196 genes were recurrent oral ulcer related genes. Of them, 76 genes up-regulated, 62 genes down-regulated, and 58 genes up-/down-regulated. Total expression level up-regulated 752 times (60%) and down-regulated 485 times (40%). IL-2 plays an important role in the occurrence, development and recurrence of recurrent oral ulcer on the mRNA and protein levels. Gene microarray can be used to analyze potential genes and pathways in recurrent oral ulcer. IL-2 may be involved in the pathogenesis of recurrent oral ulcer.

A preliminary result of three-dimensional microarray technology to gene analysis with endoscopic ultrasound-guided fine-needle aspiration specimens and pancreatic juices

PubMed Central

2010-01-01

Background Analysis of gene expression and gene mutation may add information to be different from ordinary pathological tissue diagnosis. Since samples obtained endoscopically are very small, it is desired that more sensitive technology is developed for gene analysis. We investigated whether gene expression and gene mutation analysis by newly developed ultra-sensitive three-dimensional (3D) microarray is possible using small amount samples from endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) specimens and pancreatic juices. Methods Small amount samples from 17 EUS-FNA specimens and 16 pancreatic juices were obtained. After nucleic acid extraction, the samples were amplified with labeling and analyzed by the 3D microarray. Results The analyzable rate with the microarray was 46% (6/13) in EUS-FNA specimens of RNAlater® storage, and RNA degradations were observed in all the samples of frozen storage. In pancreatic juices, the analyzable rate was 67% (4/6) in frozen storage samples and 20% (2/10) in RNAlater® storage. EUS-FNA specimens were classified into cancer and non-cancer by gene expression analysis and K-ras codon 12 mutations were also detected using the 3D microarray. Conclusions Gene analysis from small amount samples obtained endoscopically was possible by newly developed 3D microarray technology. High quality RNA from EUS-FNA samples were obtained and remained in good condition only using RNA stabilizer. In contrast, high quality RNA from pancreatic juice samples were obtained only in frozen storage without RNA stabilizer. PMID:20416107

MAAMD: a workflow to standardize meta-analyses and comparison of affymetrix microarray data

PubMed Central

2014-01-01

Background Mandatory deposit of raw microarray data files for public access, prior to study publication, provides significant opportunities to conduct new bioinformatics analyses within and across multiple datasets. Analysis of raw microarray data files (e.g. Affymetrix CEL files) can be time consuming, complex, and requires fundamental computational and bioinformatics skills. The development of analytical workflows to automate these tasks simplifies the processing of, improves the efficiency of, and serves to standardize multiple and sequential analyses. Once installed, workflows facilitate the tedious steps required to run rapid intra- and inter-dataset comparisons. Results We developed a workflow to facilitate and standardize Meta-Analysis of Affymetrix Microarray Data analysis (MAAMD) in Kepler. Two freely available stand-alone software tools, R and AltAnalyze were embedded in MAAMD. The inputs of MAAMD are user-editable csv files, which contain sample information and parameters describing the locations of input files and required tools. MAAMD was tested by analyzing 4 different GEO datasets from mice and drosophila. MAAMD automates data downloading, data organization, data quality control assesment, differential gene expression analysis, clustering analysis, pathway visualization, gene-set enrichment analysis, and cross-species orthologous-gene comparisons. MAAMD was utilized to identify gene orthologues responding to hypoxia or hyperoxia in both mice and drosophila. The entire set of analyses for 4 datasets (34 total microarrays) finished in ~ one hour. Conclusions MAAMD saves time, minimizes the required computer skills, and offers a standardized procedure for users to analyze microarray datasets and make new intra- and inter-dataset comparisons. PMID:24621103

Differential expression profiles and pathways of genes in sugarcane leaf at elongation stage in response to drought stress

PubMed Central

Li, Changning; Nong, Qian; Solanki, Manoj Kumar; Liang, Qiang; Xie, Jinlan; Liu, Xiaoyan; Li, Yijie; Wang, Weizan; Yang, Litao; Li, Yangrui

2016-01-01

Water stress causes considerable yield losses in sugarcane. To investigate differentially expressed genes under water stress, a pot experiment was performed with the sugarcane variety GT21 at three water-deficit levels (mild, moderate, and severe) during the elongation stage and gene expression was analyzed using microarray technology. Physiological parameters of sugarcane showed significant alterations in response to drought stress. Based on the expression profile of 15,593 sugarcane genes, 1,501 (9.6%) genes were differentially expressed under different water-level treatments; 821 genes were upregulated and 680 genes were downregulated. A gene similarity analysis showed that approximately 62.6% of the differentially expressed genes shared homology with functional proteins. In a Gene Ontology (GO) analysis, 901 differentially expressed genes were assigned to 36 GO categories. Moreover, 325 differentially expressed genes were classified into 101 pathway categories involved in various processes, such as the biosynthesis of secondary metabolites, ribosomes, carbon metabolism, etc. In addition, some unannotated genes were detected; these may provide a basis for studies of water-deficit tolerance. The reliability of the observed expression patterns was confirmed by RT-PCR. The results of this study may help identify useful genes for improving drought tolerance in sugarcane. PMID:27170459

Gene Expression Browser: Large-Scale and Cross-Experiment Microarray Data Management, Search & Visualization

USDA-ARS?s Scientific Manuscript database

The amount of microarray gene expression data in public repositories has been increasing exponentially for the last couple of decades. High-throughput microarray data integration and analysis has become a critical step in exploring the large amount of expression data for biological discovery. Howeve...

Exogenous isoprene modulates gene expression in unstressed Arabidopsis thaliana plants.

PubMed

Harvey, Christopher M; Sharkey, Thomas D

2016-06-01

Isoprene is a well-studied volatile hemiterpene that protects plants from abiotic stress through mechanisms that are not fully understood. The antioxidant and membrane stabilizing potential of isoprene are the two most commonly invoked mechanisms. However, isoprene also affects phenylpropanoid metabolism, suggesting an additional role as a signalling molecule. In this study, microarray-based gene expression profiling reveals transcriptional reprogramming of Arabidopsis thaliana plants fumigated for 24 h with a physiologically relevant concentration of isoprene. Functional enrichment analysis of fumigated plants revealed enhanced heat- and light-stress-responsive processes in response to isoprene. Isoprene induced a network enriched in ERF and WRKY transcription factors, which may play a role in stress tolerance. The isoprene-induced up-regulation of phenylpropanoid biosynthetic genes was specifically confirmed using quantitative reverse transcription polymerase chain reaction. These results support a role for isoprene as a signalling molecule, in addition to its possible roles as an antioxidant and membrane thermoprotectant. © 2015 John Wiley & Sons Ltd.

Transcriptome analysis of intraspecific competition in Arabidopsis thaliana reveals organ-specific signatures related to nutrient acquisition and general stress response pathways

PubMed Central

2012-01-01

Background Plants are sessile and therefore have to perceive and adjust to changes in their environment. The presence of neighbours leads to a competitive situation where resources and space will be limited. Complex adaptive responses to such situation are poorly understood at the molecular level. Results Using microarrays, we analysed whole-genome expression changes in Arabidopsis thaliana plants subjected to intraspecific competition. The leaf and root transcriptome was strongly altered by competition. Differentially expressed genes were enriched in genes involved in nutrient deficiency (mainly N, P, K), perception of light quality, and responses to abiotic and biotic stresses. Interestingly, performance of the generalist insect Spodoptera littoralis on densely grown plants was significantly reduced, suggesting that plants under competition display enhanced resistance to herbivory. Conclusions This study provides a comprehensive list of genes whose expression is affected by intraspecific competition in Arabidopsis. The outcome is a unique response that involves genes related to light, nutrient deficiency, abiotic stress, and defence responses. PMID:23194435

Parallel, confocal, and complete spectrum imager for fluorescent detection of high-density microarray

NASA Astrophysics Data System (ADS)

Bogdanov, Valery L.; Boyce-Jacino, Michael

1999-05-01

Confined arrays of biochemical probes deposited on a solid support surface (analytical microarray or 'chip') provide an opportunity to analysis multiple reactions simultaneously. Microarrays are increasingly used in genetics, medicine and environment scanning as research and analytical instruments. A power of microarray technology comes from its parallelism which grows with array miniaturization, minimization of reagent volume per reaction site and reaction multiplexing. An optical detector of microarray signals should combine high sensitivity, spatial and spectral resolution. Additionally, low-cost and a high processing rate are needed to transfer microarray technology into biomedical practice. We designed an imager that provides confocal and complete spectrum detection of entire fluorescently-labeled microarray in parallel. Imager uses microlens array, non-slit spectral decomposer, and high- sensitive detector (cooled CCD). Two imaging channels provide a simultaneous detection of localization, integrated and spectral intensities for each reaction site in microarray. A dimensional matching between microarray and imager's optics eliminates all in moving parts in instrumentation, enabling highly informative, fast and low-cost microarray detection. We report theory of confocal hyperspectral imaging with microlenses array and experimental data for implementation of developed imager to detect fluorescently labeled microarray with a density approximately 103 sites per cm2.

Multiplexed Analysis of Serum Breast and Ovarian Cancer Markers by Means of Suspension Bead-quantum Dot Microarrays

NASA Astrophysics Data System (ADS)

Brazhnik, Kristina; Sokolova, Zinaida; Baryshnikova, Maria; Bilan, Regina; Nabiev, Igor; Sukhanova, Alyona

Multiplexed analysis of cancer markers is crucial for early tumor diagnosis and screening. We have designed lab-on-a-bead microarray for quantitative detection of three breast cancer markers in human serum. Quantum dots were used as bead-bound fluorescent tags for identifying each marker by means of flow cytometry. Antigen-specific beads reliably detected CA 15-3, CEA, and CA 125 in serum samples, providing clear discrimination between the samples with respect to the antigen levels. The novel microarray is advantageous over the routine single-analyte ones due to the simultaneous detection of various markers. Therefore the developed microarray is a promising tool for serum tumor marker profiling.

Genome-Wide Identification, Evolution and Expression Analysis of mTERF Gene Family in Maize

PubMed Central

Zhao, Yanxin; Cai, Manjun; Zhang, Xiaobo; Li, Yurong; Zhang, Jianhua; Zhao, Hailiang; Kong, Fei; Zheng, Yonglian; Qiu, Fazhan

2014-01-01

Plant mitochondrial transcription termination factor (mTERF) genes comprise a large family with important roles in regulating organelle gene expression. In this study, a comprehensive database search yielded 31 potential mTERF genes in maize (Zea mays L.) and most of them were targeted to mitochondria or chloroplasts. Maize mTERF were divided into nine main groups based on phylogenetic analysis, and group IX represented the mitochondria and species-specific clade that diverged from other groups. Tandem and segmental duplication both contributed to the expansion of the mTERF gene family in the maize genome. Comprehensive expression analysis of these genes, using microarray data and RNA-seq data, revealed that these genes exhibit a variety of expression patterns. Environmental stimulus experiments revealed differential up or down-regulation expression of maize mTERF genes in seedlings exposed to light/dark, salts and plant hormones, respectively, suggesting various important roles of maize mTERF genes in light acclimation and stress-related responses. These results will be useful for elucidating the roles of mTERF genes in the growth, development and stress response of maize. PMID:24718683

Transcriptome Analysis of Arabidopsis GCR1 Mutant Reveals Its Roles in Stress, Hormones, Secondary Metabolism and Phosphate Starvation

PubMed Central

Chakraborty, Navjyoti; Sharma, Priyanka; Kanyuka, Kostya; Pathak, Ravi R.; Choudhury, Devapriya; Hooley, Richard A.; Raghuram, Nandula

2015-01-01

The controversy over the existence or the need for G-protein coupled receptors (GPCRs) in plant G-protein signalling has overshadowed a more fundamental quest for the role of AtGCR1, the most studied and often considered the best candidate for GPCR in plants. Our whole transcriptome microarray analysis of the GCR1-knock-out mutant (gcr1-5) in Arabidopsis thaliana revealed 350 differentially expressed genes spanning all chromosomes. Many of them were hitherto unknown in the context of GCR1 or G-protein signalling, such as in phosphate starvation, storage compound and fatty acid biosynthesis, cell fate, etc. We also found some GCR1-responsive genes/processes that are reported to be regulated by heterotrimeric G-proteins, such as biotic and abiotic stress, hormone response and secondary metabolism. Thus, GCR1 could have G-protein-mediated as well as independent roles and regardless of whether it works as a GPCR, further analysis of the organism-wide role of GCR1 has a significance of its own. PMID:25668726

Altered gene expression of the innate immune, neuroendocrine, and nuclear factor-kappa B (NF-κB) systems is associated with posttraumatic stress disorder in military personnel.

PubMed

Guardado, Pedro; Olivera, Anlys; Rusch, Heather L; Roy, Michael; Martin, Christiana; Lejbman, Natasha; Lee, Hwyunhwa; Gill, Jessica M

2016-03-01

Whole transcriptome analysis provides an unbiased examination of biological activity, and likely, unique insight into the mechanisms underlying posttraumatic stress disorder (PTSD) and comorbid depression and traumatic brain injury. This study compared gene-expression profiles in military personnel with PTSD (n=28) and matched controls without PTSD (n=27) using HG-U133 Plus 2.0 microarrays (Affymetrix), which contain 54,675 probe sets representing more than 38,500 genes. Analysis of expression profiles revealed 203 differentially expressed genes in PTSD, of which 72% were upregulated. Using Partek Genomics Suite 6.6, differentially expressed transcription clusters were filtered based on a selection criterion of ≥1.5 relative fold change at a false discovery rate of ≤5%. Ingenuity Pathway Analysis (Qiagen) of the differentially expressed genes indicated a dysregulation of genes associated with the innate immune, neuroendocrine, and NF-κB systems. These findings provide novel insights that may lead to new pharmaceutical agents for PTSD treatments and help mitigate mental and physical comorbidity risk. Copyright © 2016. Published by Elsevier Ltd.

Transcriptional homeostasis of a mangrove species, Ceriops tagal, in saline environments, as revealed by microarray analysis.

PubMed

Liang, Shan; Fang, Lu; Zhou, Renchao; Tang, Tian; Deng, Shulin; Dong, Suisui; Huang, Yelin; Zhong, Cairong; Shi, Suhua

2012-01-01

Differential responses to the environmental stresses at the level of transcription play a critical role in adaptation. Mangrove species compose a dominant community in intertidal zones and form dense forests at the sea-land interface, and although the anatomical and physiological features associated with their salt-tolerant lifestyles have been well characterized, little is known about the impact of transcriptional phenotypes on their adaptation to these saline environments. We report the time-course transcript profiles in the roots of a true mangrove species, Ceriops tagal, as revealed by a series of microarray experiments. The expression of a total of 432 transcripts changed significantly in the roots of C. tagal under salt shock, of which 83 had a more than 2-fold change and were further assembled into 59 unigenes. Global transcription was stable at the early stage of salt stress and then was gradually dysregulated with the increased duration of the stress. Importantly, a pair-wise comparison of predicted homologous gene pairs revealed that the transcriptional regulations of most of the differentially expressed genes were highly divergent in C. tagal from that in salt-sensitive species, Arabidopsis thaliana. This work suggests that transcriptional homeostasis and specific transcriptional regulation are major events in the roots of C. tagal when subjected to salt shock, which could contribute to the establishment of adaptation to saline environments and, thus, facilitate the salt-tolerant lifestyle of this mangrove species. Furthermore, the candidate genes underlying the adaptation were identified through comparative analyses. This study provides a foundation for dissecting the genetic basis of the adaptation of mangroves to intertidal environments.

Construction of a cDNA microarray derived from the ascidian Ciona intestinalis.

PubMed

Azumi, Kaoru; Takahashi, Hiroki; Miki, Yasufumi; Fujie, Manabu; Usami, Takeshi; Ishikawa, Hisayoshi; Kitayama, Atsusi; Satou, Yutaka; Ueno, Naoto; Satoh, Nori

2003-10-01

A cDNA microarray was constructed from a basal chordate, the ascidian Ciona intestinalis. The draft genome of Ciona has been read and inferred to contain approximately 16,000 protein-coding genes, and cDNAs for transcripts of 13,464 genes have been characterized and compiled as the "Ciona intestinalis Gene Collection Release I". In the present study, we constructed a cDNA microarray of these 13,464 Ciona genes. A preliminary experiment with Cy3- and Cy5-labeled probes showed extensive differential gene expression between fertilized eggs and larvae. In addition, there was a good correlation between results obtained by the present microarray analysis and those from previous EST analyses. This first microarray of a large collection of Ciona intestinalis cDNA clones should facilitate the analysis of global gene expression and gene networks during the embryogenesis of basal chordates.

Network analysis of oyster transcriptome revealed a cascade of cellular responses during recovery after heat shock.

PubMed

Zhang, Lingling; Hou, Rui; Su, Hailin; Hu, Xiaoli; Wang, Shi; Bao, Zhenmin

2012-01-01

Oysters, as a major group of marine bivalves, can tolerate a wide range of natural and anthropogenic stressors including heat stress. Recent studies have shown that oysters pretreated with heat shock can result in induced heat tolerance. A systematic study of cellular recovery from heat shock may provide insights into the mechanism of acquired thermal tolerance. In this study, we performed the first network analysis of oyster transcriptome by reanalyzing microarray data from a previous study. Network analysis revealed a cascade of cellular responses during oyster recovery after heat shock and identified responsive gene modules and key genes. Our study demonstrates the power of network analysis in a non-model organism with poor gene annotations, which can lead to new discoveries that go beyond the focus on individual genes.

Implementation of GenePattern within the Stanford Microarray Database.

PubMed

Hubble, Jeremy; Demeter, Janos; Jin, Heng; Mao, Maria; Nitzberg, Michael; Reddy, T B K; Wymore, Farrell; Zachariah, Zachariah K; Sherlock, Gavin; Ball, Catherine A

2009-01-01

Hundreds of researchers across the world use the Stanford Microarray Database (SMD; http://smd.stanford.edu/) to store, annotate, view, analyze and share microarray data. In addition to providing registered users at Stanford access to their own data, SMD also provides access to public data, and tools with which to analyze those data, to any public user anywhere in the world. Previously, the addition of new microarray data analysis tools to SMD has been limited by available engineering resources, and in addition, the existing suite of tools did not provide a simple way to design, execute and share analysis pipelines, or to document such pipelines for the purposes of publication. To address this, we have incorporated the GenePattern software package directly into SMD, providing access to many new analysis tools, as well as a plug-in architecture that allows users to directly integrate and share additional tools through SMD. In this article, we describe our implementation of the GenePattern microarray analysis software package into the SMD code base. This extension is available with the SMD source code that is fully and freely available to others under an Open Source license, enabling other groups to create a local installation of SMD with an enriched data analysis capability.

Analysis and Characterization of Proteins Associated with Outer Membrane Vesicles Secreted by Cronobacter spp.

PubMed Central

Kothary, Mahendra H.; Gopinath, Gopal R.; Gangiredla, Jayanthi; Rallabhandi, Prasad V.; Harrison, Lisa M.; Yan, Qiong Q.; Chase, Hannah R.; Lee, Boram; Park, Eunbi; Yoo, YeonJoo; Chung, Taejung; Finkelstein, Samantha B.; Negrete, Flavia J.; Patel, Isha R.; Carter, Laurenda; Sathyamoorthy, Venugopal; Fanning, Séamus; Tall, Ben D.

2017-01-01

Little is known about secretion of outer membrane vesicles (OMVs) by Cronobacter. In this study, OMVs isolated from Cronobacter sakazakii, Cronobacter turicensis, and Cronobacter malonaticus were examined by electron microscopy (EM) and their associated outer membrane proteins (OMP) and genes were analyzed by SDS-PAGE, protein sequencing, BLAST, PCR, and DNA microarray. EM of stained cells revealed that the OMVs are secreted as pleomorphic micro-vesicles which cascade from the cell's surface. SDS-PAGE analysis identified protein bands with molecular weights of 18 kDa to >100 kDa which had homologies to OMPs such as GroEL; OmpA, C, E, F, and X; MipA proteins; conjugative plasmid transfer protein; and an outer membrane auto-transporter protein (OMATP). PCR analyses showed that most of the OMP genes were present in all seven Cronobacter species while a few genes (OMATP gene, groEL, ompC, mipA, ctp, and ompX) were absent in some phylogenetically-related species. Microarray analysis demonstrated sequence divergence among the OMP genes that was not captured by PCR. These results support previous findings that OmpA and OmpX may be involved in virulence of Cronobacter, and are packaged within secreted OMVs. These results also suggest that other OMV-packaged OMPs may be involved in roles such as stress response, cell wall and plasmid maintenance, and extracellular transport. PMID:28232819

Gene expression microarray analysis encompassing metamorphosis and the onset of calcification in the scleractinian coral Montastraea faveolata.

PubMed

Reyes-Bermudez, Alejandro; Desalvo, Michael K; Voolstra, Christian R; Sunagawa, Shinichi; Szmant, Alina M; Iglesias-Prieto, Roberto; Medina, Mónica

2009-01-01

Similar to many marine invertebrates, scleractinian corals experience a dramatic morphological transformation, as well as a habitat switch, upon settlement and metamorphosis. At this time, planula larvae transform from non-calcifying, demersal, motile organisms into sessile, calcifying, benthic juvenile polyps. We performed gene expression microarray analyses between planulae, aposymbiotic primary polyps, and symbiotic adult tissue to elucidate the molecular mechanisms underlying coral metamorphosis and early stages of calcification in the Robust/Short clade scleractinian coral Montastraea faveolata. Among the annotated genes, the most abundant upregulated transcripts in the planula stage are involved in protein synthesis, chromatin assembly and mitochondrial metabolism; the polyp stage, morphogenesis, protein catabolism and organic matrix synthesis; and the adult stage, sexual reproduction, stress response and symbiosis. We also present evidence showing that the planula and adult transcriptomes are more similar to each other than to the polyp transcriptome. Our results also point to a large number of uncharacterized adult coral-specific genes likely involved in coral-specific functions such as symbiosis and calcification.

Molecular response to water stress in two contrasting Mediterranean pines (Pinus pinaster and Pinus pinea).

PubMed

Perdiguero, Pedro; Barbero, María Del Carmen; Cervera, María Teresa; Collada, Carmen; Soto, Alvaro

2013-06-01

Adaptation to water stress has determined the evolution and diversification of vascular plants. Water stress is forecasted to increase drastically in the next decades in certain regions, such as in the Mediterranean basin. Consequently, a proper knowledge of the response and adaptations to drought stress is essential for the correct management of plant genetic resources. However, most of the advances in the understanding of the molecular response to water stress have been attained in angiosperms, and are not always applicable to gymnosperms. In this work we analyse the transcriptional response of two emblematic Mediterranean pines, Pinus pinaster and Pinus pinea, which show noticeable differences in their performance under water stress. Using microarray analysis, up to 113 genes have been detected as significantly induced by drought in both species. Reliability of expression patterns has been confirmed by RT-PCR. While induced genes with similar profiles in both species can be considered as general candidate genes for the study of drought response in conifers, genes with diverging expression patterns can underpin the differences displayed by these species under water stress. Most promising candidate genes for drought stress response include genes related to carbohydrate metabolism, such as glycosyltransferases or galactosidases, sugar transporters, dehydrins and transcription factors. Additionally, differences in the molecular response to drought and polyethylene-glycol-induced water stress are also discussed. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Role of Arabidopsis ABF1/3/4 during det1 germination in salt and osmotic stress conditions.

PubMed

Fernando, V C Dilukshi; Al Khateeb, Wesam; Belmonte, Mark F; Schroeder, Dana F

2018-05-01

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4. While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

Response of Desulfovibrio vulgaris to Alkaline Stress▿ †

PubMed Central

Stolyar, Sergey; He, Qiang; Joachimiak, Marcin P.; He, Zhili; Yang, Zamin Koo; Borglin, Sharon E.; Joyner, Dominique C.; Huang, Katherine; Alm, Eric; Hazen, Terry C.; Zhou, Jizhong; Wall, Judy D.; Arkin, Adam P.; Stahl, David A.

2007-01-01

The response of exponentially growing Desulfovibrio vulgaris Hildenborough to pH 10 stress was studied using oligonucleotide microarrays and a study set of mutants with genes suggested by microarray data to be involved in the alkaline stress response deleted. The data showed that the response of D. vulgaris to increased pH is generally similar to that of Escherichia coli but is apparently controlled by unique regulatory circuits since the alternative sigma factors (sigma S and sigma E) contributing to this stress response in E. coli appear to be absent in D. vulgaris. Genes previously reported to be up-regulated in E. coli were up-regulated in D. vulgaris; these genes included three ATPase genes and a tryptophan synthase gene. Transcription of chaperone and protease genes (encoding ATP-dependent Clp and La proteases and DnaK) was also elevated in D. vulgaris. As in E. coli, genes involved in flagellum synthesis were down-regulated. The transcriptional data also identified regulators, distinct from sigma S and sigma E, that are likely part of a D. vulgaris Hildenborough-specific stress response system. Characterization of a study set of mutants with genes implicated in alkaline stress response deleted confirmed that there was protective involvement of the sodium/proton antiporter NhaC-2, tryptophanase A, and two putative regulators/histidine kinases (DVU0331 and DVU2580). PMID:17921288

Genome-scale cluster analysis of replicated microarrays using shrinkage correlation coefficient.

PubMed

Yao, Jianchao; Chang, Chunqi; Salmi, Mari L; Hung, Yeung Sam; Loraine, Ann; Roux, Stanley J

2008-06-18

Currently, clustering with some form of correlation coefficient as the gene similarity metric has become a popular method for profiling genomic data. The Pearson correlation coefficient and the standard deviation (SD)-weighted correlation coefficient are the two most widely-used correlations as the similarity metrics in clustering microarray data. However, these two correlations are not optimal for analyzing replicated microarray data generated by most laboratories. An effective correlation coefficient is needed to provide statistically sufficient analysis of replicated microarray data. In this study, we describe a novel correlation coefficient, shrinkage correlation coefficient (SCC), that fully exploits the similarity between the replicated microarray experimental samples. The methodology considers both the number of replicates and the variance within each experimental group in clustering expression data, and provides a robust statistical estimation of the error of replicated microarray data. The value of SCC is revealed by its comparison with two other correlation coefficients that are currently the most widely-used (Pearson correlation coefficient and SD-weighted correlation coefficient) using statistical measures on both synthetic expression data as well as real gene expression data from Saccharomyces cerevisiae. Two leading clustering methods, hierarchical and k-means clustering were applied for the comparison. The comparison indicated that using SCC achieves better clustering performance. Applying SCC-based hierarchical clustering to the replicated microarray data obtained from germinating spores of the fern Ceratopteris richardii, we discovered two clusters of genes with shared expression patterns during spore germination. Functional analysis suggested that some of the genetic mechanisms that control germination in such diverse plant lineages as mosses and angiosperms are also conserved among ferns. This study shows that SCC is an alternative to the Pearson correlation coefficient and the SD-weighted correlation coefficient, and is particularly useful for clustering replicated microarray data. This computational approach should be generally useful for proteomic data or other high-throughput analysis methodology.

Fabrication of Carbohydrate Microarrays by Boronate Formation.

PubMed

Adak, Avijit K; Lin, Ting-Wei; Li, Ben-Yuan; Lin, Chun-Cheng

2017-01-01

The interactions between soluble carbohydrates and/or surface displayed glycans and protein receptors are essential to many biological processes and cellular recognition events. Carbohydrate microarrays provide opportunities for high-throughput quantitative analysis of carbohydrate-protein interactions. Over the past decade, various techniques have been implemented for immobilizing glycans on solid surfaces in a microarray format. Herein, we describe a detailed protocol for fabricating carbohydrate microarrays that capitalizes on the intrinsic reactivity of boronic acid toward carbohydrates to form stable boronate diesters. A large variety of unprotected carbohydrates ranging in structure from simple disaccharides and trisaccharides to considerably more complex human milk and blood group (oligo)saccharides have been covalently immobilized in a single step on glass slides, which were derivatized with high-affinity boronic acid ligands. The immobilized ligands in these microarrays maintain the receptor-binding activities including those of lectins and antibodies according to the structures of their pendant carbohydrates for rapid analysis of a number of carbohydrate-recognition events within 30 h. This method facilitates the direct construction of otherwise difficult to obtain carbohydrate microarrays from underivatized glycans.

The Glycan Microarray Story from Construction to Applications.

PubMed

Hyun, Ji Young; Pai, Jaeyoung; Shin, Injae

2017-04-18

Not only are glycan-mediated binding processes in cells and organisms essential for a wide range of physiological processes, but they are also implicated in various pathological processes. As a result, elucidation of glycan-associated biomolecular interactions and their consequences is of great importance in basic biological research and biomedical applications. In 2002, we and others were the first to utilize glycan microarrays in efforts aimed at the rapid analysis of glycan-associated recognition events. Because they contain a number of glycans immobilized in a dense and orderly manner on a solid surface, glycan microarrays enable multiple parallel analyses of glycan-protein binding events while utilizing only small amounts of glycan samples. Therefore, this microarray technology has become a leading edge tool in studies aimed at elucidating roles played by glycans and glycan binding proteins in biological systems. In this Account, we summarize our efforts on the construction of glycan microarrays and their applications in studies of glycan-associated interactions. Immobilization strategies of functionalized and unmodified glycans on derivatized glass surfaces are described. Although others have developed immobilization techniques, our efforts have focused on improving the efficiencies and operational simplicity of microarray construction. The microarray-based technology has been most extensively used for rapid analysis of the glycan binding properties of proteins. In addition, glycan microarrays have been employed to determine glycan-protein interactions quantitatively, detect pathogens, and rapidly assess substrate specificities of carbohydrate-processing enzymes. More recently, the microarrays have been employed to identify functional glycans that elicit cell surface lectin-mediated cellular responses. Owing to these efforts, it is now possible to use glycan microarrays to expand the understanding of roles played by glycans and glycan binding proteins in biological systems.

EDGE3: A web-based solution for management and analysis of Agilent two color microarray experiments

PubMed Central

Vollrath, Aaron L; Smith, Adam A; Craven, Mark; Bradfield, Christopher A

2009-01-01

Background The ability to generate transcriptional data on the scale of entire genomes has been a boon both in the improvement of biological understanding and in the amount of data generated. The latter, the amount of data generated, has implications when it comes to effective storage, analysis and sharing of these data. A number of software tools have been developed to store, analyze, and share microarray data. However, a majority of these tools do not offer all of these features nor do they specifically target the commonly used two color Agilent DNA microarray platform. Thus, the motivating factor for the development of EDGE3 was to incorporate the storage, analysis and sharing of microarray data in a manner that would provide a means for research groups to collaborate on Agilent-based microarray experiments without a large investment in software-related expenditures or extensive training of end-users. Results EDGE3 has been developed with two major functions in mind. The first function is to provide a workflow process for the generation of microarray data by a research laboratory or a microarray facility. The second is to store, analyze, and share microarray data in a manner that doesn't require complicated software. To satisfy the first function, EDGE3 has been developed as a means to establish a well defined experimental workflow and information system for microarray generation. To satisfy the second function, the software application utilized as the user interface of EDGE3 is a web browser. Within the web browser, a user is able to access the entire functionality, including, but not limited to, the ability to perform a number of bioinformatics based analyses, collaborate between research groups through a user-based security model, and access to the raw data files and quality control files generated by the software used to extract the signals from an array image. Conclusion Here, we present EDGE3, an open-source, web-based application that allows for the storage, analysis, and controlled sharing of transcription-based microarray data generated on the Agilent DNA platform. In addition, EDGE3 provides a means for managing RNA samples and arrays during the hybridization process. EDGE3 is freely available for download at . PMID:19732451

EDGE(3): a web-based solution for management and analysis of Agilent two color microarray experiments.

PubMed

Vollrath, Aaron L; Smith, Adam A; Craven, Mark; Bradfield, Christopher A

2009-09-04

The ability to generate transcriptional data on the scale of entire genomes has been a boon both in the improvement of biological understanding and in the amount of data generated. The latter, the amount of data generated, has implications when it comes to effective storage, analysis and sharing of these data. A number of software tools have been developed to store, analyze, and share microarray data. However, a majority of these tools do not offer all of these features nor do they specifically target the commonly used two color Agilent DNA microarray platform. Thus, the motivating factor for the development of EDGE(3) was to incorporate the storage, analysis and sharing of microarray data in a manner that would provide a means for research groups to collaborate on Agilent-based microarray experiments without a large investment in software-related expenditures or extensive training of end-users. EDGE(3) has been developed with two major functions in mind. The first function is to provide a workflow process for the generation of microarray data by a research laboratory or a microarray facility. The second is to store, analyze, and share microarray data in a manner that doesn't require complicated software. To satisfy the first function, EDGE3 has been developed as a means to establish a well defined experimental workflow and information system for microarray generation. To satisfy the second function, the software application utilized as the user interface of EDGE(3) is a web browser. Within the web browser, a user is able to access the entire functionality, including, but not limited to, the ability to perform a number of bioinformatics based analyses, collaborate between research groups through a user-based security model, and access to the raw data files and quality control files generated by the software used to extract the signals from an array image. Here, we present EDGE(3), an open-source, web-based application that allows for the storage, analysis, and controlled sharing of transcription-based microarray data generated on the Agilent DNA platform. In addition, EDGE(3) provides a means for managing RNA samples and arrays during the hybridization process. EDGE(3) is freely available for download at http://edge.oncology.wisc.edu/.

MAPPI-DAT: data management and analysis for protein-protein interaction data from the high-throughput MAPPIT cell microarray platform.

PubMed

Gupta, Surya; De Puysseleyr, Veronic; Van der Heyden, José; Maddelein, Davy; Lemmens, Irma; Lievens, Sam; Degroeve, Sven; Tavernier, Jan; Martens, Lennart

2017-05-01

Protein-protein interaction (PPI) studies have dramatically expanded our knowledge about cellular behaviour and development in different conditions. A multitude of high-throughput PPI techniques have been developed to achieve proteome-scale coverage for PPI studies, including the microarray based Mammalian Protein-Protein Interaction Trap (MAPPIT) system. Because such high-throughput techniques typically report thousands of interactions, managing and analysing the large amounts of acquired data is a challenge. We have therefore built the MAPPIT cell microArray Protein Protein Interaction-Data management & Analysis Tool (MAPPI-DAT) as an automated data management and analysis tool for MAPPIT cell microarray experiments. MAPPI-DAT stores the experimental data and metadata in a systematic and structured way, automates data analysis and interpretation, and enables the meta-analysis of MAPPIT cell microarray data across all stored experiments. MAPPI-DAT is developed in Python, using R for data analysis and MySQL as data management system. MAPPI-DAT is cross-platform and can be ran on Microsoft Windows, Linux and OS X/macOS. The source code and a Microsoft Windows executable are freely available under the permissive Apache2 open source license at https://github.com/compomics/MAPPI-DAT. jan.tavernier@vib-ugent.be or lennart.martens@vib-ugent.be. Supplementary data are available at Bioinformatics online. © The Author 2017. Published by Oxford University Press.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster.

PubMed

Landis, Gary; Shen, Jie; Tower, John

2012-11-01

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging.

Gene expression changes in response to aging compared to heat stress, oxidative stress and ionizing radiation in Drosophila melanogaster

PubMed Central

Landis, Gary; Shen, Jie; Tower, John

2012-01-01

Gene expression changes in response to aging, heat stress, hyperoxia, hydrogen peroxide, and ionizing radiation were compared using microarrays. A set of 18 genes were up-regulated across all conditions, indicating a general stress response shared with aging, including the heat shock protein (Hsp) genes Hsp70, Hsp83 and l(2)efl, the glutathione-S-transferase gene GstD2, and the mitochondrial unfolded protein response (mUPR) gene ref(2)P. Selected gene expression changes were confirmed using quantitative PCR, Northern analysis and GstD-GFP reporter constructs. Certain genes were altered in only a subset of the conditions, for example, up-regulation of numerous developmental pathway and signaling genes in response to hydrogen peroxide. While aging shared features with each stress, aging was more similar to the stresses most associated with oxidative stress (hyperoxia, hydrogen peroxide, ionizing radiation) than to heat stress. Aging is associated with down-regulation of numerous mitochondrial genes, including electron-transport-chain (ETC) genes and mitochondrial metabolism genes, and a sub-set of these changes was also observed upon hydrogen peroxide stress and ionizing radiation stress. Aging shared the largest number of gene expression changes with hyperoxia. The extensive down-regulation of mitochondrial and ETC genes during aging is consistent with an aging-associated failure in mitochondrial maintenance, which may underlie the oxidative stress-like and proteotoxic stress-like responses observed during aging. PMID:23211361

TOXICOGENOMIC ANALYSIS INCORPORATING OPERON-TRANSCRIPTIONAL COUPLING AND TOXICANT CONCENTRATRION-EXPRESSION RESPONSE: Analysis of MX-Treated Salmonella

EPA Science Inventory

What is the study? This study is the first to use microarray analysis in the Ames strains of Salmonella. The microarray chips were custom-designed for this study and are not commercially available, and we evaluated the well-studied drinking water mutagen, MX. Because much inform...

MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

EPA Science Inventory

MICROARRAY ANALYSIS OF DICHLOROACETIC ACID-INDUCED CHANGES IN GENE EXPRESSION

Dichloroacetic acid (DCA) is a major by-product of water disinfection by chlorination. Several studies have demonstrated the hepatocarcinogenicity of DCA in rodents when administered in dri...

Robust gene selection methods using weighting schemes for microarray data analysis.

PubMed

Kang, Suyeon; Song, Jongwoo

2017-09-02

A common task in microarray data analysis is to identify informative genes that are differentially expressed between two different states. Owing to the high-dimensional nature of microarray data, identification of significant genes has been essential in analyzing the data. However, the performances of many gene selection techniques are highly dependent on the experimental conditions, such as the presence of measurement error or a limited number of sample replicates. We have proposed new filter-based gene selection techniques, by applying a simple modification to significance analysis of microarrays (SAM). To prove the effectiveness of the proposed method, we considered a series of synthetic datasets with different noise levels and sample sizes along with two real datasets. The following findings were made. First, our proposed methods outperform conventional methods for all simulation set-ups. In particular, our methods are much better when the given data are noisy and sample size is small. They showed relatively robust performance regardless of noise level and sample size, whereas the performance of SAM became significantly worse as the noise level became high or sample size decreased. When sufficient sample replicates were available, SAM and our methods showed similar performance. Finally, our proposed methods are competitive with traditional methods in classification tasks for microarrays. The results of simulation study and real data analysis have demonstrated that our proposed methods are effective for detecting significant genes and classification tasks, especially when the given data are noisy or have few sample replicates. By employing weighting schemes, we can obtain robust and reliable results for microarray data analysis.

Transcriptome Analysis Reveals Genes Commonly Induced by Botrytis cinerea Infection, Cold, Drought and Oxidative Stresses in Arabidopsis

PubMed Central

Al-Ameri, Salma; Al-Mahmoud, Bassam; Awwad, Falah; Al-Rawashdeh, Ahmed; Iratni, Rabah; AbuQamar, Synan

2014-01-01

Signaling pathways controlling biotic and abiotic stress responses may interact synergistically or antagonistically. To identify the similarities and differences among responses to diverse stresses, we analyzed previously published microarray data on the transcriptomic responses of Arabidopsis to infection with Botrytis cinerea (a biotic stress), and to cold, drought, and oxidative stresses (abiotic stresses). Our analyses showed that at early stages after B. cinerea inoculation, 1498 genes were up-regulated (B. cinerea up-regulated genes; BUGs) and 1138 genes were down-regulated (B. cinerea down-regulated genes; BDGs). We showed a unique program of gene expression was activated in response each biotic and abiotic stress, but that some genes were similarly induced or repressed by all of the tested stresses. Of the identified BUGs, 25%, 6% and 12% were also induced by cold, drought and oxidative stress, respectively; whereas 33%, 7% and 5.5% of the BDGs were also down-regulated by the same abiotic stresses. Coexpression and protein-protein interaction network analyses revealed a dynamic range in the expression levels of genes encoding regulatory proteins. Analysis of gene expression in response to electrophilic oxylipins suggested that these compounds are involved in mediating responses to B. cinerea infection and abiotic stress through TGA transcription factors. Our results suggest an overlap among genes involved in the responses to biotic and abiotic stresses in Arabidopsis. Changes in the transcript levels of genes encoding components of the cyclopentenone signaling pathway in response to biotic and abiotic stresses suggest that the oxylipin signal transduction pathway plays a role in plant defense. Identifying genes that are commonly expressed in response to environmental stresses, and further analyzing the functions of their encoded products, will increase our understanding of the plant stress response. This information could identify targets for genetic modification to improve plant resistance to multiple stresses. PMID:25422934

The application of DNA microarrays in gene expression analysis.

PubMed

van Hal, N L; Vorst, O; van Houwelingen, A M; Kok, E J; Peijnenburg, A; Aharoni, A; van Tunen, A J; Keijer, J

2000-03-31

DNA microarray technology is a new and powerful technology that will substantially increase the speed of molecular biological research. This paper gives a survey of DNA microarray technology and its use in gene expression studies. The technical aspects and their potential improvements are discussed. These comprise array manufacturing and design, array hybridisation, scanning, and data handling. Furthermore, it is discussed how DNA microarrays can be applied in the working fields of: safety, functionality and health of food and gene discovery and pathway engineering in plants.

Implementation of mutual information and bayes theorem for classification microarray data

NASA Astrophysics Data System (ADS)

Dwifebri Purbolaksono, Mahendra; Widiastuti, Kurnia C.; Syahrul Mubarok, Mohamad; Adiwijaya; Aminy Ma’ruf, Firda

2018-03-01

Microarray Technology is one of technology which able to read the structure of gen. The analysis is important for this technology. It is for deciding which attribute is more important than the others. Microarray technology is able to get cancer information to diagnose a person’s gen. Preparation of microarray data is a huge problem and takes a long time. That is because microarray data contains high number of insignificant and irrelevant attributes. So, it needs a method to reduce the dimension of microarray data without eliminating important information in every attribute. This research uses Mutual Information to reduce dimension. System is built with Machine Learning approach specifically Bayes Theorem. This theorem uses a statistical and probability approach. By combining both methods, it will be powerful for Microarray Data Classification. The experiment results show that system is good to classify Microarray data with highest F1-score using Bayesian Network by 91.06%, and Naïve Bayes by 88.85%.

Gene ARMADA: an integrated multi-analysis platform for microarray data implemented in MATLAB.

PubMed

Chatziioannou, Aristotelis; Moulos, Panagiotis; Kolisis, Fragiskos N

2009-10-27

The microarray data analysis realm is ever growing through the development of various tools, open source and commercial. However there is absence of predefined rational algorithmic analysis workflows or batch standardized processing to incorporate all steps, from raw data import up to the derivation of significantly differentially expressed gene lists. This absence obfuscates the analytical procedure and obstructs the massive comparative processing of genomic microarray datasets. Moreover, the solutions provided, heavily depend on the programming skills of the user, whereas in the case of GUI embedded solutions, they do not provide direct support of various raw image analysis formats or a versatile and simultaneously flexible combination of signal processing methods. We describe here Gene ARMADA (Automated Robust MicroArray Data Analysis), a MATLAB implemented platform with a Graphical User Interface. This suite integrates all steps of microarray data analysis including automated data import, noise correction and filtering, normalization, statistical selection of differentially expressed genes, clustering, classification and annotation. In its current version, Gene ARMADA fully supports 2 coloured cDNA and Affymetrix oligonucleotide arrays, plus custom arrays for which experimental details are given in tabular form (Excel spreadsheet, comma separated values, tab-delimited text formats). It also supports the analysis of already processed results through its versatile import editor. Besides being fully automated, Gene ARMADA incorporates numerous functionalities of the Statistics and Bioinformatics Toolboxes of MATLAB. In addition, it provides numerous visualization and exploration tools plus customizable export data formats for seamless integration by other analysis tools or MATLAB, for further processing. Gene ARMADA requires MATLAB 7.4 (R2007a) or higher and is also distributed as a stand-alone application with MATLAB Component Runtime. Gene ARMADA provides a highly adaptable, integrative, yet flexible tool which can be used for automated quality control, analysis, annotation and visualization of microarray data, constituting a starting point for further data interpretation and integration with numerous other tools.

Microarray data mining using Bioconductor packages.

PubMed

Nie, Haisheng; Neerincx, Pieter B T; van der Poel, Jan; Ferrari, Francesco; Bicciato, Silvio; Leunissen, Jack A M; Groenen, Martien A M

2009-07-16

This paper describes the results of a Gene Ontology (GO) term enrichment analysis of chicken microarray data using the Bioconductor packages. By checking the enriched GO terms in three contrasts, MM8-PM8, MM8-MA8, and MM8-MM24, of the provided microarray data during this workshop, this analysis aimed to investigate the host reactions in chickens occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria. The results of GO enrichment analysis using GO terms annotated to chicken genes and GO terms annotated to chicken-human orthologous genes were also compared. Furthermore, a locally adaptive statistical procedure (LAP) was performed to test differentially expressed chromosomal regions, rather than individual genes, in the chicken genome after Eimeria challenge. GO enrichment analysis identified significant (raw p-value < 0.05) GO terms for all three contrasts included in the analysis. Some of the GO terms linked to, generally, primary immune responses or secondary immune responses indicating the GO enrichment analysis is a useful approach to analyze microarray data. The comparisons of GO enrichment results using chicken gene information and chicken-human orthologous gene information showed more refined GO terms related to immune responses when using chicken-human orthologous gene information, this suggests that using chicken-human orthologous gene information has higher power to detect significant GO terms with more refined functionality. Furthermore, three chromosome regions were identified to be significantly up-regulated in contrast MM8-PM8 (q-value < 0.01). Overall, this paper describes a practical approach to analyze microarray data in farm animals where the genome information is still incomplete. For farm animals, such as chicken, with currently limited gene annotation, borrowing gene annotation information from orthologous genes in well-annotated species, such as human, will help improve the pathway analysis results substantially. Furthermore, LAP analysis approach is a relatively new and very useful way to be applied in microarray analysis.

Polyadenylation state microarray (PASTA) analysis.

PubMed

Beilharz, Traude H; Preiss, Thomas

2011-01-01

Nearly all eukaryotic mRNAs terminate in a poly(A) tail that serves important roles in mRNA utilization. In the cytoplasm, the poly(A) tail promotes both mRNA stability and translation, and these functions are frequently regulated through changes in tail length. To identify the scope of poly(A) tail length control in a transcriptome, we developed the polyadenylation state microarray (PASTA) method. It involves the purification of mRNA based on poly(A) tail length using thermal elution from poly(U) sepharose, followed by microarray analysis of the resulting fractions. In this chapter we detail our PASTA approach and describe some methods for bulk and mRNA-specific poly(A) tail length measurements of use to monitor the procedure and independently verify the microarray data.

The Use of Atomic Force Microscopy for 3D Analysis of Nucleic Acid Hybridization on Microarrays.

PubMed

Dubrovin, E V; Presnova, G V; Rubtsova, M Yu; Egorov, A M; Grigorenko, V G; Yaminsky, I V

2015-01-01

Oligonucleotide microarrays are considered today to be one of the most efficient methods of gene diagnostics. The capability of atomic force microscopy (AFM) to characterize the three-dimensional morphology of single molecules on a surface allows one to use it as an effective tool for the 3D analysis of a microarray for the detection of nucleic acids. The high resolution of AFM offers ways to decrease the detection threshold of target DNA and increase the signal-to-noise ratio. In this work, we suggest an approach to the evaluation of the results of hybridization of gold nanoparticle-labeled nucleic acids on silicon microarrays based on an AFM analysis of the surface both in air and in liquid which takes into account of their three-dimensional structure. We suggest a quantitative measure of the hybridization results which is based on the fraction of the surface area occupied by the nanoparticles.

The Utility of Chromosomal Microarray Analysis in Developmental and Behavioral Pediatrics

ERIC Educational Resources Information Center

Beaudet, Arthur L.

2013-01-01

Chromosomal microarray analysis (CMA) has emerged as a powerful new tool to identify genomic abnormalities associated with a wide range of developmental disabilities including congenital malformations, cognitive impairment, and behavioral abnormalities. CMA includes array comparative genomic hybridization (CGH) and single nucleotide polymorphism…

Evaluation of chronic lymphocytic leukemia by oligonucleotide-based microarray analysis uncovers novel aberrations not detected by FISH or cytogenetic analysis

PubMed Central

2011-01-01

Background Cytogenetic evaluation is a key component of the diagnosis and prognosis of chronic lymphocytic leukemia (CLL). We performed oligonucleotide-based comparative genomic hybridization microarray analysis on 34 samples with CLL and known abnormal karyotypes previously determined by cytogenetics and/or fluorescence in situ hybridization (FISH). Results Using a custom designed microarray that targets >1800 genes involved in hematologic disease and other malignancies, we identified additional cryptic aberrations and novel findings in 59% of cases. These included gains and losses of genes associated with cell cycle regulation, apoptosis and susceptibility loci on 3p21.31, 5q35.2q35.3, 10q23.31q23.33, 11q22.3, and 22q11.23. Conclusions Our results show that microarray analysis will detect known aberrations, including microscopic and cryptic alterations. In addition, novel genomic changes will be uncovered that may become important prognostic predictors or treatment targets for CLL in the future. PMID:22087757

Microbial community analysis of an Alabama coastal salt marsh impacted by the Deepwater Horizon Oil Spill

NASA Astrophysics Data System (ADS)

Beazley, M. J.; Martinez, R.; Rajan, S.; Powell, J.; Piceno, Y.; Tom, L.; Andersen, G. L.; Hazen, T. C.; Van Nostrand, J. D.; Zhou, J.; Mortazavi, B.; Sobecky, P. A.

2011-12-01

Microbial community responses of an Alabama coastal salt marsh environment to the Deepwater Horizon oil spill were studied by 16S rRNA (PhyloChip) and functional gene (GeoChip) microarray-based analysis. Oil and tar balls associated with the oil spill arrived along the Alabama coast in June 2010. Marsh and inlet sediment samples collected in June, July, and September 2010 from a salt marsh ecosystem at Point Aux Pines Alabama were analyzed to determine if bacterial community structure changed as a result of oil perturbation. Sediment total petroleum hydrocarbon (TPH) concentrations ranged from below detection to 189 mg kg-1 and were randomly dispersed throughout the salt marsh sediments. Total DNA extracted from sediment and particulates were used for PhyloChip and GeoChip hybridization. A total of 4000 to 8000 operational taxonomic units (OTUs) were detected in marsh and inlet samples. Distinctive changes in the number of detectable OTUs were observed between June, July, and September 2010. Surficial inlet sediments demonstrated a significant increase in the total number of OTUs between June and September that correlated with TPH concentrations. The most significant increases in bacterial abundance were observed in the phyla Actinobacteria, Firmicutes, Gemmatimonadetes, Proteobacteria, and Verrucomicrobia. Bacterial richness in marsh sediments also correlated with TPH concentrations with significant changes primarily in Acidobacteria, Actinobacteria, Firmicutes, Fusobacteria, Nitrospirae, and Proteobacteria. GeoChip microarray analysis detected 5000 to 8300 functional genes in marsh and inlet samples. Surficial inlet sediments demonstrated distinctive increases in the number of detectable genes and gene signal intensities in July samples compared to June. Signal intensities increased (> 1.5-fold) in genes associated with petroleum degradation. Genes related to metal resistance, stress, and carbon cycling also demonstrated increases in oiled sediments. This study demonstrates the value of applying phylogenetic and functional gene microarray technology to characterize the extensive microbial diversity of marsh environments. Moreover, this technology provides significant insight into bacterial community responses to anthropogenic oil events.

Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans

PubMed Central

Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

2016-01-01

Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development. PMID:26791749

Homeodomain-Interacting Protein Kinase (HPK-1) regulates stress responses and ageing in C. elegans.

PubMed

Berber, Slavica; Wood, Mallory; Llamosas, Estelle; Thaivalappil, Priya; Lee, Karen; Liao, Bing Mana; Chew, Yee Lian; Rhodes, Aaron; Yucel, Duygu; Crossley, Merlin; Nicholas, Hannah R

2016-01-21

Proteins of the Homeodomain-Interacting Protein Kinase (HIPK) family regulate an array of processes in mammalian systems, such as the DNA damage response, cellular proliferation and apoptosis. The nematode Caenorhabditis elegans has a single HIPK homologue called HPK-1. Previous studies have implicated HPK-1 in longevity control and suggested that this protein may be regulated in a stress-dependent manner. Here we set out to expand these observations by investigating the role of HPK-1 in longevity and in the response to heat and oxidative stress. We find that levels of HPK-1 are regulated by heat stress, and that HPK-1 contributes to survival following heat or oxidative stress. Additionally, we show that HPK-1 is required for normal longevity, with loss of HPK-1 function leading to a faster decline of physiological processes that reflect premature ageing. Through microarray analysis, we have found that HPK-1-regulated genes include those encoding proteins that serve important functions in stress responses such as Phase I and Phase II detoxification enzymes. Consistent with a role in longevity assurance, HPK-1 also regulates the expression of age-regulated genes. Lastly, we show that HPK-1 functions in the same pathway as DAF-16 to regulate longevity and reveal a new role for HPK-1 in development.

Effects of temperature on gene expression in embryos of the coral Montastraea faveolata

PubMed Central

2009-01-01

Background Coral reefs are expected to be severely impacted by rising seawater temperatures associated with climate change. This study used cDNA microarrays to investigate transcriptional effects of thermal stress in embryos of the coral Montastraea faveolata. Embryos were exposed to 27.5°C, 29.0°C, and 31.5°C directly after fertilization. Differences in gene expression were measured after 12 and 48 hours. Results Analysis of differentially expressed genes indicated that increased temperatures may lead to oxidative stress, apoptosis, and a structural reconfiguration of the cytoskeletal network. Metabolic processes were downregulated, and the action of histones and zinc finger-containing proteins may have played a role in the long-term regulation upon heat stress. Conclusions Embryos responded differently depending on exposure time and temperature level. Embryos showed expression of stress-related genes already at a temperature of 29.0°C, but seemed to be able to counteract the initial response over time. By contrast, embryos at 31.5°C displayed continuous expression of stress genes. The genes that played a role in the response to elevated temperatures consisted of both highly conserved and coral-specific genes. These genes might serve as a basis for research into coral-specific adaptations to stress responses and global climate change. PMID:20030803

Domestication Effects on Stress Induced Steroid Secretion and Adrenal Gene Expression in Chickens.

PubMed

Fallahsharoudi, Amir; de Kock, Neil; Johnsson, Martin; Ubhayasekera, S J Kumari A; Bergquist, Jonas; Wright, Dominic; Jensen, Per

2015-10-16

Understanding the genetic basis of phenotypic diversity is a challenge in contemporary biology. Domestication provides a model for unravelling aspects of the genetic basis of stress sensitivity. The ancestral Red Junglefowl (RJF) exhibits greater fear-related behaviour and a more pronounced HPA-axis reactivity than its domesticated counterpart, the White Leghorn (WL). By comparing hormones (plasmatic) and adrenal global gene transcription profiles between WL and RJF in response to an acute stress event, we investigated the molecular basis for the altered physiological stress responsiveness in domesticated chickens. Basal levels of pregnenolone and dehydroepiandrosterone as well as corticosterone response were lower in WL. Microarray analysis of gene expression in adrenal glands showed a significant breed effect in a large number of transcripts with over-representation of genes in the channel activity pathway. The expression of the best-known steroidogenesis genes were similar across the breeds used. Transcription levels of acute stress response genes such as StAR, CH25 and POMC were upregulated in response to acute stress. Dampened HPA reactivity in domesticated chickens was associated with changes in the expression of several genes that presents potentially minor regulatory effects rather than by means of change in expression of critical steroidogenic genes in the adrenal.

Identification of water stress genes in Pinus pinaster Ait. by controlled progressive stress and suppression-subtractive hybridization.

PubMed

Perdiguero, Pedro; Collada, Carmen; Barbero, María Del Carmen; García Casado, Gloria; Cervera, María Teresa; Soto, Alvaro

2012-01-01

Climate change is a major challenge particularly for forest tree species, which will have to face the severe alterations of environmental conditions with their current genetic pool. Thus, an understanding of their adaptive responses is of the utmost interest. In this work we have selected Pinus pinaster as a model species. This pine is one of the most important conifers (for which molecular tools and knowledge are far more scarce than for angiosperms) in the Mediterranean Basin, which is characterised in all foreseen scenarios as one of the regions most drastically affected by climate change, mainly because of increasing temperature and, particularly, by increasing drought. We have induced a controlled, increasing water stress by adding PEG to a hydroponic culture. We have generated a subtractive library, with the aim of identifying the genes induced by this stress and have searched for the most reliable expressional candidate genes, based on their overexpression during water stress, as revealed by microarray analysis and confirmed by RT-PCR. We have selected a set of 67 candidate genes belonging to different functional groups that will be useful molecular tools for further studies on drought stress responses, adaptation, and population genomics in conifers, as well as in breeding programs. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

NASA Technical Reports Server (NTRS)

Wilson, James W.; Ramamurthy, Rajee; Porwollik, Steffen; McClelland, Michael; Hammond, Timothy; Allen, Pat; Ott, C. Mark; Pierson, Duane L.; Nickerson, Cheryl A.

2002-01-01

The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 x g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies.

Interim report on updated microarray probes for the LLNL Burkholderia pseudomallei SNP array

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gardner, S; Jaing, C

2012-03-27

The overall goal of this project is to forensically characterize 100 unknown Burkholderia isolates in the US-Australia collaboration. We will identify genome-wide single nucleotide polymorphisms (SNPs) from B. pseudomallei and near neighbor species including B. mallei, B. thailandensis and B. oklahomensis. We will design microarray probes to detect these SNP markers and analyze 100 Burkholderia genomic DNAs extracted from environmental, clinical and near neighbor isolates from Australian collaborators on the Burkholderia SNP microarray. We will analyze the microarray genotyping results to characterize the genetic diversity of these new isolates and triage the samples for whole genome sequencing. In this interimmore » report, we described the SNP analysis and the microarray probe design for the Burkholderia SNP microarray.« less

Generalized Correlation Coefficient for Non-Parametric Analysis of Microarray Time-Course Data.

PubMed

Tan, Qihua; Thomassen, Mads; Burton, Mark; Mose, Kristian Fredløv; Andersen, Klaus Ejner; Hjelmborg, Jacob; Kruse, Torben

2017-06-06

Modeling complex time-course patterns is a challenging issue in microarray study due to complex gene expression patterns in response to the time-course experiment. We introduce the generalized correlation coefficient and propose a combinatory approach for detecting, testing and clustering the heterogeneous time-course gene expression patterns. Application of the method identified nonlinear time-course patterns in high agreement with parametric analysis. We conclude that the non-parametric nature in the generalized correlation analysis could be an useful and efficient tool for analyzing microarray time-course data and for exploring the complex relationships in the omics data for studying their association with disease and health.

Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

PubMed Central

2009-01-01

Background The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 ± 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense. PMID:20025733

MGDB: crossing the marker genes of a user microarray with a database of public-microarrays marker genes.

PubMed

Huerta, Mario; Munyi, Marc; Expósito, David; Querol, Enric; Cedano, Juan

2014-06-15

The microarrays performed by scientific teams grow exponentially. These microarray data could be useful for researchers around the world, but unfortunately they are underused. To fully exploit these data, it is necessary (i) to extract these data from a repository of the high-throughput gene expression data like Gene Expression Omnibus (GEO) and (ii) to make the data from different microarrays comparable with tools easy to use for scientists. We have developed these two solutions in our server, implementing a database of microarray marker genes (Marker Genes Data Base). This database contains the marker genes of all GEO microarray datasets and it is updated monthly with the new microarrays from GEO. Thus, researchers can see whether the marker genes of their microarray are marker genes in other microarrays in the database, expanding the analysis of their microarray to the rest of the public microarrays. This solution helps not only to corroborate the conclusions regarding a researcher's microarray but also to identify the phenotype of different subsets of individuals under investigation, to frame the results with microarray experiments from other species, pathologies or tissues, to search for drugs that promote the transition between the studied phenotypes, to detect undesirable side effects of the treatment applied, etc. Thus, the researcher can quickly add relevant information to his/her studies from all of the previous analyses performed in other studies as long as they have been deposited in public repositories. Marker-gene database tool: http://ibb.uab.es/mgdb © The Author 2014. Published by Oxford University Press.

Identifying molecular features for prostate cancer with Gleason 7 based on microarray gene expression profiles.

PubMed

Bălăcescu, Loredana; Bălăcescu, O; Crişan, N; Fetica, B; Petruţ, B; Bungărdean, Cătălina; Rus, Meda; Tudoran, Oana; Meurice, G; Irimie, Al; Dragoş, N; Berindan-Neagoe, Ioana

2011-01-01

Prostate cancer represents the first leading cause of cancer among western male population, with different clinical behavior ranging from indolent to metastatic disease. Although many molecules and deregulated pathways are known, the molecular mechanisms involved in the development of prostate cancer are not fully understood. The aim of this study was to explore the molecular variation underlying the prostate cancer, based on microarray analysis and bioinformatics approaches. Normal and prostate cancer tissues were collected by macrodissection from prostatectomy pieces. All prostate cancer specimens used in our study were Gleason score 7. Gene expression microarray (Agilent Technologies) was used for Whole Human Genome evaluation. The bioinformatics and functional analysis were based on Limma and Ingenuity software. The microarray analysis identified 1119 differentially expressed genes between prostate cancer and normal prostate, which were up- or down-regulated at least 2-fold. P-values were adjusted for multiple testing using Benjamini-Hochberg method with a false discovery rate of 0.01. These genes were analyzed with Ingenuity Pathway Analysis software and were established 23 genetic networks. Our microarray results provide new information regarding the molecular networks in prostate cancer stratified as Gleason 7. These data highlighted gene expression profiles for better understanding of prostate cancer progression.

Porous Silicon Antibody Microarrays for Quantitative Analysis: Measurement of Free and Total PSA in Clinical Plasma Samples

PubMed Central

Tojo, Axel; Malm, Johan; Marko-Varga, György; Lilja, Hans; Laurell, Thomas

2014-01-01

The antibody microarrays have become widespread, but their use for quantitative analyses in clinical samples has not yet been established. We investigated an immunoassay based on nanoporous silicon antibody microarrays for quantification of total prostate-specific-antigen (PSA) in 80 clinical plasma samples, and provide quantitative data from a duplex microarray assay that simultaneously quantifies free and total PSA in plasma. To further develop the assay the porous silicon chips was placed into a standard 96-well microtiter plate for higher throughput analysis. The samples analyzed by this quantitative microarray were 80 plasma samples obtained from men undergoing clinical PSA testing (dynamic range: 0.14-44ng/ml, LOD: 0.14ng/ml). The second dataset, measuring free PSA (dynamic range: 0.40-74.9ng/ml, LOD: 0.47ng/ml) and total PSA (dynamic range: 0.87-295ng/ml, LOD: 0.76ng/ml), was also obtained from the clinical routine. The reference for the quantification was a commercially available assay, the ProStatus PSA Free/Total DELFIA. In an analysis of 80 plasma samples the microarray platform performs well across the range of total PSA levels. This assay might have the potential to substitute for the large-scale microtiter plate format in diagnostic applications. The duplex assay paves the way for a future quantitative multiplex assay, which analyses several prostate cancer biomarkers simultaneously. PMID:22921878

Systematic evaluation of RNA quality, microarray data reliability and pathway analysis in fresh, fresh frozen and formalin-fixed paraffin-embedded tissue samples.

PubMed

Wimmer, Isabella; Tröscher, Anna R; Brunner, Florian; Rubino, Stephen J; Bien, Christian G; Weiner, Howard L; Lassmann, Hans; Bauer, Jan

2018-04-20

Formalin-fixed paraffin-embedded (FFPE) tissues are valuable resources commonly used in pathology. However, formalin fixation modifies nucleic acids challenging the isolation of high-quality RNA for genetic profiling. Here, we assessed feasibility and reliability of microarray studies analysing transcriptome data from fresh, fresh-frozen (FF) and FFPE tissues. We show that reproducible microarray data can be generated from only 2 ng FFPE-derived RNA. For RNA quality assessment, fragment size distribution (DV200) and qPCR proved most suitable. During RNA isolation, extending tissue lysis time to 10 hours reduced high-molecular-weight species, while additional incubation at 70 °C markedly increased RNA yields. Since FF- and FFPE-derived microarrays constitute different data entities, we used indirect measures to investigate gene signal variation and relative gene expression. Whole-genome analyses revealed high concordance rates, while reviewing on single-genes basis showed higher data variation in FFPE than FF arrays. Using an experimental model, gene set enrichment analysis (GSEA) of FFPE-derived microarrays and fresh tissue-derived RNA-Seq datasets yielded similarly affected pathways confirming the applicability of FFPE tissue in global gene expression analysis. Our study provides a workflow comprising RNA isolation, quality assessment and microarray profiling using minimal RNA input, thus enabling hypothesis-generating pathway analyses from limited amounts of precious, pathologically significant FFPE tissues.

Comparative study of joint analysis of microarray gene expression data in survival prediction and risk assessment of breast cancer patients

PubMed Central

2016-01-01

Abstract Microarray gene expression data sets are jointly analyzed to increase statistical power. They could either be merged together or analyzed by meta-analysis. For a given ensemble of data sets, it cannot be foreseen which of these paradigms, merging or meta-analysis, works better. In this article, three joint analysis methods, Z -score normalization, ComBat and the inverse normal method (meta-analysis) were selected for survival prognosis and risk assessment of breast cancer patients. The methods were applied to eight microarray gene expression data sets, totaling 1324 patients with two clinical endpoints, overall survival and relapse-free survival. The performance derived from the joint analysis methods was evaluated using Cox regression for survival analysis and independent validation used as bias estimation. Overall, Z -score normalization had a better performance than ComBat and meta-analysis. Higher Area Under the Receiver Operating Characteristic curve and hazard ratio were also obtained when independent validation was used as bias estimation. With a lower time and memory complexity, Z -score normalization is a simple method for joint analysis of microarray gene expression data sets. The derived findings suggest further assessment of this method in future survival prediction and cancer classification applications. PMID:26504096

Derivation of Tissue-specific Functional Gene Sets to Aid Transcriptomic Analysis of Chemical Impacts on the Teleost Reproductive Axis.

EPA Science Inventory

Oligonucleotide microarrays are a powerful tool for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the poor power of microarray-based analyses to detect diffe...

A probabilistic framework for microarray data analysis: fundamental probability models and statistical inference.

PubMed

Ogunnaike, Babatunde A; Gelmi, Claudio A; Edwards, Jeremy S

2010-05-21

Gene expression studies generate large quantities of data with the defining characteristic that the number of genes (whose expression profiles are to be determined) exceed the number of available replicates by several orders of magnitude. Standard spot-by-spot analysis still seeks to extract useful information for each gene on the basis of the number of available replicates, and thus plays to the weakness of microarrays. On the other hand, because of the data volume, treating the entire data set as an ensemble, and developing theoretical distributions for these ensembles provides a framework that plays instead to the strength of microarrays. We present theoretical results that under reasonable assumptions, the distribution of microarray intensities follows the Gamma model, with the biological interpretations of the model parameters emerging naturally. We subsequently establish that for each microarray data set, the fractional intensities can be represented as a mixture of Beta densities, and develop a procedure for using these results to draw statistical inference regarding differential gene expression. We illustrate the results with experimental data from gene expression studies on Deinococcus radiodurans following DNA damage using cDNA microarrays. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

Microarray-based screening of heat shock protein inhibitors.

PubMed

Schax, Emilia; Walter, Johanna-Gabriela; Märzhäuser, Helene; Stahl, Frank; Scheper, Thomas; Agard, David A; Eichner, Simone; Kirschning, Andreas; Zeilinger, Carsten

2014-06-20

Based on the importance of heat shock proteins (HSPs) in diseases such as cancer, Alzheimer's disease or malaria, inhibitors of these chaperons are needed. Today's state-of-the-art techniques to identify HSP inhibitors are performed in microplate format, requiring large amounts of proteins and potential inhibitors. In contrast, we have developed a miniaturized protein microarray-based assay to identify novel inhibitors, allowing analysis with 300 pmol of protein. The assay is based on competitive binding of fluorescence-labeled ATP and potential inhibitors to the ATP-binding site of HSP. Therefore, the developed microarray enables the parallel analysis of different ATP-binding proteins on a single microarray. We have demonstrated the possibility of multiplexing by immobilizing full-length human HSP90α and HtpG of Helicobacter pylori on microarrays. Fluorescence-labeled ATP was competed by novel geldanamycin/reblastatin derivatives with IC50 values in the range of 0.5 nM to 4 μM and Z(*)-factors between 0.60 and 0.96. Our results demonstrate the potential of a target-oriented multiplexed protein microarray to identify novel inhibitors for different members of the HSP90 family. Copyright © 2014 Elsevier B.V. All rights reserved.

Genome-wide analysis of DUF221 domain-containing gene family in Oryza species and identification of its salinity stress-responsive members in rice.

PubMed

Ganie, Showkat Ahmad; Pani, Dipti Ranjan; Mondal, Tapan Kumar

2017-01-01

DUF221 domain-containing genes (DDP genes) play important roles in developmental biology, hormone signalling transduction, and responses to abiotic stress. Therefore to understand their structural and evolutionary relationship, we did a genome-wide analysis of this important gene family in rice. Further, through comparative genomics, DDP genes from Oryza sativa subsp. (indica), nine different wild species of rice and Arabidopsis were also identified. We also found an expansion of the DDP gene families in rice and Arabidopsis which is due to the segmental duplication events in some of the gene family members. In general, a highly purifying selection was found acting on all the deduced paralogous and orthologous DDP gene pairs. The data from microarray and subsequent qRT-PCR analysis revealed that although several OsDDPs were differentially regulated under salinity stress, yet OsDDP6 was upregulated at all the developmental stages in salt tolerant rice genotype, FL478. Interestingly, OsDDP6 was found to be involved in proline metabolism pathway as indicated by protein network analysis. The diverse gene structures, varied transmembrane topologies and the differential expression patterns implied the functional diversity in DDP genes. Therefore, the comprehensive evolutionary analysis of DDP genes from different Oryza species and Arabidopsis performed in this study will provide the basis for further functional validation studies vis-à-vis DDP genes of rice and other plant species.

Genome-wide analysis of DUF221 domain-containing gene family in Oryza species and identification of its salinity stress-responsive members in rice

PubMed Central

Ganie, Showkat Ahmad; Pani, Dipti Ranjan

2017-01-01

DUF221 domain-containing genes (DDP genes) play important roles in developmental biology, hormone signalling transduction, and responses to abiotic stress. Therefore to understand their structural and evolutionary relationship, we did a genome-wide analysis of this important gene family in rice. Further, through comparative genomics, DDP genes from Oryza sativa subsp. (indica), nine different wild species of rice and Arabidopsis were also identified. We also found an expansion of the DDP gene families in rice and Arabidopsis which is due to the segmental duplication events in some of the gene family members. In general, a highly purifying selection was found acting on all the deduced paralogous and orthologous DDP gene pairs. The data from microarray and subsequent qRT-PCR analysis revealed that although several OsDDPs were differentially regulated under salinity stress, yet OsDDP6 was upregulated at all the developmental stages in salt tolerant rice genotype, FL478. Interestingly, OsDDP6 was found to be involved in proline metabolism pathway as indicated by protein network analysis. The diverse gene structures, varied transmembrane topologies and the differential expression patterns implied the functional diversity in DDP genes. Therefore, the comprehensive evolutionary analysis of DDP genes from different Oryza species and Arabidopsis performed in this study will provide the basis for further functional validation studies vis-à-vis DDP genes of rice and other plant species. PMID:28846681

Rapid Detection of Rare Deleterious Variants by Next Generation Sequencing with Optional Microarray SNP Genotype Data

PubMed Central

Watson, Christopher M.; Crinnion, Laura A.; Gurgel‐Gianetti, Juliana; Harrison, Sally M.; Daly, Catherine; Antanavicuite, Agne; Lascelles, Carolina; Markham, Alexander F.; Pena, Sergio D. J.; Bonthron, David T.

2015-01-01

ABSTRACT Autozygosity mapping is a powerful technique for the identification of rare, autosomal recessive, disease‐causing genes. The ease with which this category of disease gene can be identified has greatly increased through the availability of genome‐wide SNP genotyping microarrays and subsequently of exome sequencing. Although these methods have simplified the generation of experimental data, its analysis, particularly when disparate data types must be integrated, remains time consuming. Moreover, the huge volume of sequence variant data generated from next generation sequencing experiments opens up the possibility of using these data instead of microarray genotype data to identify disease loci. To allow these two types of data to be used in an integrated fashion, we have developed AgileVCFMapper, a program that performs both the mapping of disease loci by SNP genotyping and the analysis of potentially deleterious variants using exome sequence variant data, in a single step. This method does not require microarray SNP genotype data, although analysis with a combination of microarray and exome genotype data enables more precise delineation of disease loci, due to superior marker density and distribution. PMID:26037133

Automatic Identification and Quantification of Extra-Well Fluorescence in Microarray Images.

PubMed

Rivera, Robert; Wang, Jie; Yu, Xiaobo; Demirkan, Gokhan; Hopper, Marika; Bian, Xiaofang; Tahsin, Tasnia; Magee, D Mitchell; Qiu, Ji; LaBaer, Joshua; Wallstrom, Garrick

2017-11-03

In recent studies involving NAPPA microarrays, extra-well fluorescence is used as a key measure for identifying disease biomarkers because there is evidence to support that it is better correlated with strong antibody responses than statistical analysis involving intraspot intensity. Because this feature is not well quantified by traditional image analysis software, identification and quantification of extra-well fluorescence is performed manually, which is both time-consuming and highly susceptible to variation between raters. A system that could automate this task efficiently and effectively would greatly improve the process of data acquisition in microarray studies, thereby accelerating the discovery of disease biomarkers. In this study, we experimented with different machine learning methods, as well as novel heuristics, for identifying spots exhibiting extra-well fluorescence (rings) in microarray images and assigning each ring a grade of 1-5 based on its intensity and morphology. The sensitivity of our final system for identifying rings was found to be 72% at 99% specificity and 98% at 92% specificity. Our system performs this task significantly faster than a human, while maintaining high performance, and therefore represents a valuable tool for microarray image analysis.

Thermodynamically optimal whole-genome tiling microarray design and validation.

PubMed

Cho, Hyejin; Chou, Hui-Hsien

2016-06-13

Microarray is an efficient apparatus to interrogate the whole transcriptome of species. Microarray can be designed according to annotated gene sets, but the resulted microarrays cannot be used to identify novel transcripts and this design method is not applicable to unannotated species. Alternatively, a whole-genome tiling microarray can be designed using only genomic sequences without gene annotations, and it can be used to detect novel RNA transcripts as well as known genes. The difficulty with tiling microarray design lies in the tradeoff between probe-specificity and coverage of the genome. Sequence comparison methods based on BLAST or similar software are commonly employed in microarray design, but they cannot precisely determine the subtle thermodynamic competition between probe targets and partially matched probe nontargets during hybridizations. Using the whole-genome thermodynamic analysis software PICKY to design tiling microarrays, we can achieve maximum whole-genome coverage allowable under the thermodynamic constraints of each target genome. The resulted tiling microarrays are thermodynamically optimal in the sense that all selected probes share the same melting temperature separation range between their targets and closest nontargets, and no additional probes can be added without violating the specificity of the microarray to the target genome. This new design method was used to create two whole-genome tiling microarrays for Escherichia coli MG1655 and Agrobacterium tumefaciens C58 and the experiment results validated the design.

Identification of candidate genes in osteoporosis by integrated microarray analysis.

PubMed

Li, J J; Wang, B Q; Fei, Q; Yang, Y; Li, D

2016-12-01

In order to screen the altered gene expression profile in peripheral blood mononuclear cells of patients with osteoporosis, we performed an integrated analysis of the online microarray studies of osteoporosis. We searched the Gene Expression Omnibus (GEO) database for microarray studies of peripheral blood mononuclear cells in patients with osteoporosis. Subsequently, we integrated gene expression data sets from multiple microarray studies to obtain differentially expressed genes (DEGs) between patients with osteoporosis and normal controls. Gene function analysis was performed to uncover the functions of identified DEGs. A total of three microarray studies were selected for integrated analysis. In all, 1125 genes were found to be significantly differentially expressed between osteoporosis patients and normal controls, with 373 upregulated and 752 downregulated genes. Positive regulation of the cellular amino metabolic process (gene ontology (GO): 0033240, false discovery rate (FDR) = 1.00E + 00) was significantly enriched under the GO category for biological processes, while for molecular functions, flavin adenine dinucleotide binding (GO: 0050660, FDR = 3.66E-01) and androgen receptor binding (GO: 0050681, FDR = 6.35E-01) were significantly enriched. DEGs were enriched in many osteoporosis-related signalling pathways, including those of mitogen-activated protein kinase (MAPK) and calcium. Protein-protein interaction (PPI) network analysis showed that the significant hub proteins contained ubiquitin specific peptidase 9, X-linked (Degree = 99), ubiquitin specific peptidase 19 (Degree = 57) and ubiquitin conjugating enzyme E2 B (Degree = 57). Analysis of gene function of identified differentially expressed genes may expand our understanding of fundamental mechanisms leading to osteoporosis. Moreover, significantly enriched pathways, such as MAPK and calcium, may involve in osteoporosis through osteoblastic differentiation and bone formation.Cite this article: J. J. Li, B. Q. Wang, Q. Fei, Y. Yang, D. Li. Identification of candidate genes in osteoporosis by integrated microarray analysis. Bone Joint Res 2016;5:594-601. DOI: 10.1302/2046-3758.512.BJR-2016-0073.R1. © 2016 Fei et al.

Metabolic Shift of Escherichia coli under Salt Stress in the Presence of Glycine Betaine

PubMed Central

Metris, A.; George, S. M.; Mulholland, F.; Carter, A. T.

2014-01-01

An important area of food safety focuses on bacterial survival and growth in unfavorable environments. In order to understand how bacteria adapt to stresses other than nutrient limitation in batch cultures, we need to develop mechanistic models of intracellular regulation and metabolism under stress. We studied the growth of Escherichia coli in minimal medium with added salt and different osmoprotectants. To characterize the metabolic efficiency with a robust parameter, we identified the optical density (OD) values at the inflection points of measured “OD versus time” growth curves and described them as a function of glucose concentration. We found that the metabolic efficiency parameter did not necessarily follow the trend of decreasing specific growth rate as the salt concentration increased. In the absence of osmoprotectant, or in the presence of proline, the metabolic efficiency decreased with increasing NaCl concentration. However, in the presence of choline or glycine betaine, it increased between 2 and 4.5% NaCl before declining at 5% NaCl and above. Microarray analysis of the transcriptional network and proteomics analysis with glycine betaine in the medium indicated that between 4.5 and 5% NaCl, the metabolism switched from aerobic to fermentative pathways and that the response to osmotic stress is similar to that for oxidative stress. We conclude that, although the growth rate appeared to decrease smoothly with increasing NaCl, the metabolic strategy of cells changed abruptly at a threshold concentration of NaCl. PMID:24858086

Genome scale transcriptional response diversity among ten ecotypes of Arabidopsis thaliana during heat stress

PubMed Central

Barah, Pankaj; Jayavelu, Naresh D.; Mundy, John; Bones, Atle M.

2013-01-01

In the scenario of global warming and climate change, heat stress is a serious threat to crop production worldwide. Being sessile, plants cannot escape from heat. Plants have developed various adaptive mechanisms to survive heat stress. Several studies have focused on diversity of heat tolerance levels in divergent Arabidopsis thaliana (A. thaliana) ecotypes, but comprehensive genome scale understanding of heat stress response in plants is still lacking. Here we report the genome scale transcript responses to heat stress of 10 A. thaliana ecotypes (Col, Ler, C24, Cvi, Kas1, An1, Sha, Kyo2, Eri, and Kond) originated from different geographical locations. During the experiment, A. thaliana plants were subjected to heat stress (38°C) and transcript responses were monitored using Arabidopsis NimbleGen ATH6 microarrays. The responses of A. thaliana ecotypes exhibited considerable variation in the transcript abundance levels. In total, 3644 transcripts were significantly heat regulated (p < 0.01) in the 10 ecotypes, including 244 transcription factors and 203 transposable elements. By employing a systems genetics approach- Network Component Analysis (NCA), we have constructed an in silico transcript regulatory network model for 35 heat responsive transcription factors during cellular responses to heat stress in A. thaliana. The computed activities of the 35 transcription factors showed ecotype specific responses to the heat treatment. PMID:24409190

Microarray analysis of Pseudomonas aeruginosa reveals induction of pyocin genes in response to hydrogen peroxide

PubMed Central

Chang, Wook; Small, David A; Toghrol, Freshteh; Bentley, William E

2005-01-01

Background Pseudomonas aeruginosa, a pathogen infecting those with cystic fibrosis, encounters toxicity from phagocyte-derived reactive oxidants including hydrogen peroxide during active infection. P. aeruginosa responds with adaptive and protective strategies against these toxic species to effectively infect humans. Despite advances in our understanding of the responses to oxidative stress in many specific cases, the connectivity between targeted protective genes and the rest of cell metabolism remains obscure. Results Herein, we performed a genome-wide transcriptome analysis of the cellular responses to hydrogen peroxide in order to determine a more complete picture of how oxidative stress-induced genes are related and regulated. Our data reinforce the previous conclusion that DNA repair proteins and catalases may be among the most vital antioxidant defense systems of P. aeruginosa. Our results also suggest that sublethal oxidative damage reduces active and/or facilitated transport and that intracellular iron might be a key factor for a relationship between oxidative stress and iron regulation. Perhaps most intriguingly, we revealed that the transcription of all F-, R-, and S-type pyocins was upregulated by oxidative stress and at the same time, a cell immunity protein (pyocin S2 immunity protein) was downregulated, possibly leading to self-killing activity. Conclusion This finding proposes that pyocin production might be another novel defensive scheme against oxidative attack by host cells. PMID:16150148

Genome-wide identification and expression analysis of the B-box gene family in the Apple (Malus domestica Borkh.) genome.

PubMed

Liu, Xin; Li, Rong; Dai, Yaqing; Chen, Xuesen; Wang, Xiaoyun

2018-04-01

The B-box proteins (BBXs) are a family of zinc finger proteins containing one/two B-box domain(s). Compared with intensive studies of animal BBXs, investigations of the plant BBX family are limited, though some specific plant BBXs have been demonstrated to act as transcription factors in the regulation of flowering and photomorphogenesis. In this study, using a global search of the apple (Malus domestica Borkh.) genome, a total of 64 members of BBX (MdBBX) were identified. All the MdBBXs were divided into five groups based on the phylogenetic relationship, numbers of B-boxes contained and whether there was with an additional CCT domain. According to the characteristics of organ-specific expression, MdBBXs were divided into three groups based on the microarray information. An analysis of cis-acting elements showed that elements related to the stress response were prevalent in the promoter sequences of most MdBBXs. Twelve MdBBX members from different groups were randomly selected and exposed to abiotic stresses. Their expressions were up-regulated to some extent in the roots and leaves. Six among 12 MdBBXs were sensitive to osmotic pressure, salt, cold stress and exogenous abscisic acid treatment, with their expressions enhanced more than 20-fold. Our results suggested that MdBBXs may take part in response to abiotic stress.

Workflows for microarray data processing in the Kepler environment.

PubMed

Stropp, Thomas; McPhillips, Timothy; Ludäscher, Bertram; Bieda, Mark

2012-05-17

Microarray data analysis has been the subject of extensive and ongoing pipeline development due to its complexity, the availability of several options at each analysis step, and the development of new analysis demands, including integration with new data sources. Bioinformatics pipelines are usually custom built for different applications, making them typically difficult to modify, extend and repurpose. Scientific workflow systems are intended to address these issues by providing general-purpose frameworks in which to develop and execute such pipelines. The Kepler workflow environment is a well-established system under continual development that is employed in several areas of scientific research. Kepler provides a flexible graphical interface, featuring clear display of parameter values, for design and modification of workflows. It has capabilities for developing novel computational components in the R, Python, and Java programming languages, all of which are widely used for bioinformatics algorithm development, along with capabilities for invoking external applications and using web services. We developed a series of fully functional bioinformatics pipelines addressing common tasks in microarray processing in the Kepler workflow environment. These pipelines consist of a set of tools for GFF file processing of NimbleGen chromatin immunoprecipitation on microarray (ChIP-chip) datasets and more comprehensive workflows for Affymetrix gene expression microarray bioinformatics and basic primer design for PCR experiments, which are often used to validate microarray results. Although functional in themselves, these workflows can be easily customized, extended, or repurposed to match the needs of specific projects and are designed to be a toolkit and starting point for specific applications. These workflows illustrate a workflow programming paradigm focusing on local resources (programs and data) and therefore are close to traditional shell scripting or R/BioConductor scripting approaches to pipeline design. Finally, we suggest that microarray data processing task workflows may provide a basis for future example-based comparison of different workflow systems. We provide a set of tools and complete workflows for microarray data analysis in the Kepler environment, which has the advantages of offering graphical, clear display of conceptual steps and parameters and the ability to easily integrate other resources such as remote data and web services.

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays

PubMed Central

Goodman, Corey W.; Major, Heather J.; Walls, William D.; Sheffield, Val C.; Casavant, Thomas L.; Darbro, Benjamin W.

2016-01-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. PMID:25595567

SSHscreen and SSHdb, generic software for microarray based gene discovery: application to the stress response in cowpea

PubMed Central

2010-01-01

Background Suppression subtractive hybridization is a popular technique for gene discovery from non-model organisms without an annotated genome sequence, such as cowpea (Vigna unguiculata (L.) Walp). We aimed to use this method to enrich for genes expressed during drought stress in a drought tolerant cowpea line. However, current methods were inefficient in screening libraries and management of the sequence data, and thus there was a need to develop software tools to facilitate the process. Results Forward and reverse cDNA libraries enriched for cowpea drought response genes were screened on microarrays, and the R software package SSHscreen 2.0.1 was developed (i) to normalize the data effectively using spike-in control spot normalization, and (ii) to select clones for sequencing based on the calculation of enrichment ratios with associated statistics. Enrichment ratio 3 values for each clone showed that 62% of the forward library and 34% of the reverse library clones were significantly differentially expressed by drought stress (adjusted p value < 0.05). Enrichment ratio 2 calculations showed that > 88% of the clones in both libraries were derived from rare transcripts in the original tester samples, thus supporting the notion that suppression subtractive hybridization enriches for rare transcripts. A set of 118 clones were chosen for sequencing, and drought-induced cowpea genes were identified, the most interesting encoding a late embryogenesis abundant Lea5 protein, a glutathione S-transferase, a thaumatin, a universal stress protein, and a wound induced protein. A lipid transfer protein and several components of photosynthesis were down-regulated by the drought stress. Reverse transcriptase quantitative PCR confirmed the enrichment ratio values for the selected cowpea genes. SSHdb, a web-accessible database, was developed to manage the clone sequences and combine the SSHscreen data with sequence annotations derived from BLAST and Blast2GO. The self-BLAST function within SSHdb grouped redundant clones together and illustrated that the SSHscreen plots are a useful tool for choosing anonymous clones for sequencing, since redundant clones cluster together on the enrichment ratio plots. Conclusions We developed the SSHscreen-SSHdb software pipeline, which greatly facilitates gene discovery using suppression subtractive hybridization by improving the selection of clones for sequencing after screening the library on a small number of microarrays. Annotation of the sequence information and collaboration was further enhanced through a web-based SSHdb database, and we illustrated this through identification of drought responsive genes from cowpea, which can now be investigated in gene function studies. SSH is a popular and powerful gene discovery tool, and therefore this pipeline will have application for gene discovery in any biological system, particularly non-model organisms. SSHscreen 2.0.1 and a link to SSHdb are available from http://microarray.up.ac.za/SSHscreen. PMID:20359330

Comprehensive Genomic Analysis and Expression Profiling of Phospholipase C Gene Family during Abiotic Stresses and Development in Rice

PubMed Central

Singh, Amarjeet; Kanwar, Poonam; Pandey, Amita; Tyagi, Akhilesh K.; Sopory, Sudhir K.; Kapoor, Sanjay; Pandey, Girdhar K.

2013-01-01

Background Phospholipase C (PLC) is one of the major lipid hydrolysing enzymes, implicated in lipid mediated signaling. PLCs have been found to play a significant role in abiotic stress triggered signaling and developmental processes in various plant species. Genome wide identification and expression analysis have been carried out for this gene family in Arabidopsis, yet not much has been accomplished in crop plant rice. Methodology/Principal Findings An exhaustive in-silico exploration of rice genome using various online databases and tools resulted in the identification of nine PLC encoding genes. Based on sequence, motif and phylogenetic analysis rice PLC gene family could be divided into phosphatidylinositol-specific PLCs (PI-PLCs) and phosphatidylcholine- PLCs (PC-PLC or NPC) classes with four and five members, respectively. A comparative analysis revealed that PLCs are conserved in Arabidopsis (dicots) and rice (monocot) at gene structure and protein level but they might have evolved through a separate evolutionary path. Transcript profiling using gene chip microarray and quantitative RT-PCR showed that most of the PLC members expressed significantly and differentially under abiotic stresses (salt, cold and drought) and during various developmental stages with condition/stage specific and overlapping expression. This finding suggested an important role of different rice PLC members in abiotic stress triggered signaling and plant development, which was also supported by the presence of relevant cis-regulatory elements in their promoters. Sub-cellular localization of few selected PLC members in Nicotiana benthamiana and onion epidermal cells has provided a clue about their site of action and functional behaviour. Conclusion/Significance The genome wide identification, structural and expression analysis and knowledge of sub-cellular localization of PLC gene family envisage the functional characterization of these genes in crop plants in near future. PMID:23638098

Comprehensive genomic analysis and expression profiling of phospholipase C gene family during abiotic stresses and development in rice.

PubMed

Singh, Amarjeet; Kanwar, Poonam; Pandey, Amita; Tyagi, Akhilesh K; Sopory, Sudhir K; Kapoor, Sanjay; Pandey, Girdhar K

2013-01-01

Phospholipase C (PLC) is one of the major lipid hydrolysing enzymes, implicated in lipid mediated signaling. PLCs have been found to play a significant role in abiotic stress triggered signaling and developmental processes in various plant species. Genome wide identification and expression analysis have been carried out for this gene family in Arabidopsis, yet not much has been accomplished in crop plant rice. An exhaustive in-silico exploration of rice genome using various online databases and tools resulted in the identification of nine PLC encoding genes. Based on sequence, motif and phylogenetic analysis rice PLC gene family could be divided into phosphatidylinositol-specific PLCs (PI-PLCs) and phosphatidylcholine- PLCs (PC-PLC or NPC) classes with four and five members, respectively. A comparative analysis revealed that PLCs are conserved in Arabidopsis (dicots) and rice (monocot) at gene structure and protein level but they might have evolved through a separate evolutionary path. Transcript profiling using gene chip microarray and quantitative RT-PCR showed that most of the PLC members expressed significantly and differentially under abiotic stresses (salt, cold and drought) and during various developmental stages with condition/stage specific and overlapping expression. This finding suggested an important role of different rice PLC members in abiotic stress triggered signaling and plant development, which was also supported by the presence of relevant cis-regulatory elements in their promoters. Sub-cellular localization of few selected PLC members in Nicotiana benthamiana and onion epidermal cells has provided a clue about their site of action and functional behaviour. The genome wide identification, structural and expression analysis and knowledge of sub-cellular localization of PLC gene family envisage the functional characterization of these genes in crop plants in near future.

TaSK5, an abiotic stress-inducible GSK3/shaggy-like kinase from wheat, confers salt and drought tolerance in transgenic Arabidopsis.

PubMed

Christov, Nikolai Kirilov; Christova, Petya Koeva; Kato, Hideki; Liu, Yuelin; Sasaki, Kentaro; Imai, Ryozo

2014-11-01

A novel cold-inducible GSK3/shaggy-like kinase, TaSK5, was isolated from winter wheat using a macroarray-based differential screening approach. TaSK5 showed high similarity to Arabidopsis subgroup I GSK3/shaggy-like kinases ASK-alpha, AtSK-gamma and ASK-epsilon. RNA gel blot analyses revealed TaSK5 induction by cold and NaCl treatments and to a lesser extent by drought treatment. TaSK5 functionally complemented the cold- and salt-sensitive phenotypes of a yeast GSK3/shaggy-like kinase mutant, △mck1. Transgenic Arabidopsis plants overexpressing TaSK5 cDNA showed enhanced tolerance to salt and drought stresses. By contrast, the tolerance of the transgenic plants to freezing stress was not altered. Microarray analysis revealed that a number of abiotic stress-inducible genes were constitutively induced in the transgenic Arabidopsis plants, suggesting that TaSK5 may function in a novel signal transduction pathway that appears to be unrelated to DREB1/CBF regulon and may involve crosstalk between abiotic and hormonal signals. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

MICROARRAY DATA ANALYSIS USING MULTIPLE STATISTICAL MODELS

EPA Science Inventory

Microarray Data Analysis Using Multiple Statistical Models

Wenjun Bao1, Judith E. Schmid1, Amber K. Goetz1, Ming Ouyang2, William J. Welsh2,Andrew I. Brooks3,4, ChiYi Chu3,Mitsunori Ogihara3,4, Yinhe Cheng5, David J. Dix1. 1National Health and Environmental Effects Researc...

Parents' Perceptions of the Usefulness of Chromosomal Microarray Analysis for Children with Autism Spectrum Disorders

ERIC Educational Resources Information Center

Reiff, Marian; Giarelli, Ellen; Bernhardt, Barbara A.; Easley, Ebony; Spinner, Nancy B.; Sankar, Pamela L.; Mulchandani, Surabhi

2015-01-01

Clinical guidelines recommend chromosomal microarray analysis (CMA) for all children with autism spectrum disorders (ASDs). We explored the test's perceived usefulness among parents of children with ASD who had undergone CMA, and received a result categorized as pathogenic, variant of uncertain significance, or negative. Fifty-seven parents…

A Graphical Systems Model and Tissue-specific Functional Gene Sets to Aid Transcriptomic Analysis of Chemical Impacts on the Female Teleost Reproductive Axis

EPA Science Inventory

Oligonucleotide microarrays and other ‘omics’ approaches are powerful tools for unsupervised analysis of chemical impacts on biological systems. However, the lack of well annotated biological pathways for many aquatic organisms, including fish, and the poor power of microarray-b...

CGO: utilizing and integrating gene expression microarray data in clinical research and data management.

PubMed

Bumm, Klaus; Zheng, Mingzhong; Bailey, Clyde; Zhan, Fenghuang; Chiriva-Internati, M; Eddlemon, Paul; Terry, Julian; Barlogie, Bart; Shaughnessy, John D

2002-02-01

Clinical GeneOrganizer (CGO) is a novel windows-based archiving, organization and data mining software for the integration of gene expression profiling in clinical medicine. The program implements various user-friendly tools and extracts data for further statistical analysis. This software was written for Affymetrix GeneChip *.txt files, but can also be used for any other microarray-derived data. The MS-SQL server version acts as a data mart and links microarray data with clinical parameters of any other existing database and therefore represents a valuable tool for combining gene expression analysis and clinical disease characteristics.

cluML: A markup language for clustering and cluster validity assessment of microarray data.

PubMed

Bolshakova, Nadia; Cunningham, Pádraig

2005-01-01

cluML is a new markup language for microarray data clustering and cluster validity assessment. The XML-based format has been designed to address some of the limitations observed in traditional formats, such as inability to store multiple clustering (including biclustering) and validation results within a dataset. cluML is an effective tool to support biomedical knowledge representation in gene expression data analysis. Although cluML was developed for DNA microarray analysis applications, it can be effectively used for the representation of clustering and for the validation of other biomedical and physical data that has no limitations.

Analysis of developmental gene conservation in the Actinomycetales using DNA/DNA microarray comparisons.

PubMed

Kirby, Ralph; Herron, Paul; Hoskisson, Paul

2011-02-01

Based on available genome sequences, Actinomycetales show significant gene synteny across a wide range of species and genera. In addition, many genera show varying degrees of complex morphological development. Using the presence of gene synteny as a basis, it is clear that an analysis of gene conservation across the Streptomyces and various other Actinomycetales will provide information on both the importance of genes and gene clusters and the evolution of morphogenesis in these bacteria. Genome sequencing, although becoming cheaper, is still relatively expensive for comparing large numbers of strains. Thus, a heterologous DNA/DNA microarray hybridization dataset based on a Streptomyces coelicolor microarray allows a cheaper and greater depth of analysis of gene conservation. This study, using both bioinformatical and microarray approaches, was able to classify genes previously identified as involved in morphogenesis in Streptomyces into various subgroups in terms of conservation across species and genera. This will allow the targeting of genes for further study based on their importance at the species level and at higher evolutionary levels.

Cross species analysis of microarray expression data

PubMed Central

Lu, Yong; Huggins, Peter; Bar-Joseph, Ziv

2009-01-01

Motivation: Many biological systems operate in a similar manner across a large number of species or conditions. Cross-species analysis of sequence and interaction data is often applied to determine the function of new genes. In contrast to these static measurements, microarrays measure the dynamic, condition-specific response of complex biological systems. The recent exponential growth in microarray expression datasets allows researchers to combine expression experiments from multiple species to identify genes that are not only conserved in sequence but also operated in a similar way in the different species studied. Results: In this review we discuss the computational and technical challenges associated with these studies, the approaches that have been developed to address these challenges and the advantages of cross-species analysis of microarray data. We show how successful application of these methods lead to insights that cannot be obtained when analyzing data from a single species. We also highlight current open problems and discuss possible ways to address them. Contact: zivbj@cs.cmu.edu PMID:19357096

Separation and Analysis of Adherent and Non-Adherent Cancer Cells Using a Single-Cell Microarray Chip.

PubMed

Yamamura, Shohei; Yamada, Eriko; Kimura, Fukiko; Miyajima, Kumiko; Shigeto, Hajime

2017-10-21

A new single-cell microarray chip was designed and developed to separate and analyze single adherent and non-adherent cancer cells. The single-cell microarray chip is made of polystyrene with over 60,000 microchambers of 10 different size patterns (31-40 µm upper diameter, 11-20 µm lower diameter). A drop of suspension of adherent carcinoma (NCI-H1650) and non-adherent leukocyte (CCRF-CEM) cells was placed onto the chip, and single-cell occupancy of NCI-H1650 and CCRF-CEM was determined to be 79% and 84%, respectively. This was achieved by controlling the chip design and surface treatment. Analysis of protein expression in single NCI-H1650 and CCRF-CEM cells was performed on the single-cell microarray chip by multi-antibody staining. Additionally, with this system, we retrieved positive single cells from the microchambers by a micromanipulator. Thus, this system demonstrates the potential for easy and accurate separation and analysis of various types of single cells.

Global Expression Profiling of Low Temperature Induced Genes in the Chilling Tolerant Japonica Rice Jumli Marshi

PubMed Central

Chawade, Aakash; Lindlöf, Angelica; Olsson, Björn; Olsson, Olof

2013-01-01

Low temperature is a key factor that limits growth and productivity of many important agronomical crops worldwide. Rice (Oryza sativa L.) is negatively affected already at temperatures below +10°C and is therefore denoted as chilling sensitive. However, chilling tolerant rice cultivars exist and can be commercially cultivated at altitudes up to 3,050 meters with temperatures reaching as low as +4°C. In this work, the global transcriptional response to cold stress (+4°C) was studied in the Nepalese highland variety Jumli Marshi (spp. japonica) and 4,636 genes were identified as significantly differentially expressed within 24 hours of cold stress. Comparison with previously published microarray data from one chilling tolerant and two sensitive rice cultivars identified 182 genes differentially expressed (DE) upon cold stress in all four rice cultivars and 511 genes DE only in the chilling tolerant rice. Promoter analysis of the 182 genes suggests a complex cross-talk between ABRE and CBF regulons. Promoter analysis of the 511 genes identified over-represented ABRE motifs but not DRE motifs, suggesting a role for ABA signaling in cold tolerance. Moreover, 2,101 genes were DE in Jumli Marshi alone. By chromosomal localization analysis, 473 of these cold responsive genes were located within 13 different QTLs previously identified as cold associated. PMID:24349120

Soil microbial community successional patterns during forest ecosystem restoration.

PubMed

Banning, Natasha C; Gleeson, Deirdre B; Grigg, Andrew H; Grant, Carl D; Andersen, Gary L; Brodie, Eoin L; Murphy, D V

2011-09-01

Soil microbial community characterization is increasingly being used to determine the responses of soils to stress and disturbances and to assess ecosystem sustainability. However, there is little experimental evidence to indicate that predictable patterns in microbial community structure or composition occur during secondary succession or ecosystem restoration. This study utilized a chronosequence of developing jarrah (Eucalyptus marginata) forest ecosystems, rehabilitated after bauxite mining (up to 18 years old), to examine changes in soil bacterial and fungal community structures (by automated ribosomal intergenic spacer analysis [ARISA]) and changes in specific soil bacterial phyla by 16S rRNA gene microarray analysis. This study demonstrated that mining in these ecosystems significantly altered soil bacterial and fungal community structures. The hypothesis that the soil microbial community structures would become more similar to those of the surrounding nonmined forest with rehabilitation age was broadly supported by shifts in the bacterial but not the fungal community. Microarray analysis enabled the identification of clear successional trends in the bacterial community at the phylum level and supported the finding of an increase in similarity to nonmined forest soil with rehabilitation age. Changes in soil microbial community structure were significantly related to the size of the microbial biomass as well as numerous edaphic variables (including pH and C, N, and P nutrient concentrations). These findings suggest that soil bacterial community dynamics follow a pattern in developing ecosystems that may be predictable and can be conceptualized as providing an integrated assessment of numerous edaphic variables.

Soil Microbial Community Successional Patterns during Forest Ecosystem Restoration ▿†

PubMed Central

Banning, Natasha C.; Gleeson, Deirdre B.; Grigg, Andrew H.; Grant, Carl D.; Andersen, Gary L.; Brodie, Eoin L.; Murphy, D. V.

2011-01-01

Soil microbial community characterization is increasingly being used to determine the responses of soils to stress and disturbances and to assess ecosystem sustainability. However, there is little experimental evidence to indicate that predictable patterns in microbial community structure or composition occur during secondary succession or ecosystem restoration. This study utilized a chronosequence of developing jarrah (Eucalyptus marginata) forest ecosystems, rehabilitated after bauxite mining (up to 18 years old), to examine changes in soil bacterial and fungal community structures (by automated ribosomal intergenic spacer analysis [ARISA]) and changes in specific soil bacterial phyla by 16S rRNA gene microarray analysis. This study demonstrated that mining in these ecosystems significantly altered soil bacterial and fungal community structures. The hypothesis that the soil microbial community structures would become more similar to those of the surrounding nonmined forest with rehabilitation age was broadly supported by shifts in the bacterial but not the fungal community. Microarray analysis enabled the identification of clear successional trends in the bacterial community at the phylum level and supported the finding of an increase in similarity to nonmined forest soil with rehabilitation age. Changes in soil microbial community structure were significantly related to the size of the microbial biomass as well as numerous edaphic variables (including pH and C, N, and P nutrient concentrations). These findings suggest that soil bacterial community dynamics follow a pattern in developing ecosystems that may be predictable and can be conceptualized as providing an integrated assessment of numerous edaphic variables. PMID:21724890

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarrays. 22-24 January 2001, Zurich, Switzerland.

PubMed

Jain, K K

2001-02-01

Cambridge Healthtech Institute's Third Annual Conference on Lab-on-a-Chip and Microarray technology covered the latest advances in this technology and applications in life sciences. Highlights of the meetings are reported briefly with emphasis on applications in genomics, drug discovery and molecular diagnostics. There was an emphasis on microfluidics because of the wide applications in laboratory and drug discovery. The lab-on-a-chip provides the facilities of a complete laboratory in a hand-held miniature device. Several microarray systems have been used for hybridisation and detection techniques. Oligonucleotide scanning arrays provide a versatile tool for the analysis of nucleic acid interactions and provide a platform for improving the array-based methods for investigation of antisense therapeutics. A method for analysing combinatorial DNA arrays using oligonucleotide-modified gold nanoparticle probes and a conventional scanner has considerable potential in molecular diagnostics. Various applications of microarray technology for high-throughput screening in drug discovery and single nucleotide polymorphisms (SNP) analysis were discussed. Protein chips have important applications in proteomics. With the considerable amount of data generated by the different technologies using microarrays, it is obvious that the reading of the information and its interpretation and management through the use of bioinformatics is essential. Various techniques for data analysis were presented. Biochip and microarray technology has an essential role to play in the evolving trends in healthcare, which integrate diagnosis with prevention/treatment and emphasise personalised medicines.

Differential gene expression detection and sample classification using penalized linear regression models.

PubMed

Wu, Baolin

2006-02-15

Differential gene expression detection and sample classification using microarray data have received much research interest recently. Owing to the large number of genes p and small number of samples n (p > n), microarray data analysis poses big challenges for statistical analysis. An obvious problem owing to the 'large p small n' is over-fitting. Just by chance, we are likely to find some non-differentially expressed genes that can classify the samples very well. The idea of shrinkage is to regularize the model parameters to reduce the effects of noise and produce reliable inferences. Shrinkage has been successfully applied in the microarray data analysis. The SAM statistics proposed by Tusher et al. and the 'nearest shrunken centroid' proposed by Tibshirani et al. are ad hoc shrinkage methods. Both methods are simple, intuitive and prove to be useful in empirical studies. Recently Wu proposed the penalized t/F-statistics with shrinkage by formally using the (1) penalized linear regression models for two-class microarray data, showing good performance. In this paper we systematically discussed the use of penalized regression models for analyzing microarray data. We generalize the two-class penalized t/F-statistics proposed by Wu to multi-class microarray data. We formally derive the ad hoc shrunken centroid used by Tibshirani et al. using the (1) penalized regression models. And we show that the penalized linear regression models provide a rigorous and unified statistical framework for sample classification and differential gene expression detection.

Best practices for hybridization design in two-colour microarray analysis.

PubMed

Knapen, Dries; Vergauwen, Lucia; Laukens, Kris; Blust, Ronny

2009-07-01

Two-colour microarrays are a popular platform of choice in gene expression studies. Because two different samples are hybridized on a single microarray, and several microarrays are usually needed in a given experiment, there are many possible ways to combine samples on different microarrays. The actual combination employed is commonly referred to as the 'hybridization design'. Different types of hybridization designs have been developed, all aimed at optimizing the experimental setup for the detection of differentially expressed genes while coping with technical noise. Here, we first provide an overview of the different classes of hybridization designs, discussing their advantages and limitations, and then we illustrate the current trends in the use of different hybridization design types in contemporary research.

Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B–Treated Cervical Cancer HeLa Cells

PubMed Central

Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

2016-01-01

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H2O2–induced cell death is via neutralization of reactive oxygen species. PMID:27458249

Gene Expression Analysis Reveals the Concurrent Activation of Proapoptotic and Antioxidant-Defensive Mechanisms in Flavokawain B-Treated Cervical Cancer HeLa Cells.

PubMed

Yeap, Swee Keong; Abu, Nadiah; Akthar, Nadeem; Ho, Wan Yong; Ky, Huynh; Tan, Sheau Wei; Alitheen, Noorjahan Banu; Kamarul, Tunku

2017-09-01

Flavokawain B (FKB) is known to possess promising anticancer abilities. This is demonstrated in various cancer cell lines including HeLa cells. Cervical cancer is among the most widely diagnosed cancer among women today. Though FKB has been shown to be effective in treating cancer cells, the exact molecular mechanism is still unknown. This study is aimed at understanding the effects of FKB on HeLa cells using a microarray-based mRNA expression profiling and proteome profiling of stress-related proteins. The results of this study suggest that FKB induced cell death through p21-mediated cell cycle arrest and activation of p38. However, concurrent activation of antioxidant-related pathways and iron sequestration pathway followed by activation of ER-resident stress proteins clearly indicate that FKB failed to induce apoptosis in HeLa cells via oxidative stress. This effect implies that the protection of HeLa cells by FKB from H 2 O 2 -induced cell death is via neutralization of reactive oxygen species.

[Diagnosis of a case with Williams-Beuren syndrome with nephrocalcinosis using chromosome microarray analysis].

PubMed

Jin, S J; Liu, M; Long, W J; Luo, X P

2016-12-02

Objective: To explore the clinical phenotypes and the genetic cause for a boy with unexplained growth retardation, nephrocalcinosis, auditory anomalies and multi-organ/system developmental disorders. Method: Routine G-banding and chromosome microarray analysis were applied to a child with unexplained growth retardation, nephrocalcinosis, auditory anomalies and multi-organ/system developmental disorders treated in the Department of Pediatrics of Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology in September 2015 and his parents to conduct the chromosomal karyotype analysis and the whole genome scanning. Deleted genes were searched in the Decipher and NCBI databases, and their relationships with the clinical phenotypes were analyzed. Result: A six-month-old boy was refered to us because of unexplained growth retardation and feeding intolerance.The affected child presented with abnormal manifestation such as special face, umbilical hernia, growth retardation, hypothyroidism, congenital heart disease, right ear sensorineural deafness, hypercalcemia and nephrocalcinosis. The child's karyotype was 46, XY, 16qh + , and his parents' karyotypes were normal. Chromosome microarray analysis revealed a 1 436 kb deletion on the 7q11.23(72701098_74136633) region of the child. This region included 23 protein-coding genes, which were reported to be corresponding to Williams-Beuren syndrome and its certain clinical phenotypes. His parents' results of chromosome microarray analysis were normal. Conclusion: A boy with characteristic manifestation of Williams-Beuren syndrome and rare nephrocalcinosis was diagnosed using chromosome microarray analysis. The deletion on the 7q11.23 might be related to the clinical phenotypes of Williams-Beuren syndrome, yet further studies are needed.

AFM 4.0: a toolbox for DNA microarray analysis

PubMed Central

Breitkreutz, Bobby-Joe; Jorgensen, Paul; Breitkreutz, Ashton; Tyers, Mike

2001-01-01

We have developed a series of programs, collectively packaged as Array File Maker 4.0 (AFM), that manipulate and manage DNA microarray data. AFM 4.0 is simple to use, applicable to any organism or microarray, and operates within the familiar confines of Microsoft Excel. Given a database of expression ratios, AFM 4.0 generates input files for clustering, helps prepare colored figures and Venn diagrams, and can uncover aneuploidy in yeast microarray data. AFM 4.0 should be especially useful to laboratories that do not have access to specialized commercial or in-house software. PMID:11532221

Fully Automated Complementary DNA Microarray Segmentation using a Novel Fuzzy-based Algorithm.

PubMed

Saberkari, Hamidreza; Bahrami, Sheyda; Shamsi, Mousa; Amoshahy, Mohammad Javad; Ghavifekr, Habib Badri; Sedaaghi, Mohammad Hossein

2015-01-01

DNA microarray is a powerful approach to study simultaneously, the expression of 1000 of genes in a single experiment. The average value of the fluorescent intensity could be calculated in a microarray experiment. The calculated intensity values are very close in amount to the levels of expression of a particular gene. However, determining the appropriate position of every spot in microarray images is a main challenge, which leads to the accurate classification of normal and abnormal (cancer) cells. In this paper, first a preprocessing approach is performed to eliminate the noise and artifacts available in microarray cells using the nonlinear anisotropic diffusion filtering method. Then, the coordinate center of each spot is positioned utilizing the mathematical morphology operations. Finally, the position of each spot is exactly determined through applying a novel hybrid model based on the principle component analysis and the spatial fuzzy c-means clustering (SFCM) algorithm. Using a Gaussian kernel in SFCM algorithm will lead to improving the quality in complementary DNA microarray segmentation. The performance of the proposed algorithm has been evaluated on the real microarray images, which is available in Stanford Microarray Databases. Results illustrate that the accuracy of microarray cells segmentation in the proposed algorithm reaches to 100% and 98% for noiseless/noisy cells, respectively.

Where statistics and molecular microarray experiments biology meet.

PubMed

Kelmansky, Diana M

2013-01-01

This review chapter presents a statistical point of view to microarray experiments with the purpose of understanding the apparent contradictions that often appear in relation to their results. We give a brief introduction of molecular biology for nonspecialists. We describe microarray experiments from their construction and the biological principles the experiments rely on, to data acquisition and analysis. The role of epidemiological approaches and sample size considerations are also discussed.

Self-directed student research through analysis of microarray datasets: a computer-based functional genomics practical class for masters-level students.

PubMed

Grenville-Briggs, Laura J; Stansfield, Ian

2011-01-01

This report describes a linked series of Masters-level computer practical workshops. They comprise an advanced functional genomics investigation, based upon analysis of a microarray dataset probing yeast DNA damage responses. The workshops require the students to analyse highly complex transcriptomics datasets, and were designed to stimulate active learning through experience of current research methods in bioinformatics and functional genomics. They seek to closely mimic a realistic research environment, and require the students first to propose research hypotheses, then test those hypotheses using specific sections of the microarray dataset. The complexity of the microarray data provides students with the freedom to propose their own unique hypotheses, tested using appropriate sections of the microarray data. This research latitude was highly regarded by students and is a strength of this practical. In addition, the focus on DNA damage by radiation and mutagenic chemicals allows them to place their results in a human medical context, and successfully sparks broad interest in the subject material. In evaluation, 79% of students scored the practical workshops on a five-point scale as 4 or 5 (totally effective) for student learning. More broadly, the general use of microarray data as a "student research playground" is also discussed. Copyright © 2011 Wiley Periodicals, Inc.

Correlation of gene expression and contaminat concentrations in wild largescale suckers: a field-based study

USGS Publications Warehouse

Christiansen, Helena E.; Mehinto, Alvine C.; Yu, Fahong; Perry, Russell W.; Denslow, Nancy D.; Maule, Alec G.; Mesa, Matthew G.

2014-01-01

Toxic compounds such as organochlorine pesticides (OCs), polychlorinated biphenyls (PCBs), and polybrominated diphenyl ether flame retardants (PBDEs) have been detected in fish, birds, and aquatic mammals that live in the Columbia River or use food resources from within the river. We developed a custom microarray for largescale suckers (Catostomus macrocheilus) and used it to investigate the molecular effects of contaminant exposure on wild fish in the Columbia River. Using Significance Analysis of Microarrays (SAM) we identified 72 probes representing 69 unique genes with expression patterns that correlated with hepatic tissue levels of OCs, PCBs, or PBDEs. These genes were involved in many biological processes previously shown to respond to contaminant exposure, including drug and lipid metabolism, apoptosis, cellular transport, oxidative stress, and cellular chaperone function. The relation between gene expression and contaminant concentration suggests that these genes may respond to environmental contaminant exposure and are promising candidates for further field and laboratory studies to develop biomarkers for monitoring exposure of wild fish to contaminant mixtures found in the Columbia River Basin. The array developed in this study could also be a useful tool for studies involving endangered sucker species and other sucker species used in contaminant research.

Quantitative Proteomics Analysis of the cAMP/Protein Kinase A Signaling Pathway

PubMed Central

2012-01-01

To define the proteins whose expression is regulated by cAMP and protein kinase A (PKA), we used a quantitative proteomics approach in studies of wild-type (WT) and kin- (PKA-null) S49 murine T lymphoma cells. We also compared the impact of endogenous increases in the level of cAMP [by forskolin (Fsk) and the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX)] or by a cAMP analogue (8-CPT-cAMP). We identified 1056 proteins in WT and kin- S49 cells and found that 8-CPT-cAMP and Fsk with IBMX produced differences in protein expression. WT S49 cells had a correlation coefficient of 0.41 between DNA microarray data and the proteomics analysis in cells incubated with 8-CPT-cAMP for 24 h and a correlation coefficient of 0.42 between the DNA microarray data obtained at 6 h and the changes in protein expression after incubation with 8-CPT-cAMP for 24 h. Glutathione reductase (Gsr) had a higher level of basal expression in kin- S49 cells than in WT cells. Consistent with this finding, kin- cells are less sensitive to cell killing and generation of malondialdehyde than are WT cells incubated with H2O2. Cyclic AMP acting via PKA thus has a broad impact on protein expression in mammalian cells, including in the regulation of Gsr and oxidative stress. PMID:23110364

Changes in trophoblasts gene expression in response to perchlorate exposition.

PubMed

la Peña Sol, De; Isela, Santiago-Roque; Zendy, Olivo-Vidal; Mónica, Navarro-Meza; Irene, Xochihua-Rosas; Omar, Arroyo-Helguera

2018-04-17

Contaminated water with chlorates is a public health problem associated with iodine deficiency. Epidemiological evidence shows that iodine deficiency is a risk factor for preeclampsia (PE). In this study we use human BeWo trophoblast cells exposed to perchlorate (KClO 4 ) and changes in gene expression were analyzed by microarrays, quantitative RT-PCR (qRT-PCR) and immunoblot. The microarray analysis identified 48 transcripts up-regulated and 112 down-regulated in comparison with non-exposed trophoblast. The qRT-PCR analysis confirmed changes in GAS7, PKP2, Emilin, Dynatic 3, protocadherins 11, 15, gamma A12, EGFR, SAFB1, ACE2, ANXA2, Apoliprotein E, SREBF1, and C/EBP-β. KClO 4 exposition decreased the mRNA and protein of C/EBP-β and GPX4. Also, we observed a nuclear translocation of HIF1α protein, and increase in both Snail and ACE2 protein by immunoblot. These effects were accompanied by an increases in ROS and nitric oxide. In conclusion, our results show that exposure to KClO 4 alters genes involved in migration, adhesion, differentiation, and correlate with the increase of oxidative stress and nitric oxide production in trophoblast cells. It is possible that iodine deficiency is associated with these processes. However, further studies are required to corroborate the role of iodine in trophoblast cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Identification and handling of artifactual gene expression profiles emerging in microarray hybridization experiments

PubMed Central

Brodsky, Leonid; Leontovich, Andrei; Shtutman, Michael; Feinstein, Elena

2004-01-01

Mathematical methods of analysis of microarray hybridizations deal with gene expression profiles as elementary units. However, some of these profiles do not reflect a biologically relevant transcriptional response, but rather stem from technical artifacts. Here, we describe two technically independent but rationally interconnected methods for identification of such artifactual profiles. Our diagnostics are based on detection of deviations from uniformity, which is assumed as the main underlying principle of microarray design. Method 1 is based on detection of non-uniformity of microarray distribution of printed genes that are clustered based on the similarity of their expression profiles. Method 2 is based on evaluation of the presence of gene-specific microarray spots within the slides’ areas characterized by an abnormal concentration of low/high differential expression values, which we define as ‘patterns of differentials’. Applying two novel algorithms, for nested clustering (method 1) and for pattern detection (method 2), we can make a dual estimation of the profile’s quality for almost every printed gene. Genes with artifactual profiles detected by method 1 may then be removed from further analysis. Suspicious differential expression values detected by method 2 may be either removed or weighted according to the probabilities of patterns that cover them, thus diminishing their input in any further data analysis. PMID:14999086

Novel calibration tools and validation concepts for microarray-based platforms used in molecular diagnostics and food safety control.

PubMed

Brunner, C; Hoffmann, K; Thiele, T; Schedler, U; Jehle, H; Resch-Genger, U

2015-04-01

Commercial platforms consisting of ready-to-use microarrays printed with target-specific DNA probes, a microarray scanner, and software for data analysis are available for different applications in medical diagnostics and food analysis, detecting, e.g., viral and bacteriological DNA sequences. The transfer of these tools from basic research to routine analysis, their broad acceptance in regulated areas, and their use in medical practice requires suitable calibration tools for regular control of instrument performance in addition to internal assay controls. Here, we present the development of a novel assay-adapted calibration slide for a commercialized DNA-based assay platform, consisting of precisely arranged fluorescent areas of various intensities obtained by incorporating different concentrations of a "green" dye and a "red" dye in a polymer matrix. These dyes present "Cy3" and "Cy5" analogues with improved photostability, chosen based upon their spectroscopic properties closely matching those of common labels for the green and red channel of microarray scanners. This simple tool allows to efficiently and regularly assess and control the performance of the microarray scanner provided with the biochip platform and to compare different scanners. It will be eventually used as fluorescence intensity scale for referencing of assays results and to enhance the overall comparability of diagnostic tests.

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

AccuTyping: new algorithms for automated analysis of data from high-throughput genotyping with oligonucleotide microarrays

PubMed Central

Hu, Guohong; Wang, Hui-Yun; Greenawalt, Danielle M.; Azaro, Marco A.; Luo, Minjie; Tereshchenko, Irina V.; Cui, Xiangfeng; Yang, Qifeng; Gao, Richeng; Shen, Li; Li, Honghua

2006-01-01

Microarray-based analysis of single nucleotide polymorphisms (SNPs) has many applications in large-scale genetic studies. To minimize the influence of experimental variation, microarray data usually need to be processed in different aspects including background subtraction, normalization and low-signal filtering before genotype determination. Although many algorithms are sophisticated for these purposes, biases are still present. In the present paper, new algorithms for SNP microarray data analysis and the software, AccuTyping, developed based on these algorithms are described. The algorithms take advantage of a large number of SNPs included in each assay, and the fact that the top and bottom 20% of SNPs can be safely treated as homozygous after sorting based on their ratios between the signal intensities. These SNPs are then used as controls for color channel normalization and background subtraction. Genotype calls are made based on the logarithms of signal intensity ratios using two cutoff values, which were determined after training the program with a dataset of ∼160 000 genotypes and validated by non-microarray methods. AccuTyping was used to determine >300 000 genotypes of DNA and sperm samples. The accuracy was shown to be >99%. AccuTyping can be downloaded from . PMID:16982644

Isolation, structural analysis, and expression characteristics of the maize TIFY gene family.

PubMed

Zhang, Zhongbao; Li, Xianglong; Yu, Rong; Han, Meng; Wu, Zhongyi

2015-10-01

TIFY, previously known as ZIM, comprises a plant-specific family annotated as transcription factors that might play important roles in stress response. Despite TIFY proteins have been reported in Arabidopsis and rice, a comprehensive and systematic survey of ZmTIFY genes has not yet been conducted. To investigate the functions of ZmTIFY genes in this family, we isolated and characterized 30 ZmTIFY (1 TIFY, 3 ZML, and 26 JAZ) genes in an analysis of the maize (Zea mays L.) genome in this study. The 30 ZmTIFY genes were distributed over eight chromosomes. Multiple alignment and motif display results indicated that all ZmTIFY proteins share two conserved TIFY and Jas domains. Phylogenetic analysis revealed that the ZmTIFY family could be divided into two groups. Putative cis-elements, involved in abiotic stress response, phytohormones, pollen grain, and seed development, were detected in the promoters of maize TIFY genes. Microarray data showed that the ZmTIFY genes had tissue-specific expression patterns in various maize developmental stages and in response to biotic and abiotic stresses. The results indicated that ZmTIFY4, 5, 8, 26, and 28 were induced, while ZmTIFY16, 13, 24, 27, 18, and 30 were suppressed, by drought stress in the maize inbred lines Han21 and Ye478. ZmTIFY1, 19, and 28 were upregulated after infection by three pathogens, whereas ZmTIFY4, 13, 21, 23, 24, and 26 were suppressed. These results indicate that the ZmTIFY family may have vital roles in response to abiotic and biotic stresses. The data presented in this work provide vital clues for further investigating the functions of the genes in the ZmTIFY family.

The statistics of identifying differentially expressed genes in Expresso and TM4: a comparison

PubMed Central

Sioson, Allan A; Mane, Shrinivasrao P; Li, Pinghua; Sha, Wei; Heath, Lenwood S; Bohnert, Hans J; Grene, Ruth

2006-01-01

Background Analysis of DNA microarray data takes as input spot intensity measurements from scanner software and returns differential expression of genes between two conditions, together with a statistical significance assessment. This process typically consists of two steps: data normalization and identification of differentially expressed genes through statistical analysis. The Expresso microarray experiment management system implements these steps with a two-stage, log-linear ANOVA mixed model technique, tailored to individual experimental designs. The complement of tools in TM4, on the other hand, is based on a number of preset design choices that limit its flexibility. In the TM4 microarray analysis suite, normalization, filter, and analysis methods form an analysis pipeline. TM4 computes integrated intensity values (IIV) from the average intensities and spot pixel counts returned by the scanner software as input to its normalization steps. By contrast, Expresso can use either IIV data or median intensity values (MIV). Here, we compare Expresso and TM4 analysis of two experiments and assess the results against qRT-PCR data. Results The Expresso analysis using MIV data consistently identifies more genes as differentially expressed, when compared to Expresso analysis with IIV data. The typical TM4 normalization and filtering pipeline corrects systematic intensity-specific bias on a per microarray basis. Subsequent statistical analysis with Expresso or a TM4 t-test can effectively identify differentially expressed genes. The best agreement with qRT-PCR data is obtained through the use of Expresso analysis and MIV data. Conclusion The results of this research are of practical value to biologists who analyze microarray data sets. The TM4 normalization and filtering pipeline corrects microarray-specific systematic bias and complements the normalization stage in Expresso analysis. The results of Expresso using MIV data have the best agreement with qRT-PCR results. In one experiment, MIV is a better choice than IIV as input to data normalization and statistical analysis methods, as it yields as greater number of statistically significant differentially expressed genes; TM4 does not support the choice of MIV input data. Overall, the more flexible and extensive statistical models of Expresso achieve more accurate analytical results, when judged by the yardstick of qRT-PCR data, in the context of an experimental design of modest complexity. PMID:16626497

Diagnostic Yield of Chromosomal Microarray Analysis in an Autism Primary Care Practice: Which Guidelines to Implement?

ERIC Educational Resources Information Center

McGrew, Susan G.; Peters, Brittany R.; Crittendon, Julie A.; Veenstra-VanderWeele, Jeremy

2012-01-01

Genetic testing is recommended for patients with ASD; however specific recommendations vary by specialty. American Academy of Pediatrics and American Academy of Neurology guidelines recommend G-banded karyotype and Fragile X DNA. The American College of Medical Genetics recommends Chromosomal Microarray Analysis (CMA). We determined the yield of…

Self-Directed Student Research through Analysis of Microarray Datasets: A Computer-Based Functional Genomics Practical Class for Masters-Level Students

ERIC Educational Resources Information Center

Grenville-Briggs, Laura J.; Stansfield, Ian

2011-01-01

This report describes a linked series of Masters-level computer practical workshops. They comprise an advanced functional genomics investigation, based upon analysis of a microarray dataset probing yeast DNA damage responses. The workshops require the students to analyse highly complex transcriptomics datasets, and were designed to stimulate…

A MICROARRAY ANALYSIS OF GENE EXPRESSION IN THE EMBRYONIC FORELIMB OF THE C57BL/6J MOUSE REVEALS SIGNIFICANT ALTERATIONS METABOLIC AND DEVELOPMENTAL REGULATION FOLLOWING ETHANOL EXPOSURE.

EPA Science Inventory

The observation of transcriptional changes following embryonic ethanol exposure may provide significant insights into the biological response to ethanol exposure. In this study, we used microarray analysis to examine the transcriptional response of the developing limb to a dose ...

Pathway modeling of microarray data: A case study of pathway activity changes in the testis following in utero exposure to dibutyl phthalate (DBP)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ovacik, Meric A.; Sen, Banalata; Euling, Susan Y.

Pathway activity level analysis, the approach pursued in this study, focuses on all genes that are known to be members of metabolic and signaling pathways as defined by the KEGG database. The pathway activity level analysis entails singular value decomposition (SVD) of the expression data of the genes constituting a given pathway. We explore an extension of the pathway activity methodology for application to time-course microarray data. We show that pathway analysis enhances our ability to detect biologically relevant changes in pathway activity using synthetic data. As a case study, we apply the pathway activity level formulation coupled with significancemore » analysis to microarray data from two different rat testes exposed in utero to Dibutyl Phthalate (DBP). In utero DBP exposure in the rat results in developmental toxicity of a number of male reproductive organs, including the testes. One well-characterized mode of action for DBP and the male reproductive developmental effects is the repression of expression of genes involved in cholesterol transport, steroid biosynthesis and testosterone synthesis that lead to a decreased fetal testicular testosterone. Previous analyses of DBP testes microarray data focused on either individual gene expression changes or changes in the expression of specific genes that are hypothesized, or known, to be important in testicular development and testosterone synthesis. However, a pathway analysis may inform whether there are additional affected pathways that could inform additional modes of action linked to DBP developmental toxicity. We show that Pathway activity analysis may be considered for a more comprehensive analysis of microarray data.« less

Inflammatory Pathways in Parkinson's Disease; A BNE Microarray Study

PubMed Central

Durrenberger, Pascal. F.; Grünblatt, Edna; Fernando, Francesca S.; Monoranu, Camelia Maria; Evans, Jordan; Riederer, Peter; Reynolds, Richard; Dexter, David T.

2012-01-01

The aetiology of Parkinson's disease (PD) is yet to be fully understood but it is becoming more and more evident that neuronal cell death may be multifactorial in essence. The main focus of PD research is to better understand substantia nigra homeostasis disruption, particularly in relation to the wide-spread deposition of the aberrant protein α-synuclein. Microarray technology contributed towards PD research with several studies to date and one gene, ALDH1A1 (Aldehyde dehydrogenase 1 family, member A1), consistently reappeared across studies including the present study, highlighting dopamine (DA) metabolism dysfunction resulting in oxidative stress and most probably leading to neuronal cell death. Neuronal cell death leads to increased inflammation through the activation of astrocytes and microglia. Using our dataset, we aimed to isolate some of these pathways so to offer potential novel neuroprotective therapeutic avenues. To that effect our study has focused on the upregulation of P2X7 (purinergic receptor P2X, ligand-gated ion channel, 7) receptor pathway (microglial activation) and on the NOS3 (nitric oxide synthase 3) pathway (angiogenesis). In summary, although the exact initiator of striatal DA neuronal cell death remains to be determined, based on our analysis, this event does not remain without consequence. Extracellular ATP and reactive astrocytes appear to be responsible for the activation of microglia which in turn release proinflammatory cytokines contributing further to the parkinsonian condition. In addition to tackling oxidative stress pathways we also suggest to reduce microglial and endothelial activation to support neuronal outgrowth. PMID:22548201

Effects of oral contraception with ethinylestradiol and drospirenone on oxidative stress in women 18-35 years old.

PubMed

De Groote, Donat; Perrier d'Hauterive, Sophie; Pintiaux, Axelle; Balteau, Bénédicte; Gerday, Colette; Claesen, Jürgen; Foidart, Jean-Michel

2009-08-01

Oral contraceptives (OCs) with estrogens and progestins may affect oxidative stress (OS) status. A group of 32 women using oral contraceptives (OCU) containing 0.03 mg ethinylestradiol and 3 mg drospirenone have been compared to a matched control group of 30 noncontraception users (NCU). Blood levels of antioxidants, trace elements and markers of lipid peroxidation were assessed by biochemical methods. A microarray analysis of whole blood mRNA levels of 200 genes involved in OS-dependant pathway was also performed. Levels of zinc, vitamin E and antibodies to oxidized low-density lipoproteins (LDLs) were not significantly different between the two groups. On the other hand, significant increases in the mean levels of lipid peroxides (+176%, p<.001), oxidized LDLs (+145%, p<.002), copper (+103%, p<.001), Cu/Zn ratio (+100%, p<.001) and a significant decrease in the mean level of beta-carotene (-41%, p<.01) were observed in the OCU compared to NCU. There was a highly significant positive correlation between the lipid peroxide levels and the copper-to-zinc ratio. From the 200 genes tested by microarray, one coding for HSP70 was significantly up-regulated (log(2) fold change=+ 0.45, p<.02) and one coding for inducible nitric oxide synthase significantly down-regulated (log(2) fold change=-0.24, p<.05) in the OCU compared to the NCU. The recently introduced combination of ethinylestradiol and drospirenone induced the heightening of lipid peroxidation correlated with high levels of copper, a situation that could be associated with increased cardiovascular risk.

Microarray analysis identifies Salmonella genes belonging to the low-shear modeled microgravity regulon

PubMed Central

Wilson, James W.; Ramamurthy, Rajee; Porwollik, Steffen; McClelland, Michael; Hammond, Timothy; Allen, Pat; Ott, C. Mark; Pierson, Duane L.; Nickerson, Cheryl A.

2002-01-01

The low-shear environment of optimized rotation suspension culture allows both eukaryotic and prokaryotic cells to assume physiologically relevant phenotypes that have led to significant advances in fundamental investigations of medical and biological importance. This culture environment has also been used to model microgravity for ground-based studies regarding the impact of space flight on eukaryotic and prokaryotic physiology. We have previously demonstrated that low-shear modeled microgravity (LSMMG) under optimized rotation suspension culture is a novel environmental signal that regulates the virulence, stress resistance, and protein expression levels of Salmonella enterica serovar Typhimurium. However, the mechanisms used by the cells of any species, including Salmonella, to sense and respond to LSMMG and identities of the genes involved are unknown. In this study, we used DNA microarrays to elucidate the global transcriptional response of Salmonella to LSMMG. When compared with identical growth conditions under normal gravity (1 × g), LSMMG differentially regulated the expression of 163 genes distributed throughout the chromosome, representing functionally diverse groups including transcriptional regulators, virulence factors, lipopolysaccharide biosynthetic enzymes, iron-utilization enzymes, and proteins of unknown function. Many of the LSMMG-regulated genes were organized in clusters or operons. The microarray results were further validated by RT-PCR and phenotypic analyses, and they indicate that the ferric uptake regulator is involved in the LSMMG response. The results provide important insight about the Salmonella LSMMG response and could provide clues for the functioning of known Salmonella virulence systems or the identification of uncharacterized bacterial virulence strategies. PMID:12370447

Analyses of Catharanthus roseus and Arabidopsis thaliana WRKY transcription factors reveal involvement in jasmonate signaling.

PubMed

Schluttenhofer, Craig; Pattanaik, Sitakanta; Patra, Barunava; Yuan, Ling

2014-06-20

To combat infection to biotic stress plants elicit the biosynthesis of numerous natural products, many of which are valuable pharmaceutical compounds. Jasmonate is a central regulator of defense response to pathogens and accumulation of specialized metabolites. Catharanthus roseus produces a large number of terpenoid indole alkaloids (TIAs) and is an excellent model for understanding the regulation of this class of valuable compounds. Recent work illustrates a possible role for the Catharanthus WRKY transcription factors (TFs) in regulating TIA biosynthesis. In Arabidopsis and other plants, the WRKY TF family is also shown to play important role in controlling tolerance to biotic and abiotic stresses, as well as secondary metabolism. Here, we describe the WRKY TF families in response to jasmonate in Arabidopsis and Catharanthus. Publically available Arabidopsis microarrays revealed at least 30% (22 of 72) of WRKY TFs respond to jasmonate treatments. Microarray analysis identified at least six jasmonate responsive Arabidopsis WRKY genes (AtWRKY7, AtWRKY20, AtWRKY26, AtWRKY45, AtWRKY48, and AtWRKY72) that have not been previously reported. The Catharanthus WRKY TF family is comprised of at least 48 members. Phylogenetic clustering reveals 11 group I, 32 group II, and 5 group III WRKY TFs. Furthermore, we found that at least 25% (12 of 48) were jasmonate responsive, and 75% (9 of 12) of the jasmonate responsive CrWRKYs are orthologs of AtWRKYs known to be regulated by jasmonate. Overall, the CrWRKY family, ascertained from transcriptome sequences, contains approximately 75% of the number of WRKYs found in other sequenced asterid species (pepper, tomato, potato, and bladderwort). Microarray and transcriptomic data indicate that expression of WRKY TFs in Arabidopsis and Catharanthus are under tight spatio-temporal and developmental control, and potentially have a significant role in jasmonate signaling. Profiling of CrWRKY expression in response to jasmonate treatment revealed potential associations with secondary metabolism. This study provides a foundation for further characterization of WRKY TFs in jasmonate responses and regulation of natural product biosynthesis.

Genome-Wide Survey on Genomic Variation, Expression Divergence, and Evolution in Two Contrasting Rice Genotypes under High Salinity Stress

PubMed Central

Jiang, Shu-Ye; Ma, Ali; Ramamoorthy, Rengasamy; Ramachandran, Srinivasan

2013-01-01

Expression profiling is one of the most important tools for dissecting biological functions of genes and the upregulation or downregulation of gene expression is sufficient for recreating phenotypic differences. Expression divergence of genes significantly contributes to phenotypic variations. However, little is known on the molecular basis of expression divergence and evolution among rice genotypes with contrasting phenotypes. In this study, we have implemented an integrative approach using bioinformatics and experimental analyses to provide insights into genomic variation, expression divergence, and evolution between salinity-sensitive rice variety Nipponbare and tolerant rice line Pokkali under normal and high salinity stress conditions. We have detected thousands of differentially expressed genes between these two genotypes and thousands of up- or downregulated genes under high salinity stress. Many genes were first detected with expression evidence using custom microarray analysis. Some gene families were preferentially regulated by high salinity stress and might play key roles in stress-responsive biological processes. Genomic variations in promoter regions resulted from single nucleotide polymorphisms, indels (1–10 bp of insertion/deletion), and structural variations significantly contributed to the expression divergence and regulation. Our data also showed that tandem and segmental duplication, CACTA and hAT elements played roles in the evolution of gene expression divergence and regulation between these two contrasting genotypes under normal or high salinity stress conditions. PMID:24121498

CNV-ROC: A cost effective, computer-aided analytical performance evaluator of chromosomal microarrays.

PubMed

Goodman, Corey W; Major, Heather J; Walls, William D; Sheffield, Val C; Casavant, Thomas L; Darbro, Benjamin W

2015-04-01

Chromosomal microarrays (CMAs) are routinely used in both research and clinical laboratories; yet, little attention has been given to the estimation of genome-wide true and false negatives during the assessment of these assays and how such information could be used to calibrate various algorithmic metrics to improve performance. Low-throughput, locus-specific methods such as fluorescence in situ hybridization (FISH), quantitative PCR (qPCR), or multiplex ligation-dependent probe amplification (MLPA) preclude rigorous calibration of various metrics used by copy number variant (CNV) detection algorithms. To aid this task, we have established a comparative methodology, CNV-ROC, which is capable of performing a high throughput, low cost, analysis of CMAs that takes into consideration genome-wide true and false negatives. CNV-ROC uses a higher resolution microarray to confirm calls from a lower resolution microarray and provides for a true measure of genome-wide performance metrics at the resolution offered by microarray testing. CNV-ROC also provides for a very precise comparison of CNV calls between two microarray platforms without the need to establish an arbitrary degree of overlap. Comparison of CNVs across microarrays is done on a per-probe basis and receiver operator characteristic (ROC) analysis is used to calibrate algorithmic metrics, such as log2 ratio threshold, to enhance CNV calling performance. CNV-ROC addresses a critical and consistently overlooked aspect of analytical assessments of genome-wide techniques like CMAs which is the measurement and use of genome-wide true and false negative data for the calculation of performance metrics and comparison of CNV profiles between different microarray experiments. Copyright © 2015 Elsevier Inc. All rights reserved.

Mining microarrays for metabolic meaning: nutritional regulation of hypothalamic gene expression.

PubMed

Mobbs, Charles V; Yen, Kelvin; Mastaitis, Jason; Nguyen, Ha; Watson, Elizabeth; Wurmbach, Elisa; Sealfon, Stuart C; Brooks, Andrew; Salton, Stephen R J

2004-06-01

DNA microarray analysis has been used to investigate relative changes in the level of gene expression in the CNS, including changes that are associated with disease, injury, psychiatric disorders, drug exposure or withdrawal, and memory formation. We have used oligonucleotide microarrays to identify hypothalamic genes that respond to nutritional manipulation. In addition to commonly used microarray analysis based on criteria such as fold-regulation, we have also found that simply carrying out multiple t tests then sorting by P value constitutes a highly reliable method to detect true regulation, as assessed by real-time polymerase chain reaction (PCR), even for relatively low abundance genes or relatively low magnitude of regulation. Such analyses directly suggested novel mechanisms that mediate effects of nutritional state on neuroendocrine function and are being used to identify regulated gene products that may elucidate the metabolic pathology of obese ob/ob, lean Vgf-/Vgf-, and other models with profound metabolic impairments.

Parallel human genome analysis: microarray-based expression monitoring of 1000 genes.

PubMed Central

Schena, M; Shalon, D; Heller, R; Chai, A; Brown, P O; Davis, R W

1996-01-01

Microarrays containing 1046 human cDNAs of unknown sequence were printed on glass with high-speed robotics. These 1.0-cm2 DNA "chips" were used to quantitatively monitor differential expression of the cognate human genes using a highly sensitive two-color hybridization assay. Array elements that displayed differential expression patterns under given experimental conditions were characterized by sequencing. The identification of known and novel heat shock and phorbol ester-regulated genes in human T cells demonstrates the sensitivity of the assay. Parallel gene analysis with microarrays provides a rapid and efficient method for large-scale human gene discovery. Images Fig. 1 Fig. 2 Fig. 3 PMID:8855227

Microarray Data Processing Techniques for Genome-Scale Network Inference from Large Public Repositories.

PubMed

Chockalingam, Sriram; Aluru, Maneesha; Aluru, Srinivas

2016-09-19

Pre-processing of microarray data is a well-studied problem. Furthermore, all popular platforms come with their own recommended best practices for differential analysis of genes. However, for genome-scale network inference using microarray data collected from large public repositories, these methods filter out a considerable number of genes. This is primarily due to the effects of aggregating a diverse array of experiments with different technical and biological scenarios. Here we introduce a pre-processing pipeline suitable for inferring genome-scale gene networks from large microarray datasets. We show that partitioning of the available microarray datasets according to biological relevance into tissue- and process-specific categories significantly extends the limits of downstream network construction. We demonstrate the effectiveness of our pre-processing pipeline by inferring genome-scale networks for the model plant Arabidopsis thaliana using two different construction methods and a collection of 11,760 Affymetrix ATH1 microarray chips. Our pre-processing pipeline and the datasets used in this paper are made available at http://alurulab.cc.gatech.edu/microarray-pp.

The regulatory network of ThbZIP1 in response to abscisic acid treatment

PubMed Central

Ji, Xiaoyu; Liu, Guifeng; Liu, Yujia; Nie, Xianguang; Zheng, Lei; Wang, Yucheng

2015-01-01

Previously, a bZIP transcription factor from Tamarix hispida, ThbZIP1, was characterized: plants overexpressing ThbZIP1 displayed improved salt stress tolerance but were sensitive to abscisic acid (ABA). In the current study, we further characterized the regulatory network of ThbZIP1 and the mechanism of ABA sensitivity mediated by ThbZIP1. An ABF transcription factor from T. hispida, ThABF1, directly regulates the expression of ThbZIP1. Microarray analysis identified 1662 and 1609 genes that were respectively significantly upregulated or downregulated by ThbZIP1 when exposed to ABA. Gene ontology (GO) analysis showed that the processes including “response to stimulus,” “catalytic activity,” “binding function,” and “metabolic process” were highly altered in ThbZIP1 expressing plants exposed to ABA. The gene expression in ThbZIP1 transformed plants were compared between exposed to ABA and salt on the genome scale. Genes differentially regulated by both salt and ABA treatment only accounted for 9.75% of total differentially regulated genes. GO analysis showed that structural molecule activity, organelle part, membrane-enclosed lumen, reproduction, and reproductive process are enhanced by ABA but inhibited by salt stress. Conversely, immune system and multi-organism process were improved by salt but inhibited by ABA. Transcription regulator activity, enzyme regulator activity, and developmental process were significantly altered by ABA but were not affected by salt stress. Our study provides insights into how ThbZIP1 mediates ABA and salt stress response at the molecular level. PMID:25713576

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data.

PubMed

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R; Del Río-Navarro, Blanca E; Mendoza-Vargas, Alfredo; Sánchez, Filiberto; Ochoa-Leyva, Adrian

2017-01-01

In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6-10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments.

Molecular Insights on Post-chemotherapy Retinoblastoma by Microarray Gene Expression Analysis

PubMed Central

Nalini, Venkatesan; Segu, Ramya; Deepa, Perinkulam Ravi; Khetan, Vikas; Vasudevan, Madavan; Krishnakumar, Subramanian

2013-01-01

Purpose Management of Retinoblastoma (RB), a pediatric ocular cancer is limited by drug-resistance and drug-dosage related side effects during chemotherapy. Molecular de-regulation in post-chemotherapy RB tumors was investigated. Materials and Methods cDNA microarray analysis of two post-chemotherapy and one pre-chemotherapy RB tumor tissues was performed, followed by Principle Component Analysis, Gene ontology, Pathway Enrichment analysis and Biological Analysis Network (BAN) modeling. The drug modulation role of two significantly up-regulated genes (p≤0.05) − Ect2 (Epithelial-cell-transforming-sequence-2), and PRAME (preferentially-expressed-Antigen-in-Melanoma) was assessed by qRT-PCR, immunohistochemistry and cell viability assays. Results Differential up-regulation of 1672 genes and down-regulation of 2538 genes was observed in RB tissues (relative to normal adult retina), while 1419 genes were commonly de-regulated between pre-chemotherapy and post- chemotherapy RB. Twenty one key gene ontology categories, pathways, biomarkers and phenotype groups harboring 250 differentially expressed genes were dys-regulated (EZH2, NCoR1, MYBL2, RB1, STAMN1, SYK, JAK1/2, STAT1/2, PLK2/4, BIRC5, LAMN1, Ect2, PRAME and ABCC4). Differential molecular expressions of PRAME and Ect2 in RB tumors with and without chemotherapy were analyzed. There was neither up- regulation of MRP1, nor any significant shift in chemotherapeutic IC50, in PRAME over-expressed versus non-transfected RB cells. Conclusion Cell cycle regulatory genes were dys-regulated post-chemotherapy. Ect2 gene was expressed in response to chemotherapy-induced stress. PRAME does not contribute to drug resistance in RB, yet its nuclear localization and BAN information, points to its possible regulatory role in RB. PMID:24092970

Chipster: user-friendly analysis software for microarray and other high-throughput data.

PubMed

Kallio, M Aleksi; Tuimala, Jarno T; Hupponen, Taavi; Klemelä, Petri; Gentile, Massimiliano; Scheinin, Ilari; Koski, Mikko; Käki, Janne; Korpelainen, Eija I

2011-10-14

The growth of high-throughput technologies such as microarrays and next generation sequencing has been accompanied by active research in data analysis methodology, producing new analysis methods at a rapid pace. While most of the newly developed methods are freely available, their use requires substantial computational skills. In order to enable non-programming biologists to benefit from the method development in a timely manner, we have created the Chipster software. Chipster (http://chipster.csc.fi/) brings a powerful collection of data analysis methods within the reach of bioscientists via its intuitive graphical user interface. Users can analyze and integrate different data types such as gene expression, miRNA and aCGH. The analysis functionality is complemented with rich interactive visualizations, allowing users to select datapoints and create new gene lists based on these selections. Importantly, users can save the performed analysis steps as reusable, automatic workflows, which can also be shared with other users. Being a versatile and easily extendable platform, Chipster can be used for microarray, proteomics and sequencing data. In this article we describe its comprehensive collection of analysis and visualization tools for microarray data using three case studies. Chipster is a user-friendly analysis software for high-throughput data. Its intuitive graphical user interface enables biologists to access a powerful collection of data analysis and integration tools, and to visualize data interactively. Users can collaborate by sharing analysis sessions and workflows. Chipster is open source, and the server installation package is freely available.

Chipster: user-friendly analysis software for microarray and other high-throughput data

PubMed Central

2011-01-01

Background The growth of high-throughput technologies such as microarrays and next generation sequencing has been accompanied by active research in data analysis methodology, producing new analysis methods at a rapid pace. While most of the newly developed methods are freely available, their use requires substantial computational skills. In order to enable non-programming biologists to benefit from the method development in a timely manner, we have created the Chipster software. Results Chipster (http://chipster.csc.fi/) brings a powerful collection of data analysis methods within the reach of bioscientists via its intuitive graphical user interface. Users can analyze and integrate different data types such as gene expression, miRNA and aCGH. The analysis functionality is complemented with rich interactive visualizations, allowing users to select datapoints and create new gene lists based on these selections. Importantly, users can save the performed analysis steps as reusable, automatic workflows, which can also be shared with other users. Being a versatile and easily extendable platform, Chipster can be used for microarray, proteomics and sequencing data. In this article we describe its comprehensive collection of analysis and visualization tools for microarray data using three case studies. Conclusions Chipster is a user-friendly analysis software for high-throughput data. Its intuitive graphical user interface enables biologists to access a powerful collection of data analysis and integration tools, and to visualize data interactively. Users can collaborate by sharing analysis sessions and workflows. Chipster is open source, and the server installation package is freely available. PMID:21999641

A database for the analysis of immunity genes in Drosophila: PADMA database.

PubMed

Lee, Mark J; Mondal, Ariful; Small, Chiyedza; Paddibhatla, Indira; Kawaguchi, Akira; Govind, Shubha

2011-01-01

While microarray experiments generate voluminous data, discerning trends that support an existing or alternative paradigm is challenging. To synergize hypothesis building and testing, we designed the Pathogen Associated Drosophila MicroArray (PADMA) database for easy retrieval and comparison of microarray results from immunity-related experiments (www.padmadatabase.org). PADMA also allows biologists to upload their microarray-results and compare it with datasets housed within PADMA. We tested PADMA using a preliminary dataset from Ganaspis xanthopoda-infected fly larvae, and uncovered unexpected trends in gene expression, reshaping our hypothesis. Thus, the PADMA database will be a useful resource to fly researchers to evaluate, revise, and refine hypotheses.

Enhancing Interdisciplinary Mathematics and Biology Education: A Microarray Data Analysis Course Bridging These Disciplines

ERIC Educational Resources Information Center

Tra, Yolande V.; Evans, Irene M.

2010-01-01

"BIO2010" put forth the goal of improving the mathematical educational background of biology students. The analysis and interpretation of microarray high-dimensional data can be very challenging and is best done by a statistician and a biologist working and teaching in a collaborative manner. We set up such a collaboration and designed a course on…

Diagnostic Yield of Chromosomal Microarray Analysis in a Cohort of Patients with Autism Spectrum Disorders from a Highly Consanguineous Population

ERIC Educational Resources Information Center

Al-Mamari, Watfa; Al-Saegh, Abeer; Al-Kindy, Adila; Bruwer, Zandre; Al-Murshedi, Fathiya; Al-Thihli, Khalid

2015-01-01

Autism Spectrum Disorders are a complicated group of disorders characterized with heterogeneous genetic etiologies. The genetic investigations for this group of disorders have expanded considerably over the past decade. In our study we designed a tired approach and studied the diagnostic yield of chromosomal microarray analysis on patients…

Immunological Targeting of Tumor Initiating Prostate Cancer Cells

DTIC Science & Technology

2014-10-01

clinically using well-accepted immuno-competent animal models. 2) Keywords: Prostate Cancer, Lymphocyte, Vaccine, Antibody 3) Overall Project Summary...castrate animals . Task 1: Identify and verify antigenic targets from CAstrate Resistant Luminal Epithelial Cells (CRLEC) (months 1-16... animals per group will be processed to derive sufficient RNA for microarray analysis; the experiment will be repeated x 3. Microarray analysis will

MiMiR – an integrated platform for microarray data sharing, mining and analysis

PubMed Central

Tomlinson, Chris; Thimma, Manjula; Alexandrakis, Stelios; Castillo, Tito; Dennis, Jayne L; Brooks, Anthony; Bradley, Thomas; Turnbull, Carly; Blaveri, Ekaterini; Barton, Geraint; Chiba, Norie; Maratou, Klio; Soutter, Pat; Aitman, Tim; Game, Laurence

2008-01-01

Background Despite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Microarray data Mining Resource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data. Results A user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package. Conclusion The new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centre's large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies. PMID:18801157

MiMiR--an integrated platform for microarray data sharing, mining and analysis.

PubMed

Tomlinson, Chris; Thimma, Manjula; Alexandrakis, Stelios; Castillo, Tito; Dennis, Jayne L; Brooks, Anthony; Bradley, Thomas; Turnbull, Carly; Blaveri, Ekaterini; Barton, Geraint; Chiba, Norie; Maratou, Klio; Soutter, Pat; Aitman, Tim; Game, Laurence

2008-09-18

Despite considerable efforts within the microarray community for standardising data format, content and description, microarray technologies present major challenges in managing, sharing, analysing and re-using the large amount of data generated locally or internationally. Additionally, it is recognised that inconsistent and low quality experimental annotation in public data repositories significantly compromises the re-use of microarray data for meta-analysis. MiMiR, the Microarray data Mining Resource was designed to tackle some of these limitations and challenges. Here we present new software components and enhancements to the original infrastructure that increase accessibility, utility and opportunities for large scale mining of experimental and clinical data. A user friendly Online Annotation Tool allows researchers to submit detailed experimental information via the web at the time of data generation rather than at the time of publication. This ensures the easy access and high accuracy of meta-data collected. Experiments are programmatically built in the MiMiR database from the submitted information and details are systematically curated and further annotated by a team of trained annotators using a new Curation and Annotation Tool. Clinical information can be annotated and coded with a clinical Data Mapping Tool within an appropriate ethical framework. Users can visualise experimental annotation, assess data quality, download and share data via a web-based experiment browser called MiMiR Online. All requests to access data in MiMiR are routed through a sophisticated middleware security layer thereby allowing secure data access and sharing amongst MiMiR registered users prior to publication. Data in MiMiR can be mined and analysed using the integrated EMAAS open source analysis web portal or via export of data and meta-data into Rosetta Resolver data analysis package. The new MiMiR suite of software enables systematic and effective capture of extensive experimental and clinical information with the highest MIAME score, and secure data sharing prior to publication. MiMiR currently contains more than 150 experiments corresponding to over 3000 hybridisations and supports the Microarray Centre's large microarray user community and two international consortia. The MiMiR flexible and scalable hardware and software architecture enables secure warehousing of thousands of datasets, including clinical studies, from microarray and potentially other -omics technologies.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, G.L.; He, Z.; DeSantis, T.Z.

Microarrays have proven to be a useful and high-throughput method to provide targeted DNA sequence information for up to many thousands of specific genetic regions in a single test. A microarray consists of multiple DNA oligonucleotide probes that, under high stringency conditions, hybridize only to specific complementary nucleic acid sequences (targets). A fluorescent signal indicates the presence and, in many cases, the abundance of genetic regions of interest. In this chapter we will look at how microarrays are used in microbial ecology, especially with the recent increase in microbial community DNA sequence data. Of particular interest to microbial ecologists, phylogeneticmore » microarrays are used for the analysis of phylotypes in a community and functional gene arrays are used for the analysis of functional genes, and, by inference, phylotypes in environmental samples. A phylogenetic microarray that has been developed by the Andersen laboratory, the PhyloChip, will be discussed as an example of a microarray that targets the known diversity within the 16S rRNA gene to determine microbial community composition. Using multiple, confirmatory probes to increase the confidence of detection and a mismatch probe for every perfect match probe to minimize the effect of cross-hybridization by non-target regions, the PhyloChip is able to simultaneously identify any of thousands of taxa present in an environmental sample. The PhyloChip is shown to reveal greater diversity within a community than rRNA gene sequencing due to the placement of the entire gene product on the microarray compared with the analysis of up to thousands of individual molecules by traditional sequencing methods. A functional gene array that has been developed by the Zhou laboratory, the GeoChip, will be discussed as an example of a microarray that dynamically identifies functional activities of multiple members within a community. The recent version of GeoChip contains more than 24,000 50mer oligonucleotide probes and covers more than 10,000 gene sequences in 150 gene categories involved in carbon, nitrogen, sulfur, and phosphorus cycling, metal resistance and reduction, and organic contaminant degradation. GeoChip can be used as a generic tool for microbial community analysis, and also link microbial community structure to ecosystem functioning. Examples of the application of both arrays in different environmental samples will be described in the two subsequent sections.« less

Evaluation of a low density DNA microarray for small B-cell non-Hodgkin lymphoma differential diagnosis.

PubMed

Gillet, Jean-Pierre; Molina, Thierry Jo; Jamart, Jacques; Gaulard, Philippe; Leroy, Karen; Briere, Josette; Theate, Ivan; Thieblemont, Catherine; Bosly, Andre; Herin, Michel; Hamels, Jacques; Remacle, Jose

2009-03-01

Lymphomas are classified according to the World Health Organisation (WHO) classification which defines subtypes on the basis of clinical, morphological, immunophenotypic, molecular and cytogenetic criteria. Differential diagnosis of the subtypes is sometimes difficult, especially for small B-cell lymphoma (SBCL). Standardisation of molecular genetic assays using multiple gene expression analysis by microarrays could be a useful complement to the current diagnosis. The aim of the present study was to develop a low density DNA microarray for the analysis of 107 genes associated with B-cell non-Hodgkin lymphoma and to evaluate its performance in the diagnosis of SBCL. A predictive tool based on Fisher discriminant analysis using a training set of 40 patients including four different subtypes (follicular lymphoma n = 15, mantle cell lymphoma n = 7, B-cell chronic lymphocytic leukemia n = 6 and splenic marginal zone lymphoma n = 12) was designed. A short additional preliminary analysis to gauge the accuracy of this signature was then performed on an external set of nine patients. Using this model, eight of nine of those samples were classified successfully. This pilot study demonstrates that such a microarray tool may be a promising diagnostic approach for small B-cell non-Hodgkin lymphoma.

MAGMA: analysis of two-channel microarrays made easy.

PubMed

Rehrauer, Hubert; Zoller, Stefan; Schlapbach, Ralph

2007-07-01

The web application MAGMA provides a simple and intuitive interface to identify differentially expressed genes from two-channel microarray data. While the underlying algorithms are not superior to those of similar web applications, MAGMA is particularly user friendly and can be used without prior training. The user interface guides the novice user through the most typical microarray analysis workflow consisting of data upload, annotation, normalization and statistical analysis. It automatically generates R-scripts that document MAGMA's entire data processing steps, thereby allowing the user to regenerate all results in his local R installation. The implementation of MAGMA follows the model-view-controller design pattern that strictly separates the R-based statistical data processing, the web-representation and the application logic. This modular design makes the application flexible and easily extendible by experts in one of the fields: statistical microarray analysis, web design or software development. State-of-the-art Java Server Faces technology was used to generate the web interface and to perform user input processing. MAGMA's object-oriented modular framework makes it easily extendible and applicable to other fields and demonstrates that modern Java technology is also suitable for rather small and concise academic projects. MAGMA is freely available at www.magma-fgcz.uzh.ch.

Tomato Expression Database (TED): a suite of data presentation and analysis tools

PubMed Central

Fei, Zhangjun; Tang, Xuemei; Alba, Rob; Giovannoni, James

2006-01-01

The Tomato Expression Database (TED) includes three integrated components. The Tomato Microarray Data Warehouse serves as a central repository for raw gene expression data derived from the public tomato cDNA microarray. In addition to expression data, TED stores experimental design and array information in compliance with the MIAME guidelines and provides web interfaces for researchers to retrieve data for their own analysis and use. The Tomato Microarray Expression Database contains normalized and processed microarray data for ten time points with nine pair-wise comparisons during fruit development and ripening in a normal tomato variety and nearly isogenic single gene mutants impacting fruit development and ripening. Finally, the Tomato Digital Expression Database contains raw and normalized digital expression (EST abundance) data derived from analysis of the complete public tomato EST collection containing >150 000 ESTs derived from 27 different non-normalized EST libraries. This last component also includes tools for the comparison of tomato and Arabidopsis digital expression data. A set of query interfaces and analysis, and visualization tools have been developed and incorporated into TED, which aid users in identifying and deciphering biologically important information from our datasets. TED can be accessed at . PMID:16381976

Tomato Expression Database (TED): a suite of data presentation and analysis tools.

PubMed

Fei, Zhangjun; Tang, Xuemei; Alba, Rob; Giovannoni, James

2006-01-01

The Tomato Expression Database (TED) includes three integrated components. The Tomato Microarray Data Warehouse serves as a central repository for raw gene expression data derived from the public tomato cDNA microarray. In addition to expression data, TED stores experimental design and array information in compliance with the MIAME guidelines and provides web interfaces for researchers to retrieve data for their own analysis and use. The Tomato Microarray Expression Database contains normalized and processed microarray data for ten time points with nine pair-wise comparisons during fruit development and ripening in a normal tomato variety and nearly isogenic single gene mutants impacting fruit development and ripening. Finally, the Tomato Digital Expression Database contains raw and normalized digital expression (EST abundance) data derived from analysis of the complete public tomato EST collection containing >150,000 ESTs derived from 27 different non-normalized EST libraries. This last component also includes tools for the comparison of tomato and Arabidopsis digital expression data. A set of query interfaces and analysis, and visualization tools have been developed and incorporated into TED, which aid users in identifying and deciphering biologically important information from our datasets. TED can be accessed at http://ted.bti.cornell.edu.

Experimental Systems-Biology Approaches for Clostridia-Based Bioenergy Production

DOE Office of Scientific and Technical Information (OSTI.GOV)

Papoutsakis, Elefterios

This is the final project report for project "Experimental Systems-Biology Approaches for Clostridia-Based Bioenergy Production" for the funding period of 9/1/12 to 2/28/2015 (three years with a 6-month no-cost extension) OVERVIEW AND PROJECT GOALS The bottleneck of achieving higher rates and titers of toxic metabolites (such as solvents and carboxylic acids that can used as biofuels or biofuel precursors) can be overcome by engineering the stress response system. Thus, understanding and modeling the response of cells to toxic metabolites is a problem of great fundamental and practical significance. In this project, our goal is to dissect at the molecular systemsmore » level and build models (conceptual and quantitative) for the stress response of C. acetobutylicum (Cac) to its two toxic metabolites: butanol (BuOH) and butyrate (BA). Transcriptional (RNAseq and microarray based), proteomic and fluxomic data and their analysis are key requirements for this goal. Transcriptional data from mid-exponential cultures of Cac under 4 different levels of BuOH and BA stress was obtained using both microarrays (Papoutsakis group) and deep sequencing (RNAseq; Meyers and Papoutsakis groups). These two sets of data do not only serve to validate each other, but are also used for identification of stress-induced changes in transcript levels, small regulatory RNAs, & in transcriptional start sites. Quantitative proteomic data (Lee group), collected using the iTRAQ technology, are essential for understanding of protein levels and turnover under stress and the various protein-protein interactions that orchestrate the stress response. Metabolic flux changes (Antoniewicz group) of core pathways, which provide important information on the re-allocation of energy and carbon resources under metabolite stress, were examined using 13C-labelled chemicals. Omics data are integrated at different levels and scales. At the metabolic-pathway level, omics data are integrated into a 2nd generation genome-scale model (GSM) (Maranas group). Omics data are also integrated using bioinformatics (Wu and Huang group), whereby regulatory details of gene and protein expression, protein-protein interactions and metabolic flux regulation are incorporated. The PI (Papoutsakis) facilitated project integration through monthly meeting and reports, conference calls, and collaborative manuscript preparation. The five groups collaborated extensively and made a large number of presentations in national and international meetings. It has also published several papers, with several more in the preparation stage. Several PhD, MS and postdoctoral students were trained as part of this collaborative and interdisciplinary project.« less

Development and application of a microarray meter tool to optimize microarray experiments

PubMed Central

Rouse, Richard JD; Field, Katrine; Lapira, Jennifer; Lee, Allen; Wick, Ivan; Eckhardt, Colleen; Bhasker, C Ramana; Soverchia, Laura; Hardiman, Gary

2008-01-01

Background Successful microarray experimentation requires a complex interplay between the slide chemistry, the printing pins, the nucleic acid probes and targets, and the hybridization milieu. Optimization of these parameters and a careful evaluation of emerging slide chemistries are a prerequisite to any large scale array fabrication effort. We have developed a 'microarray meter' tool which assesses the inherent variations associated with microarray measurement prior to embarking on large scale projects. Findings The microarray meter consists of nucleic acid targets (reference and dynamic range control) and probe components. Different plate designs containing identical probe material were formulated to accommodate different robotic and pin designs. We examined the variability in probe quality and quantity (as judged by the amount of DNA printed and remaining post-hybridization) using three robots equipped with capillary printing pins. Discussion The generation of microarray data with minimal variation requires consistent quality control of the (DNA microarray) manufacturing and experimental processes. Spot reproducibility is a measure primarily of the variations associated with printing. The microarray meter assesses array quality by measuring the DNA content for every feature. It provides a post-hybridization analysis of array quality by scoring probe performance using three metrics, a) a measure of variability in the signal intensities, b) a measure of the signal dynamic range and c) a measure of variability of the spot morphologies. PMID:18710498

VTCdb: a gene co-expression database for the crop species Vitis vinifera (grapevine).

PubMed

Wong, Darren C J; Sweetman, Crystal; Drew, Damian P; Ford, Christopher M

2013-12-16

Gene expression datasets in model plants such as Arabidopsis have contributed to our understanding of gene function and how a single underlying biological process can be governed by a diverse network of genes. The accumulation of publicly available microarray data encompassing a wide range of biological and environmental conditions has enabled the development of additional capabilities including gene co-expression analysis (GCA). GCA is based on the understanding that genes encoding proteins involved in similar and/or related biological processes may exhibit comparable expression patterns over a range of experimental conditions, developmental stages and tissues. We present an open access database for the investigation of gene co-expression networks within the cultivated grapevine, Vitis vinifera. The new gene co-expression database, VTCdb (http://vtcdb.adelaide.edu.au/Home.aspx), offers an online platform for transcriptional regulatory inference in the cultivated grapevine. Using condition-independent and condition-dependent approaches, grapevine co-expression networks were constructed using the latest publicly available microarray datasets from diverse experimental series, utilising the Affymetrix Vitis vinifera GeneChip (16 K) and the NimbleGen Grape Whole-genome microarray chip (29 K), thus making it possible to profile approximately 29,000 genes (95% of the predicted grapevine transcriptome). Applications available with the online platform include the use of gene names, probesets, modules or biological processes to query the co-expression networks, with the option to choose between Affymetrix or Nimblegen datasets and between multiple co-expression measures. Alternatively, the user can browse existing network modules using interactive network visualisation and analysis via CytoscapeWeb. To demonstrate the utility of the database, we present examples from three fundamental biological processes (berry development, photosynthesis and flavonoid biosynthesis) whereby the recovered sub-networks reconfirm established plant gene functions and also identify novel associations. Together, we present valuable insights into grapevine transcriptional regulation by developing network models applicable to researchers in their prioritisation of gene candidates, for on-going study of biological processes related to grapevine development, metabolism and stress responses.

Gene profiling reveals a role for stress hormones in the molecular and behavioral response to food restriction

PubMed Central

Guarnieri, Douglas J.; Brayton, Catherine E.; Richards, Sarah M.; Maldonado-Aviles, Jaime; Trinko, Joseph R.; Nelson, Jessica; Taylor, Jane R.; Gourley, Shannon L.; DiLeone, Ralph J.

2011-01-01

Background Food restriction is known to enhance learning and motivation. The neural mechanisms underlying these responses likely involve alterations in gene expression in brain regions mediating the motivation to feed. Methods Analysis of gene expression profiles in male C57BL6/J mice using whole-genome microarrays was completed in the medial prefrontal cortex, nucleus accumbens, ventral tegmental area, and the hypothalamus following a five day food restriction. Quantitative PCR was used to validate these findings and determine the time-course of expression changes. Plasma levels of the stress hormone corticosterone (CORT) were measured by ELISA. Expression changes were measured in adrenalectomized animals that underwent food restriction, as well as in animals receiving daily injections of CORT. Progressive ratio responding for food, a measure of motivated behavior, was assessed after CORT treatment in restricted and fed animals. Results Brief food restriction results in an upregulation of peripheral stress responsive genes in the mammalian brain. Time-course analysis demonstrated rapid and persistent expression changes in all four brain regions under study. Administration of CORT to non-restricted animals was sufficient to induce a subset of the genes, and alterations in gene expression after food restriction were dependent on intact adrenal glands. CORT can increase the motivation to work for food only in the restricted state. Conclusions These data demonstrate a central role for CORT in mediating both molecular and behavioral responses to food restriction. The stress hormone-induced alterations in gene expression described here may be relevant for both adaptive and pathological responses to stress. PMID:21855858

Genome-Wide Survey of Cold Stress Regulated Alternative Splicing in Arabidopsis thaliana with Tiling Microarray

PubMed Central

Leviatan, Noam; Alkan, Noam; Leshkowitz, Dena; Fluhr, Robert

2013-01-01

Alternative splicing plays a major role in expanding the potential informational content of eukaryotic genomes. It is an important post-transcriptional regulatory mechanism that can increase protein diversity and affect mRNA stability. Alternative splicing is often regulated in a tissue-specific and stress-responsive manner. Cold stress, which adversely affects plant growth and development, regulates the transcription and splicing of plant splicing factors. This can affect the pre-mRNA processing of many genes. To identify cold regulated alternative splicing we applied Affymetrix Arabidopsis tiling arrays to survey the transcriptome under cold treatment conditions. A novel algorithm was used for detection of statistically relevant changes in intron expression within a transcript between control and cold growth conditions. A reverse transcription polymerase chain reaction (RT-PCR) analysis of a number of randomly selected genes confirmed the changes in splicing patterns under cold stress predicted by tiling array. Our analysis revealed new types of cold responsive genes. While their expression level remains relatively unchanged under cold stress their splicing pattern shows detectable changes in the relative abundance of isoforms. The majority of cold regulated alternative splicing introduced a premature termination codon (PTC) into the transcripts creating potential targets for degradation by the nonsense mediated mRNA decay (NMD) process. A number of these genes were analyzed in NMD-defective mutants by RT-PCR and shown to evade NMD. This may result in new and truncated proteins with altered functions or dominant negative effects. The results indicate that cold affects both quantitative and qualitative aspects of gene expression. PMID:23776682

Transcriptomic characterization of temperature stress responses in larval zebrafish.

PubMed

Long, Yong; Li, Linchun; Li, Qing; He, Xiaozhen; Cui, Zongbin

2012-01-01

Temperature influences nearly all biochemical, physiological and life history activities of fish, but the molecular mechanisms underlying the temperature acclimation remains largely unknown. Previous studies have identified many temperature-regulated genes in adult tissues; however, the transcriptional responses of fish larvae to temperature stress are not well understood. In this study, we characterized the transcriptional responses in larval zebrafish exposed to cold or heat stress using microarray analysis. In comparison with genes expressed in the control at 28 °C, a total of 2680 genes were found to be affected in 96 hpf larvae exposed to cold (16 °C) or heat (34 °C) for 2 and 48h and most of these genes were expressed in a temperature-specific and temporally regulated manner. Bioinformatic analysis identified multiple temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of transcription, nucleosome assembly, chromatin organization and protein folding. However, processes such as RNA processing, cellular metal ion homeostasis and protein transport and were enriched in genes up-regulated under cold exposure for 48 h. Pathways such as mTOR signalling, p53 signalling and circadian rhythm were enriched among cold-induced genes, while adipocytokine signalling, protein export and arginine and praline metabolism were enriched among heat-induced genes. Although most of these biological processes and pathways were specifically regulated by cold or heat, common responses to both cold and heat stresses were also found. Thus, these findings provide new interesting clues for elucidation of mechanisms underlying the temperature acclimation in fish.

High-throughput DNA microarray detection of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley, Nepal.

PubMed

Inoue, Daisuke; Hinoura, Takuji; Suzuki, Noriko; Pang, Junqin; Malla, Rabin; Shrestha, Sadhana; Chapagain, Saroj Kumar; Matsuzawa, Hiroaki; Nakamura, Takashi; Tanaka, Yasuhiro; Ike, Michihiko; Nishida, Kei; Sei, Kazunari

2015-01-01

Because of heavy dependence on groundwater for drinking water and other domestic use, microbial contamination of groundwater is a serious problem in the Kathmandu Valley, Nepal. This study investigated comprehensively the occurrence of pathogenic bacteria in shallow well groundwater in the Kathmandu Valley by applying DNA microarray analysis targeting 941 pathogenic bacterial species/groups. Water quality measurements found significant coliform (fecal) contamination in 10 of the 11 investigated groundwater samples and significant nitrogen contamination in some samples. The results of DNA microarray analysis revealed the presence of 1-37 pathogen species/groups, including 1-27 biosafety level 2 ones, in 9 of the 11 groundwater samples. While the detected pathogens included several feces- and animal-related ones, those belonging to Legionella and Arthrobacter, which were considered not to be directly associated with feces, were detected prevalently. This study could provide a rough picture of overall pathogenic bacterial contamination in the Kathmandu Valley, and demonstrated the usefulness of DNA microarray analysis as a comprehensive screening tool of a wide variety of pathogenic bacteria.

Microarray Analysis of Long Noncoding RNAs in Female Diabetic Peripheral Neuropathy Patients.

PubMed

Luo, Lin; Ji, Lin-Dan; Cai, Jiang-Jia; Feng, Mei; Zhou, Mi; Hu, Su-Pei; Xu, Jin; Zhou, Wen-Hua

2018-01-01

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM). Because of its controversial pathogenesis, DPN is still not diagnosed or managed properly in most patients. In this study, human lncRNA microarrays were used to identify the differentially expressed lncRNAs in DM and DPN patients, and some of the discovered lncRNAs were further validated in additional 78 samples by quantitative realtime PCR (qRT-PCR). The microarray analysis identified 446 and 1327 differentially expressed lncRNAs in DM and DPN, respectively. The KEGG pathway analysis further revealed that the differentially expressed lncRNA-coexpressed mRNAs between DPN and DM groups were significantly enriched in the MAPK signaling pathway. The lncRNA/mRNA coexpression network indicated that BDNF and TRAF2 correlated with 6 lncRNAs. The qRT-PCR confirmed the initial microarray results. These findings demonstrated that the interplay between lncRNAs and mRNA may be involved in the pathogenesis of DPN, especially the neurotrophin-MAPK signaling pathway, thus providing relevant information for future studies. © 2018 The Author(s). Published by S. Karger AG, Basel.

MASQOT: a method for cDNA microarray spot quality control

PubMed Central

Bylesjö, Max; Eriksson, Daniel; Sjödin, Andreas; Sjöström, Michael; Jansson, Stefan; Antti, Henrik; Trygg, Johan

2005-01-01

Background cDNA microarray technology has emerged as a major player in the parallel detection of biomolecules, but still suffers from fundamental technical problems. Identifying and removing unreliable data is crucial to prevent the risk of receiving illusive analysis results. Visual assessment of spot quality is still a common procedure, despite the time-consuming work of manually inspecting spots in the range of hundreds of thousands or more. Results A novel methodology for cDNA microarray spot quality control is outlined. Multivariate discriminant analysis was used to assess spot quality based on existing and novel descriptors. The presented methodology displays high reproducibility and was found superior in identifying unreliable data compared to other evaluated methodologies. Conclusion The proposed methodology for cDNA microarray spot quality control generates non-discrete values of spot quality which can be utilized as weights in subsequent analysis procedures as well as to discard spots of undesired quality using the suggested threshold values. The MASQOT approach provides a consistent assessment of spot quality and can be considered an alternative to the labor-intensive manual quality assessment process. PMID:16223442

Clinical application of chromosomal microarray analysis for the prenatal diagnosis of chromosomal abnormalities and copy number variations in fetuses with congenital heart disease.

PubMed

Xia, Yu; Yang, Yongchao; Huang, Shufang; Wu, Yueheng; Li, Ping; Zhuang, Jian

2018-03-24

This study aimed to determine chromosomal abnormalities and copy number variations (CNVs) in fetuses with congenital heart disease (CHD) by chromosomal microarray analysis (CMA). One hundred and ten cases with CHD detected by prenatal echocardiography were enrolled in the study; 27 cases were simple CHDs, and 83 were complex CHDs. Chromosomal microarray analysis was performed on the Affymetrix CytoScan HD platform. All annotated CNVs were validated by quantitative PCR. Chromosomal microarray analysis identified 6 cases with chromosomal abnormalities, including 2 cases with trisomy 21, 2 cases with trisomy 18, 1 case with trisomy 13, and 1 unusual case of mosaic trisomy 21. Pathogenic CNVs were detected in 15.5% (17/110) of the fetuses with CHDs, including 13 cases with CHD-associated CNVs. We further identified 10 genes as likely novel CHD candidate genes through gene functional enrichment analysis. We also found that pathogenic CMA results impacted the rate of pregnancy termination. This study shows that CMA is particularly effective for identifying chromosomal abnormalities and CNVs in fetuses with CHDs as well as having an effect on obstetrical outcomes. The elucidation of the genetic basis of CHDs will continue to expand our understanding of the etiology of CHDs. © 2018 John Wiley & Sons, Ltd.

An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

PubMed Central

Karsten, Stanislav L.; Van Deerlin, Vivianna M. D.; Sabatti, Chiara; Gill, Lisa H.; Geschwind, Daniel H.

2002-01-01

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling. PMID:11788730

ALT1, a Snf2 Family Chromatin Remodeling ATPase, Negatively Regulates Alkaline Tolerance through Enhanced Defense against Oxidative Stress in Rice

PubMed Central

Guo, Mingxin; Wang, Ruci; Wang, Juan; Hua, Kai; Wang, Yueming; Liu, Xiaoqiang; Yao, Shanguo

2014-01-01

Alkaline salt stress adversely affects rice growth, productivity and grain quality. However, the mechanism underlying this process remains elusive. We characterized here an alkaline tolerant mutant, alt1 in rice. Map-based cloning revealed that alt1 harbors a mutation in a chromatin remodeling ATPase gene. ALT1-RNAi transgenic plants under different genetic background mimicked the alt1 phenotype, exhibiting tolerance to alkaline stress in a transcript dosage-dependent manner. The predicted ALT1 protein belonged to the Ris1 subgroup of the Snf2 family and was localized in the nucleus, and transcription of ALT1 was transiently suppressed after alkaline treatment. Although the absorption of several metal ions maintained well in the mutant under alkaline stress, expression level of the genes involved in metal ions homeostasis was not altered in the alt1 mutant. Classification of differentially expressed abiotic stress related genes, as revealed by microarray analysis, found that the majority (50/78) were involved in ROS production, ROS scavenging, and DNA repair. This finding was further confirmed by that alt1 exhibited lower levels of H2O2 under alkaline stress and tolerance to methyl viologen treatment. Taken together, these results suggest that ALT1 negatively functions in alkaline tolerance mainly through the defense against oxidative damage, and provide a potential two-step strategy for improving the tolerance of rice plants to alkaline stress. PMID:25473841

MIGS-GPU: Microarray Image Gridding and Segmentation on the GPU.

PubMed

Katsigiannis, Stamos; Zacharia, Eleni; Maroulis, Dimitris

2017-05-01

Complementary DNA (cDNA) microarray is a powerful tool for simultaneously studying the expression level of thousands of genes. Nevertheless, the analysis of microarray images remains an arduous and challenging task due to the poor quality of the images that often suffer from noise, artifacts, and uneven background. In this study, the MIGS-GPU [Microarray Image Gridding and Segmentation on Graphics Processing Unit (GPU)] software for gridding and segmenting microarray images is presented. MIGS-GPU's computations are performed on the GPU by means of the compute unified device architecture (CUDA) in order to achieve fast performance and increase the utilization of available system resources. Evaluation on both real and synthetic cDNA microarray images showed that MIGS-GPU provides better performance than state-of-the-art alternatives, while the proposed GPU implementation achieves significantly lower computational times compared to the respective CPU approaches. Consequently, MIGS-GPU can be an advantageous and useful tool for biomedical laboratories, offering a user-friendly interface that requires minimum input in order to run.

The MicroArray Quality Control (MAQC) project shows inter- and intraplatform reproducibility of gene expression measurements

PubMed Central

2012-01-01

Over the last decade, the introduction of microarray technology has had a profound impact on gene expression research. The publication of studies with dissimilar or altogether contradictory results, obtained using different microarray platforms to analyze identical RNA samples, has raised concerns about the reliability of this technology. The MicroArray Quality Control (MAQC) project was initiated to address these concerns, as well as other performance and data analysis issues. Expression data on four titration pools from two distinct reference RNA samples were generated at multiple test sites using a variety of microarray-based and alternative technology platforms. Here we describe the experimental design and probe mapping efforts behind the MAQC project. We show intraplatform consistency across test sites as well as a high level of interplatform concordance in terms of genes identified as differentially expressed. This study provides a resource that represents an important first step toward establishing a framework for the use of microarrays in clinical and regulatory settings. PMID:16964229

Women's experiences receiving abnormal prenatal chromosomal microarray testing results.

PubMed

Bernhardt, Barbara A; Soucier, Danielle; Hanson, Karen; Savage, Melissa S; Jackson, Laird; Wapner, Ronald J

2013-02-01

Genomic microarrays can detect copy-number variants not detectable by conventional cytogenetics. This technology is diffusing rapidly into prenatal settings even though the clinical implications of many copy-number variants are currently unknown. We conducted a qualitative pilot study to explore the experiences of women receiving abnormal results from prenatal microarray testing performed in a research setting. Participants were a subset of women participating in a multicenter prospective study "Prenatal Cytogenetic Diagnosis by Array-based Copy Number Analysis." Telephone interviews were conducted with 23 women receiving abnormal prenatal microarray results. We found that five key elements dominated the experiences of women who had received abnormal prenatal microarray results: an offer too good to pass up, blindsided by the results, uncertainty and unquantifiable risks, need for support, and toxic knowledge. As prenatal microarray testing is increasingly used, uncertain findings will be common, resulting in greater need for careful pre- and posttest counseling, and more education of and resources for providers so they can adequately support the women who are undergoing testing.

Fuzzy support vector machine: an efficient rule-based classification technique for microarrays.

PubMed

Hajiloo, Mohsen; Rabiee, Hamid R; Anooshahpour, Mahdi

2013-01-01

The abundance of gene expression microarray data has led to the development of machine learning algorithms applicable for tackling disease diagnosis, disease prognosis, and treatment selection problems. However, these algorithms often produce classifiers with weaknesses in terms of accuracy, robustness, and interpretability. This paper introduces fuzzy support vector machine which is a learning algorithm based on combination of fuzzy classifiers and kernel machines for microarray classification. Experimental results on public leukemia, prostate, and colon cancer datasets show that fuzzy support vector machine applied in combination with filter or wrapper feature selection methods develops a robust model with higher accuracy than the conventional microarray classification models such as support vector machine, artificial neural network, decision trees, k nearest neighbors, and diagonal linear discriminant analysis. Furthermore, the interpretable rule-base inferred from fuzzy support vector machine helps extracting biological knowledge from microarray data. Fuzzy support vector machine as a new classification model with high generalization power, robustness, and good interpretability seems to be a promising tool for gene expression microarray classification.

Microarray Meta-Analysis Identifies Acute Lung Injury Biomarkers in Donor Lungs That Predict Development of Primary Graft Failure in Recipients

PubMed Central

Haitsma, Jack J.; Furmli, Suleiman; Masoom, Hussain; Liu, Mingyao; Imai, Yumiko; Slutsky, Arthur S.; Beyene, Joseph; Greenwood, Celia M. T.; dos Santos, Claudia

2012-01-01

Objectives To perform a meta-analysis of gene expression microarray data from animal studies of lung injury, and to identify an injury-specific gene expression signature capable of predicting the development of lung injury in humans. Methods We performed a microarray meta-analysis using 77 microarray chips across six platforms, two species and different animal lung injury models exposed to lung injury with or/and without mechanical ventilation. Individual gene chips were classified and grouped based on the strategy used to induce lung injury. Effect size (change in gene expression) was calculated between non-injurious and injurious conditions comparing two main strategies to pool chips: (1) one-hit and (2) two-hit lung injury models. A random effects model was used to integrate individual effect sizes calculated from each experiment. Classification models were built using the gene expression signatures generated by the meta-analysis to predict the development of lung injury in human lung transplant recipients. Results Two injury-specific lists of differentially expressed genes generated from our meta-analysis of lung injury models were validated using external data sets and prospective data from animal models of ventilator-induced lung injury (VILI). Pathway analysis of gene sets revealed that both new and previously implicated VILI-related pathways are enriched with differentially regulated genes. Classification model based on gene expression signatures identified in animal models of lung injury predicted development of primary graft failure (PGF) in lung transplant recipients with larger than 80% accuracy based upon injury profiles from transplant donors. We also found that better classifier performance can be achieved by using meta-analysis to identify differentially-expressed genes than using single study-based differential analysis. Conclusion Taken together, our data suggests that microarray analysis of gene expression data allows for the detection of “injury" gene predictors that can classify lung injury samples and identify patients at risk for clinically relevant lung injury complications. PMID:23071521

Genome-wide expression profiling of maize in response to individual and combined water and nitrogen stresses

PubMed Central

2013-01-01

Background Water and nitrogen are two of the most critical inputs required to achieve the high yield potential of modern corn varieties. Under most agricultural settings however they are often scarce and costly. Fortunately, tremendous progress has been made in the past decades in terms of modeling to assist growers in the decision making process and many tools are now available to achieve more sustainable practices both environmentally and economically. Nevertheless large gaps remain between our empirical knowledge of the physiological changes observed in the field in response to nitrogen and water stresses, and our limited understanding of the molecular processes leading to those changes. Results This work examines in particular the impact of simultaneous stresses on the transcriptome. In a greenhouse setting, corn plants were grown under tightly controlled nitrogen and water conditions, allowing sampling of various tissues and stress combinations. A microarray profiling experiment was performed using this material and showed that the concomitant presence of nitrogen and water limitation affects gene expression to an extent much larger than anticipated. A clustering analysis also revealed how the interaction between the two stresses shapes the patterns of gene expression over various levels of water stresses and recovery. Conclusions Overall, this study suggests that the molecular signature of a specific combination of stresses on the transcriptome might be as unique as the impact of individual stresses, and hence underlines the difficulty to extrapolate conclusions obtained from the study of individual stress responses to more complex settings. PMID:23324127

Hyperosmotic Stress Response of Campylobacter jejuni

PubMed Central

Cameron, Andrew; Frirdich, Emilisa; Huynh, Steven; Parker, Craig T.

2012-01-01

The diarrheal pathogen Campylobacter jejuni and other gastrointestinal bacteria encounter changes in osmolarity in the environment, through exposure to food processing, and upon entering host organisms, where osmotic adaptation can be associated with virulence. In this study, growth profiles, transcriptomics, and phenotypic, mutant, and single-cell analyses were used to explore the effects of hyperosmotic stress exposure on C. jejuni. Increased growth inhibition correlated with increased osmotic concentration, with both ionic and nonionic stressors inhibiting growth at 0.620 total osmol liter−1. C. jejuni adaptation to a range of osmotic stressors and concentrations was accompanied by severe filamentation in subpopulations, with microscopy indicating septum formation and phenotypic diversity between individual cells in a filament. Population heterogeneity was also exemplified by the bifurcation of colony morphology into small and large variants on salt stress plates. Flow cytometry of C. jejuni harboring green fluorescent protein (GFP) fused to the ATP synthase promoter likewise revealed bimodal subpopulations under hyperosmotic stress. We also identified frequent hyperosmotic stress-sensitive variants within the clonal wild-type population propagated on standard laboratory medium. Microarray analysis following hyperosmotic upshift revealed enhanced expression of heat shock genes and genes encoding enzymes for synthesis of potential osmoprotectants and cross-protective induction of oxidative stress genes. The capsule export gene kpsM was also upregulated, and an acapsular mutant was defective for growth under hyperosmotic stress. For C. jejuni, an organism lacking most conventional osmotic response factors, these data suggest an unusual hyperosmotic stress response, including likely “bet-hedging” survival strategies relying on the presence of stress-fit individuals in a heterogeneous population. PMID:22961853

Patterns of gene expression associated with recovery and injury in heat-stressed rats.

PubMed

Stallings, Jonathan D; Ippolito, Danielle L; Rakesh, Vineet; Baer, Christine E; Dennis, William E; Helwig, Bryan G; Jackson, David A; Leon, Lisa R; Lewis, John A; Reifman, Jaques

2014-12-03

The in vivo gene response associated with hyperthermia is poorly understood. Here, we perform a global, multiorgan characterization of the gene response to heat stress using an in vivo conscious rat model. We heated rats until implanted thermal probes indicated a maximal core temperature of 41.8°C (Tc,Max). We then compared transcriptomic profiles of liver, lung, kidney, and heart tissues harvested from groups of experimental animals at Tc,Max, 24 hours, and 48 hours after heat stress to time-matched controls kept at an ambient temperature. Cardiac histopathology at 48 hours supported persistent cardiac injury in three out of six animals. Microarray analysis identified 78 differentially expressed genes common to all four organs at Tc,Max. Self-organizing maps identified gene-specific signatures corresponding to protein-folding disorders in heat-stressed rats with histopathological evidence of cardiac injury at 48 hours. Quantitative proteomics analysis by iTRAQ (isobaric tag for relative and absolute quantitation) demonstrated that differential protein expression most closely matched the transcriptomic profile in heat-injured animals at 48 hours. Calculation of protein supersaturation scores supported an increased propensity of proteins to aggregate for proteins that were found to be changing in abundance at 24 hours and in animals with cardiac injury at 48 hours, suggesting a mechanistic association between protein misfolding and the heat-stress response. Pathway analyses at both the transcript and protein levels supported catastrophic deficits in energetics and cellular metabolism and activation of the unfolded protein response in heat-stressed rats with histopathological evidence of persistent heat injury, providing the basis for a systems-level physiological model of heat illness and recovery.

Addressable droplet microarrays for single cell protein analysis.

PubMed

Salehi-Reyhani, Ali; Burgin, Edward; Ces, Oscar; Willison, Keith R; Klug, David R

2014-11-07

Addressable droplet microarrays are potentially attractive as a way to achieve miniaturised, reduced volume, high sensitivity analyses without the need to fabricate microfluidic devices or small volume chambers. We report a practical method for producing oil-encapsulated addressable droplet microarrays which can be used for such analyses. To demonstrate their utility, we undertake a series of single cell analyses, to determine the variation in copy number of p53 proteins in cells of a human cancer cell line.

Recurrent pregnancy loss evaluation combined with 24-chromosome microarray of miscarriage tissue provides a probable or definite cause of pregnancy loss in over 90% of patients.

PubMed

Popescu, F; Jaslow, C R; Kutteh, W H

2018-04-01

Will the addition of 24-chromosome microarray analysis on miscarriage tissue combined with the standard American Society for Reproductive Medicine (ASRM) evaluation for recurrent miscarriage explain most losses? Over 90% of patients with recurrent pregnancy loss (RPL) will have a probable or definitive cause identified when combining genetic testing on miscarriage tissue with the standard ASRM evaluation for recurrent miscarriage. RPL is estimated to occur in 2-4% of reproductive age couples. A probable cause can be identified in approximately 50% of patients after an ASRM recommended workup including an evaluation for parental chromosomal abnormalities, congenital and acquired uterine anomalies, endocrine imbalances and autoimmune factors including antiphospholipid syndrome. Single-center, prospective cohort study that included 100 patients seen in a private RPL clinic from 2014 to 2017. All 100 women had two or more pregnancy losses, a complete evaluation for RPL as defined by the ASRM, and miscarriage tissue evaluated by 24-chromosome microarray analysis after their second or subsequent miscarriage. Frequencies of abnormal results for evidence-based diagnostic tests considered definite or probable causes of RPL (karyotyping for parental chromosomal abnormalities, and 24-chromosome microarray evaluation for products of conception (POC); pelvic sonohysterography, hysterosalpingogram, or hysteroscopy for uterine anomalies; immunological tests for lupus anticoagulant and anticardiolipin antibodies; and blood tests for thyroid stimulating hormone (TSH), prolactin and hemoglobin A1c) were evaluated. We excluded cases where there was maternal cell contamination of the miscarriage tissue or if the ASRM evaluation was incomplete. A cost analysis for the evaluation of RPL was conducted to determine whether a proposed procedure of 24-chromome microarray evaluation followed by an ASRM RPL workup (for those RPL patients who had a normal 24-chromosome microarray evaluation) was more cost-efficient than conducting ASRM RPL workups on RPL patients followed by 24-chromosome microarray analysis (for those RPL patients who had a normal RPL workup). A definite or probable cause of pregnancy loss was identified in the vast majority (95/100; 95%) of RPL patients when a 24-chromosome pair microarray evaluation of POC testing is combined with the standard ASRM RPL workup evaluation at the time of the second or subsequent loss. The ASRM RPL workup identified an abnormality and a probable explanation for pregnancy loss in only 45/100 or 45% of all patients. A definite abnormality was identified in 67/100 patients or 67% when initial testing was performed using 24-chromosome microarray analyses on the miscarriage tissue. Only 5/100 (5%) patients, who had a euploid loss and a normal ASRM RPL workup, had a pregnancy loss without a probable or definitive cause identified. All other losses were explained by an abnormal 24-chromosome microarray analysis of the miscarriage tissue, an abnormal finding of the RPL workup, or a combination of both. Results from the cost analysis indicated that an initial approach of using a 24-chromosome microarray analysis on miscarriage tissue resulted in a 50% savings in cost to the health care system and to the patient. This is a single-center study on a small group of well-characterized women with RPL. There was an incomplete follow-up on subsequent pregnancy outcomes after evaluation, however this should not affect our principal results. The maternal age of patients varied from 26 to 45 years old. More aneuploid pregnancy losses would be expected in older women, particularly over the age of 35 years old. Evaluation of POC using 24-chromosome microarray analysis adds significantly to the ASRM recommended evaluation of RPL. Genetic evaluation on miscarriage tissue obtained at the time of the second and subsequent pregnancy losses should be offered to all couples with two or more consecutive pregnancy losses. The combination of a genetic evaluation on miscarriage tissue with an evidence-based evaluation for RPL will identify a probable or definitive cause in over 90% of miscarriages. No funding was received for this study and there are no conflicts of interest to declare. Not applicable.

A Platform for Combined DNA and Protein Microarrays Based on Total Internal Reflection Fluorescence

PubMed Central

Asanov, Alexander; Zepeda, Angélica; Vaca, Luis

2012-01-01

We have developed a novel microarray technology based on total internal reflection fluorescence (TIRF) in combination with DNA and protein bioassays immobilized at the TIRF surface. Unlike conventional microarrays that exhibit reduced signal-to-background ratio, require several stages of incubation, rinsing and stringency control, and measure only end-point results, our TIRF microarray technology provides several orders of magnitude better signal-to-background ratio, performs analysis rapidly in one step, and measures the entire course of association and dissociation kinetics between target DNA and protein molecules and the bioassays. In many practical cases detection of only DNA or protein markers alone does not provide the necessary accuracy for diagnosing a disease or detecting a pathogen. Here we describe TIRF microarrays that detect DNA and protein markers simultaneously, which reduces the probabilities of false responses. Supersensitive and multiplexed TIRF DNA and protein microarray technology may provide a platform for accurate diagnosis or enhanced research studies. Our TIRF microarray system can be mounted on upright or inverted microscopes or interfaced directly with CCD cameras equipped with a single objective, facilitating the development of portable devices. As proof-of-concept we applied TIRF microarrays for detecting molecular markers from Bacillus anthracis, the pathogen responsible for anthrax. PMID:22438738

Validation of MIMGO: a method to identify differentially expressed GO terms in a microarray dataset

PubMed Central

2012-01-01

Background We previously proposed an algorithm for the identification of GO terms that commonly annotate genes whose expression is upregulated or downregulated in some microarray data compared with in other microarray data. We call these “differentially expressed GO terms” and have named the algorithm “matrix-assisted identification method of differentially expressed GO terms” (MIMGO). MIMGO can also identify microarray data in which genes annotated with a differentially expressed GO term are upregulated or downregulated. However, MIMGO has not yet been validated on a real microarray dataset using all available GO terms. Findings We combined Gene Set Enrichment Analysis (GSEA) with MIMGO to identify differentially expressed GO terms in a yeast cell cycle microarray dataset. GSEA followed by MIMGO (GSEA + MIMGO) correctly identified (p < 0.05) microarray data in which genes annotated to differentially expressed GO terms are upregulated. We found that GSEA + MIMGO was slightly less effective than, or comparable to, GSEA (Pearson), a method that uses Pearson’s correlation as a metric, at detecting true differentially expressed GO terms. However, unlike other methods including GSEA (Pearson), GSEA + MIMGO can comprehensively identify the microarray data in which genes annotated with a differentially expressed GO term are upregulated or downregulated. Conclusions MIMGO is a reliable method to identify differentially expressed GO terms comprehensively. PMID:23232071

Multifaceted role of nitric oxide in an in vitro mouse neuronal injury model: transcriptomic profiling defines the temporal recruitment of death signalling cascades

PubMed Central

Peng, Zhao Feng; Chen, Minghui Jessica; Manikandan, Jayapal; Melendez, Alirio J; Shui, Guanghou; Russo-Marie, Françoise; Whiteman, Matthew; Beart, Philip M; Moore, Philip K; Cheung, Nam Sang

2012-01-01

Abstract Nitric oxide is implicated in the pathogenesis of various neuropathologies characterized by oxidative stress. Although nitric oxide has been reported to be involved in the exacerbation of oxidative stress observed in several neuropathologies, existent data fail to provide a holistic description of how nitrergic pathobiology elicits neuronal injury. Here we provide a comprehensive description of mechanisms contributing to nitric oxide induced neuronal injury by global transcriptomic profiling. Microarray analyses were undertaken on RNA from murine primary cortical neurons treated with the nitric oxide generator DETA-NONOate (NOC-18, 0.5 mM) for 8–24 hrs. Biological pathway analysis focused upon 3672 gene probes which demonstrated at least a ±1.5-fold expression in a minimum of one out of three time-points and passed statistical analysis (one-way anova, P < 0.05). Numerous enriched processes potentially determining nitric oxide mediated neuronal injury were identified from the transcriptomic profile: cell death, developmental growth and survival, cell cycle, calcium ion homeostasis, endoplasmic reticulum stress, oxidative stress, mitochondrial homeostasis, ubiquitin-mediated proteolysis, and GSH and nitric oxide metabolism. Our detailed time-course study of nitric oxide induced neuronal injury allowed us to provide the first time a holistic description of the temporal sequence of cellular events contributing to nitrergic injury. These data form a foundation for the development of screening platforms and define targets for intervention in nitric oxide neuropathologies where nitric oxide mediated injury is causative. PMID:21352476

The use of open source bioinformatics tools to dissect transcriptomic data.

PubMed

Nitsche, Benjamin M; Ram, Arthur F J; Meyer, Vera

2012-01-01

Microarrays are a valuable technology to study fungal physiology on a transcriptomic level. Various microarray platforms are available comprising both single and two channel arrays. Despite different technologies, preprocessing of microarray data generally includes quality control, background correction, normalization, and summarization of probe level data. Subsequently, depending on the experimental design, diverse statistical analysis can be performed, including the identification of differentially expressed genes and the construction of gene coexpression networks.We describe how Bioconductor, a collection of open source and open development packages for the statistical programming language R, can be used for dissecting microarray data. We provide fundamental details that facilitate the process of getting started with R and Bioconductor. Using two publicly available microarray datasets from Aspergillus niger, we give detailed protocols on how to identify differentially expressed genes and how to construct gene coexpression networks.

Integrated Microfluidic Devices for Automated Microarray-Based Gene Expression and Genotyping Analysis

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Lodes, Mike; Fuji, H. Sho; Danley, David; McShea, Andrew

Microarray assays typically involve multistage sample processing and fluidic handling, which are generally labor-intensive and time-consuming. Automation of these processes would improve robustness, reduce run-to-run and operator-to-operator variation, and reduce costs. In this chapter, a fully integrated and self-contained microfluidic biochip device that has been developed to automate the fluidic handling steps for microarray-based gene expression or genotyping analysis is presented. The device consists of a semiconductor-based CustomArray® chip with 12,000 features and a microfluidic cartridge. The CustomArray was manufactured using a semiconductor-based in situ synthesis technology. The micro-fluidic cartridge consists of microfluidic pumps, mixers, valves, fluid channels, and reagent storage chambers. Microarray hybridization and subsequent fluidic handling and reactions (including a number of washing and labeling steps) were performed in this fully automated and miniature device before fluorescent image scanning of the microarray chip. Electrochemical micropumps were integrated in the cartridge to provide pumping of liquid solutions. A micromixing technique based on gas bubbling generated by electrochemical micropumps was developed. Low-cost check valves were implemented in the cartridge to prevent cross-talk of the stored reagents. Gene expression study of the human leukemia cell line (K562) and genotyping detection and sequencing of influenza A subtypes have been demonstrated using this integrated biochip platform. For gene expression assays, the microfluidic CustomArray device detected sample RNAs with a concentration as low as 0.375 pM. Detection was quantitative over more than three orders of magnitude. Experiment also showed that chip-to-chip variability was low indicating that the integrated microfluidic devices eliminate manual fluidic handling steps that can be a significant source of variability in genomic analysis. The genotyping results showed that the device identified influenza A hemagglutinin and neuraminidase subtypes and sequenced portions of both genes, demonstrating the potential of integrated microfluidic and microarray technology for multiple virus detection. The device provides a cost-effective solution to eliminate labor-intensive and time-consuming fluidic handling steps and allows microarray-based DNA analysis in a rapid and automated fashion.

CLUSFAVOR 5.0: hierarchical cluster and principal-component analysis of microarray-based transcriptional profiles

PubMed Central

Peterson, Leif E

2002-01-01

CLUSFAVOR (CLUSter and Factor Analysis with Varimax Orthogonal Rotation) 5.0 is a Windows-based computer program for hierarchical cluster and principal-component analysis of microarray-based transcriptional profiles. CLUSFAVOR 5.0 standardizes input data; sorts data according to gene-specific coefficient of variation, standard deviation, average and total expression, and Shannon entropy; performs hierarchical cluster analysis using nearest-neighbor, unweighted pair-group method using arithmetic averages (UPGMA), or furthest-neighbor joining methods, and Euclidean, correlation, or jack-knife distances; and performs principal-component analysis. PMID:12184816

Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology

NASA Technical Reports Server (NTRS)

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Said; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis.

Parallel Characterization of Anaerobic Toluene- and Ethylbenzene-Degrading Microbial Consortia by PCR-Denaturing Gradient Gel Electrophoresis, RNA-DNA Membrane Hybridization, and DNA Microarray Technology

PubMed Central

Koizumi, Yoshikazu; Kelly, John J.; Nakagawa, Tatsunori; Urakawa, Hidetoshi; El-Fantroussi, Saïd; Al-Muzaini, Saleh; Fukui, Manabu; Urushigawa, Yoshikuni; Stahl, David A.

2002-01-01

A mesophilic toluene-degrading consortium (TDC) and an ethylbenzene-degrading consortium (EDC) were established under sulfate-reducing conditions. These consortia were first characterized by denaturing gradient gel electrophoresis (DGGE) fingerprinting of PCR-amplified 16S rRNA gene fragments, followed by sequencing. The sequences of the major bands (T-1 and E-2) belonging to TDC and EDC, respectively, were affiliated with the family Desulfobacteriaceae. Another major band from EDC (E-1) was related to an uncultured non-sulfate-reducing soil bacterium. Oligonucleotide probes specific for the 16S rRNAs of target organisms corresponding to T-1, E-1, and E-2 were designed, and hybridization conditions were optimized for two analytical formats, membrane and DNA microarray hybridization. Both formats were used to characterize the TDC and EDC, and the results of both were consistent with DGGE analysis. In order to assess the utility of the microarray format for analysis of environmental samples, oil-contaminated sediments from the coast of Kuwait were analyzed. The DNA microarray successfully detected bacterial nucleic acids from these samples, but probes targeting specific groups of sulfate-reducing bacteria did not give positive signals. The results of this study demonstrate the limitations and the potential utility of DNA microarrays for microbial community analysis. PMID:12088997

Ontology-based, Tissue MicroArray oriented, image centered tissue bank

PubMed Central

Viti, Federica; Merelli, Ivan; Caprera, Andrea; Lazzari, Barbara; Stella, Alessandra; Milanesi, Luciano

2008-01-01

Background Tissue MicroArray technique is becoming increasingly important in pathology for the validation of experimental data from transcriptomic analysis. This approach produces many images which need to be properly managed, if possible with an infrastructure able to support tissue sharing between institutes. Moreover, the available frameworks oriented to Tissue MicroArray provide good storage for clinical patient, sample treatment and block construction information, but their utility is limited by the lack of data integration with biomolecular information. Results In this work we propose a Tissue MicroArray web oriented system to support researchers in managing bio-samples and, through the use of ontologies, enables tissue sharing aimed at the design of Tissue MicroArray experiments and results evaluation. Indeed, our system provides ontological description both for pre-analysis tissue images and for post-process analysis image results, which is crucial for information exchange. Moreover, working on well-defined terms it is then possible to query web resources for literature articles to integrate both pathology and bioinformatics data. Conclusions Using this system, users associate an ontology-based description to each image uploaded into the database and also integrate results with the ontological description of biosequences identified in every tissue. Moreover, it is possible to integrate the ontological description provided by the user with a full compliant gene ontology definition, enabling statistical studies about correlation between the analyzed pathology and the most commonly related biological processes. PMID:18460177

Identifying Functional Mechanisms of Gene and Protein Regulatory Networks in Response to a Broader Range of Environmental Stresses

PubMed Central

Li, Cheng-Wei; Chen, Bor-Sen

2010-01-01

Cellular responses to sudden environmental stresses or physiological changes provide living organisms with the opportunity for final survival and further development. Therefore, it is an important topic to understand protective mechanisms against environmental stresses from the viewpoint of gene and protein networks. We propose two coupled nonlinear stochastic dynamic models to reconstruct stress-activated gene and protein regulatory networks via microarray data in response to environmental stresses. According to the reconstructed gene/protein networks, some possible mutual interactions, feedforward and feedback loops are found for accelerating response and filtering noises in these signaling pathways. A bow-tie core network is also identified to coordinate mutual interactions and feedforward loops, feedback inhibitions, feedback activations, and cross talks to cope efficiently with a broader range of environmental stresses with limited proteins and pathways. PMID:20454442

Comparison of gene expression microarray data with count-based RNA measurements informs microarray interpretation.

PubMed

Richard, Arianne C; Lyons, Paul A; Peters, James E; Biasci, Daniele; Flint, Shaun M; Lee, James C; McKinney, Eoin F; Siegel, Richard M; Smith, Kenneth G C

2014-08-04

Although numerous investigations have compared gene expression microarray platforms, preprocessing methods and batch correction algorithms using constructed spike-in or dilution datasets, there remains a paucity of studies examining the properties of microarray data using diverse biological samples. Most microarray experiments seek to identify subtle differences between samples with variable background noise, a scenario poorly represented by constructed datasets. Thus, microarray users lack important information regarding the complexities introduced in real-world experimental settings. The recent development of a multiplexed, digital technology for nucleic acid measurement enables counting of individual RNA molecules without amplification and, for the first time, permits such a study. Using a set of human leukocyte subset RNA samples, we compared previously acquired microarray expression values with RNA molecule counts determined by the nCounter Analysis System (NanoString Technologies) in selected genes. We found that gene measurements across samples correlated well between the two platforms, particularly for high-variance genes, while genes deemed unexpressed by the nCounter generally had both low expression and low variance on the microarray. Confirming previous findings from spike-in and dilution datasets, this "gold-standard" comparison demonstrated signal compression that varied dramatically by expression level and, to a lesser extent, by dataset. Most importantly, examination of three different cell types revealed that noise levels differed across tissues. Microarray measurements generally correlate with relative RNA molecule counts within optimal ranges but suffer from expression-dependent accuracy bias and precision that varies across datasets. We urge microarray users to consider expression-level effects in signal interpretation and to evaluate noise properties in each dataset independently.

Genome Expression Pathway Analysis Tool – Analysis and visualization of microarray gene expression data under genomic, proteomic and metabolic context

PubMed Central

Weniger, Markus; Engelmann, Julia C; Schultz, Jörg

2007-01-01

Background Regulation of gene expression is relevant to many areas of biology and medicine, in the study of treatments, diseases, and developmental stages. Microarrays can be used to measure the expression level of thousands of mRNAs at the same time, allowing insight into or comparison of different cellular conditions. The data derived out of microarray experiments is highly dimensional and often noisy, and interpretation of the results can get intricate. Although programs for the statistical analysis of microarray data exist, most of them lack an integration of analysis results and biological interpretation. Results We have developed GEPAT, Genome Expression Pathway Analysis Tool, offering an analysis of gene expression data under genomic, proteomic and metabolic context. We provide an integration of statistical methods for data import and data analysis together with a biological interpretation for subsets of probes or single probes on the chip. GEPAT imports various types of oligonucleotide and cDNA array data formats. Different normalization methods can be applied to the data, afterwards data annotation is performed. After import, GEPAT offers various statistical data analysis methods, as hierarchical, k-means and PCA clustering, a linear model based t-test or chromosomal profile comparison. The results of the analysis can be interpreted by enrichment of biological terms, pathway analysis or interaction networks. Different biological databases are included, to give various information for each probe on the chip. GEPAT offers no linear work flow, but allows the usage of any subset of probes and samples as a start for a new data analysis. GEPAT relies on established data analysis packages, offers a modular approach for an easy extension, and can be run on a computer grid to allow a large number of users. It is freely available under the LGPL open source license for academic and commercial users at . Conclusion GEPAT is a modular, scalable and professional-grade software integrating analysis and interpretation of microarray gene expression data. An installation available for academic users can be found at . PMID:17543125

Phytochromes A and C cooperatively regulate early and transient gene expression after red-light irradiation in rice seedlings.

PubMed

Kiyota, Seiichiro; Xie, Xianzhi; Takano, Makoto

2012-02-01

Phytochromes are red/far-red photoreceptors encoded by a small gene family in higher plants. Differences in phenotype among mutants suggest distinct functions among phytochrome subfamilies. We attempted to find distinct functions among phytochromes by oligo-microarray analysis of single, double, and triple mutants in rice. In most cases, gene expression was redundantly regulated by phytochromes A and B after irradiation by a red light pulse in etiolated rice shoots. However, we found that several genes were specifically regulated by phytochromes A and C. Most of them were expressed immediately after the red light pulse in a transient manner. They are stress-related genes that may be involved in resistance to light stress when etiolated seedlings are exposed to light. These genes were not expressed in green leaves after the red light pulse, suggesting that they have a function specific to etiolated seedlings. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Development of DNA Microarrays for Metabolic Pathway and Bioprocess Monitoring

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gregory Stephanopoulos

Transcriptional profiling experiments utilizing DNA microarrays to study the intracellular accumulation of PHB in Synechocystis has proved difficult in large part because strains that show significant differences in PHB which would justify global analysis of gene expression have not been isolated.

Identification and characterization of nuclear genes involved in photosynthesis in Populus

PubMed Central

2014-01-01

Background The gap between the real and potential photosynthetic rate under field conditions suggests that photosynthesis could potentially be improved. Nuclear genes provide possible targets for improving photosynthetic efficiency. Hence, genome-wide identification and characterization of the nuclear genes affecting photosynthetic traits in woody plants would provide key insights on genetic regulation of photosynthesis and identify candidate processes for improvement of photosynthesis. Results Using microarray and bulked segregant analysis strategies, we identified differentially expressed nuclear genes for photosynthesis traits in a segregating population of poplar. We identified 515 differentially expressed genes in this population (FC ≥ 2 or FC ≤ 0.5, P < 0.05), 163 up-regulated and 352 down-regulated. Real-time PCR expression analysis confirmed the microarray data. Singular Enrichment Analysis identified 48 significantly enriched GO terms for molecular functions (28), biological processes (18) and cell components (2). Furthermore, we selected six candidate genes for functional examination by a single-marker association approach, which demonstrated that 20 SNPs in five candidate genes significantly associated with photosynthetic traits, and the phenotypic variance explained by each SNP ranged from 2.3% to 12.6%. This revealed that regulation of photosynthesis by the nuclear genome mainly involves transport, metabolism and response to stimulus functions. Conclusions This study provides new genome-scale strategies for the discovery of potential candidate genes affecting photosynthesis in Populus, and for identification of the functions of genes involved in regulation of photosynthesis. This work also suggests that improving photosynthetic efficiency under field conditions will require the consideration of multiple factors, such as stress responses. PMID:24673936

No changes in heme synthesis in human Friedreich´s ataxia erythroid progenitor cells.

PubMed

Steinkellner, Hannes; Singh, Himanshu Narayan; Muckenthaler, Martina U; Goldenberg, Hans; Moganty, Rajeswari R; Scheiber-Mojdehkar, Barbara; Sturm, Brigitte

2017-07-20

Friedreich's ataxia (FRDA) is a neurodegenerative disease caused by reduced expression of the protein frataxin. Frataxin is thought to play a role in iron-sulfur cluster biogenesis and heme synthesis. In this study, we used erythroid progenitor stem cells obtained from FRDA patients and healthy donors to investigate the putative role, if any, of frataxin deficiency in heme synthesis. We used electrochemiluminescence and qRT-PCR for frataxin protein and mRNA quantification. We used atomic absorption spectrophotometry for iron levels and a photometric assay for hemoglobin levels. Protoporphyrin IX and Ferrochelatase were analyzed using auto-fluorescence. An "IronChip" microarray analysis followed by a protein-protein interaction analysis was performed. FRDA patient cells showed no significant changes in iron levels, hemoglobin synthesis, protoporphyrin IX levels, and ferrochelatase activity. Microarray analysis presented 11 genes that were significantly changed in all patients compared to controls. The genes are especially involved in oxidative stress, iron homeostasis and angiogenesis. The mystery about the involvement of frataxin on iron metabolism raises the question why frataxin deficiency in primary FRDA cells did not lead to changes in biochemical parameters of heme synthesis. It seems that alternative pathways can circumvent the impact of frataxin deficiency on heme synthesis. We show for the first time in primary FRDA patient cells that reduced frataxin levels are still sufficient for heme synthesis and possibly other mechanisms can overcome reduced frataxin levels in this process. Our data strongly support the fact that so far no anemia in FRDA patients was reported. Copyright © 2017 Elsevier B.V. All rights reserved.

Reuse of imputed data in microarray analysis increases imputation efficiency

PubMed Central

Kim, Ki-Yeol; Kim, Byoung-Jin; Yi, Gwan-Su

2004-01-01

Background The imputation of missing values is necessary for the efficient use of DNA microarray data, because many clustering algorithms and some statistical analysis require a complete data set. A few imputation methods for DNA microarray data have been introduced, but the efficiency of the methods was low and the validity of imputed values in these methods had not been fully checked. Results We developed a new cluster-based imputation method called sequential K-nearest neighbor (SKNN) method. This imputes the missing values sequentially from the gene having least missing values, and uses the imputed values for the later imputation. Although it uses the imputed values, the efficiency of this new method is greatly improved in its accuracy and computational complexity over the conventional KNN-based method and other methods based on maximum likelihood estimation. The performance of SKNN was in particular higher than other imputation methods for the data with high missing rates and large number of experiments. Application of Expectation Maximization (EM) to the SKNN method improved the accuracy, but increased computational time proportional to the number of iterations. The Multiple Imputation (MI) method, which is well known but not applied previously to microarray data, showed a similarly high accuracy as the SKNN method, with slightly higher dependency on the types of data sets. Conclusions Sequential reuse of imputed data in KNN-based imputation greatly increases the efficiency of imputation. The SKNN method should be practically useful to save the data of some microarray experiments which have high amounts of missing entries. The SKNN method generates reliable imputed values which can be used for further cluster-based analysis of microarray data. PMID:15504240

Enhanced Phospholipase A2 Group 3 Expression by Oxidative Stress Decreases the Insulin-Degrading Enzyme

PubMed Central

Yui, Daishi; Nishida, Yoichiro; Nishina, Tomoko; Mogushi, Kaoru; Tajiri, Mio; Ishibashi, Satoru; Ajioka, Itsuki; Ishikawa, Kinya; Mizusawa, Hidehiro; Murayama, Shigeo; Yokota, Takanori

2015-01-01

Oxidative stress has a ubiquitous role in neurodegenerative diseases and oxidative damage in specific regions of the brain is associated with selective neurodegeneration. We previously reported that Alzheimer disease (AD) model mice showed decreased insulin-degrading enzyme (IDE) levels in the cerebrum and accelerated phenotypic features of AD when crossbred with alpha-tocopherol transfer protein knockout (Ttpa -/-) mice. To further investigate the role of chronic oxidative stress in AD pathophysiology, we performed DNA microarray analysis using young and aged wild-type mice and aged Ttpa -/- mice. Among the genes whose expression changed dramatically was Phospholipase A2 group 3 (Pla2g3); Pla2g3 was identified because of its expression profile of cerebral specific up-regulation by chronic oxidative stress in silico and in aged Ttpa -/- mice. Immunohistochemical studies also demonstrated that human astrocytic Pla2g3 expression was significantly increased in human AD brains compared with control brains. Moreover, transfection of HEK293 cells with human Pla2g3 decreased endogenous IDE expression in a dose-dependent manner. Our findings show a key role of Pla2g3 on the reduction of IDE, and suggest that cerebrum specific increase of Pla2g3 is involved in the initiation and/or progression of AD. PMID:26637123

Mitochondrial Gene Expression Profiles and Metabolic Pathways in the Amygdala Associated with Exaggerated Fear in an Animal Model of PTSD.

PubMed

Li, He; Li, Xin; Smerin, Stanley E; Zhang, Lei; Jia, Min; Xing, Guoqiang; Su, Yan A; Wen, Jillian; Benedek, David; Ursano, Robert

2014-01-01

The metabolic mechanisms underlying the development of exaggerated fear in post-traumatic stress disorder (PTSD) are not well defined. In the present study, alteration in the expression of genes associated with mitochondrial function in the amygdala of an animal model of PTSD was determined. Amygdala tissue samples were excised from 10 non-stressed control rats and 10 stressed rats, 14 days post-stress treatment. Total RNA was isolated, cDNA was synthesized, and gene expression levels were determined using a cDNA microarray. During the development of the exaggerated fear associated with PTSD, 48 genes were found to be significantly upregulated and 37 were significantly downregulated in the amygdala complex based on stringent criteria (p < 0.01). Ingenuity pathway analysis revealed up- or downregulation in the amygdala complex of four signaling networks - one associated with inflammatory and apoptotic pathways, one with immune mediators and metabolism, one with transcriptional factors, and one with chromatin remodeling. Thus, informatics of a neuronal gene array allowed us to determine the expression profile of mitochondrial genes in the amygdala complex of an animal model of PTSD. The result is a further understanding of the metabolic and neuronal signaling mechanisms associated with delayed and exaggerated fear.

Detection of clonal evolution in hematopoietic malignancies by combining comparative genomic hybridization and single nucleotide polymorphism arrays.

PubMed

Hartmann, Luise; Stephenson, Christine F; Verkamp, Stephanie R; Johnson, Krystal R; Burnworth, Bettina; Hammock, Kelle; Brodersen, Lisa Eidenschink; de Baca, Monica E; Wells, Denise A; Loken, Michael R; Zehentner, Barbara K

2014-12-01

Array comparative genomic hybridization (aCGH) has become a powerful tool for analyzing hematopoietic neoplasms and identifying genome-wide copy number changes in a single assay. aCGH also has superior resolution compared with fluorescence in situ hybridization (FISH) or conventional cytogenetics. Integration of single nucleotide polymorphism (SNP) probes with microarray analysis allows additional identification of acquired uniparental disomy, a copy neutral aberration with known potential to contribute to tumor pathogenesis. However, a limitation of microarray analysis has been the inability to detect clonal heterogeneity in a sample. This study comprised 16 samples (acute myeloid leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, plasma cell neoplasm) with complex cytogenetic features and evidence of clonal evolution. We used an integrated manual peak reassignment approach combining analysis of aCGH and SNP microarray data for characterization of subclonal abnormalities. We compared array findings with results obtained from conventional cytogenetic and FISH studies. Clonal heterogeneity was detected in 13 of 16 samples by microarray on the basis of log2 values. Use of the manual peak reassignment analysis approach improved resolution of the sample's clonal composition and genetic heterogeneity in 10 of 13 (77%) patients. Moreover, in 3 patients, clonal disease progression was revealed by array analysis that was not evident by cytogenetic or FISH studies. Genetic abnormalities originating from separate clonal subpopulations can be identified and further characterized by combining aCGH and SNP hybridization results from 1 integrated microarray chip by use of the manual peak reassignment technique. Its clinical utility in comparison to conventional cytogenetic or FISH studies is demonstrated. © 2014 American Association for Clinical Chemistry.

Comprehensive Genomic Analysis and Expression Profiling of the NOX Gene Families under Abiotic Stresses and Hormones in Plants.

PubMed

Chang, Yan-Li; Li, Wen-Yan; Miao, Hai; Yang, Shuai-Qi; Li, Ri; Wang, Xiang; Li, Wen-Qiang; Chen, Kun-Ming

2016-02-23

Plasma membrane NADPH oxidases (NOXs) are key producers of reactive oxygen species under both normal and stress conditions in plants and they form functional subfamilies. Studies of these subfamilies indicated that they show considerable evolutionary selection. We performed a comparative genomic analysis that identified 50 ferric reduction oxidases (FRO) and 77 NOX gene homologs from 20 species representing the eight major plant lineages within the supergroup Plantae: glaucophytes, rhodophytes, chlorophytes, bryophytes, lycophytes, gymnosperms, monocots, and eudicots. Phylogenetic and structural analysis classified these FRO and NOX genes into four well-conserved groups represented as NOX, FRO I, FRO II, and FRO III. Further analysis of NOXs of phylogenetic and exon/intron structures showed that single intron loss and gain had occurred, yielding the diversified gene structures during the evolution of NOXs family genes and which were classified into four conserved subfamilies which are represented as Sub.I, Sub.II, Sub.III, and Sub.IV. Additionally, both available global microarray data analysis and quantitative real-time PCR experiments revealed that the NOX genes in Arabidopsis and rice (Oryza sativa) have different expression patterns in different developmental stages, various abiotic stresses and hormone treatments. Finally, coexpression network analysis of NOX genes in Arabidopsis and rice revealed that NOXs have significantly correlated expression profiles with genes which are involved in plants metabolic and resistance progresses. All these results suggest that NOX family underscores the functional diversity and divergence in plants. This finding will facilitate further studies of the NOX family and provide valuable information for functional validation of this family in plants. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

GEM2Net: from gene expression modeling to -omics networks, a new CATdb module to investigate Arabidopsis thaliana genes involved in stress response.

PubMed

Zaag, Rim; Tamby, Jean Philippe; Guichard, Cécile; Tariq, Zakia; Rigaill, Guillem; Delannoy, Etienne; Renou, Jean-Pierre; Balzergue, Sandrine; Mary-Huard, Tristan; Aubourg, Sébastien; Martin-Magniette, Marie-Laure; Brunaud, Véronique

2015-01-01

CATdb (http://urgv.evry.inra.fr/CATdb) is a database providing a public access to a large collection of transcriptomic data, mainly for Arabidopsis but also for other plants. This resource has the rare advantage to contain several thousands of microarray experiments obtained with the same technical protocol and analyzed by the same statistical pipelines. In this paper, we present GEM2Net, a new module of CATdb that takes advantage of this homogeneous dataset to mine co-expression units and decipher Arabidopsis gene functions. GEM2Net explores 387 stress conditions organized into 18 biotic and abiotic stress categories. For each one, a model-based clustering is applied on expression differences to identify clusters of co-expressed genes. To characterize functions associated with these clusters, various resources are analyzed and integrated: Gene Ontology, subcellular localization of proteins, Hormone Families, Transcription Factor Families and a refined stress-related gene list associated to publications. Exploiting protein-protein interactions and transcription factors-targets interactions enables to display gene networks. GEM2Net presents the analysis of the 18 stress categories, in which 17,264 genes are involved and organized within 681 co-expression clusters. The meta-data analyses were stored and organized to compose a dynamic Web resource. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Large-scale gene expression profiling data for the model moss Physcomitrella patens aid understanding of developmental progression, culture and stress conditions.

PubMed

Hiss, Manuel; Laule, Oliver; Meskauskiene, Rasa M; Arif, Muhammad A; Decker, Eva L; Erxleben, Anika; Frank, Wolfgang; Hanke, Sebastian T; Lang, Daniel; Martin, Anja; Neu, Christina; Reski, Ralf; Richardt, Sandra; Schallenberg-Rüdinger, Mareike; Szövényi, Peter; Tiko, Theodhor; Wiedemann, Gertrud; Wolf, Luise; Zimmermann, Philip; Rensing, Stefan A

2014-08-01

The moss Physcomitrella patens is an important model organism for studying plant evolution, development, physiology and biotechnology. Here we have generated microarray gene expression data covering the principal developmental stages, culture forms and some environmental/stress conditions. Example analyses of developmental stages and growth conditions as well as abiotic stress treatments demonstrate that (i) growth stage is dominant over culture conditions, (ii) liquid culture is not stressful for the plant, (iii) low pH might aid protoplastation by reduced expression of cell wall structure genes, (iv) largely the same gene pool mediates response to dehydration and rehydration, and (v) AP2/EREBP transcription factors play important roles in stress response reactions. With regard to the AP2 gene family, phylogenetic analysis and comparison with Arabidopsis thaliana shows commonalities as well as uniquely expressed family members under drought, light perturbations and protoplastation. Gene expression profiles for P. patens are available for the scientific community via the easy-to-use tool at https://www.genevestigator.com. By providing large-scale expression profiles, the usability of this model organism is further enhanced, for example by enabling selection of control genes for quantitative real-time PCR. Now, gene expression levels across a broad range of conditions can be accessed online for P. patens. © 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.

SpxA1 Involved in Hydrogen Peroxide Production, Stress Tolerance and Endocarditis Virulence in Streptococcus sanguinis

PubMed Central

Chen, Lei; Ge, Xiuchun; Wang, Xiaojing; Patel, Jenishkumar R.; Xu, Ping

2012-01-01

Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H2O2) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H2O2 production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses. PMID:22768210

SpxA1 involved in hydrogen peroxide production, stress tolerance and endocarditis virulence in Streptococcus sanguinis.

PubMed

Chen, Lei; Ge, Xiuchun; Wang, Xiaojing; Patel, Jenishkumar R; Xu, Ping

2012-01-01

Streptococcus sanguinis is one of the most common agents of infective endocarditis. Spx proteins are a group of global regulators that negatively or positively control global transcription initiation. In this study, we characterized the spxA1 gene in S. sanguinis SK36. The spxA1 null mutant displayed opaque colony morphology, reduced hydrogen peroxide (H(2)O(2)) production, and reduced antagonistic activity against Streptococcus mutans UA159 relative to the wild type strain. The ΔspxA1 mutant also demonstrated decreased tolerance to high temperature, acidic and oxidative stresses. Further analysis revealed that ΔspxA1 also exhibited a ∼5-fold reduction in competitiveness in an animal model of endocarditis. Microarray studies indicated that expression of several oxidative stress genes was downregulated in the ΔspxA1 mutant. The expression of spxB and nox was significantly decreased in the ΔspxA1 mutant compared with the wild type. These results indicate that spxA1 plays a major role in H(2)O(2) production, stress tolerance and endocarditis virulence in S. sanguinis SK36. The second spx gene, spxA2, was also found in S. sanguinis SK36. The spxA2 null mutant was found to be defective for growth under normal conditions and showed sensitivity to high temperature, acidic and oxidative stresses.

Gene expression profiling of three different stressors in the water flea Daphnia magna.

PubMed

Jansen, Mieke; Vergauwen, Lucia; Vandenbrouck, Tine; Knapen, Dries; Dom, Nathalie; Spanier, Katina I; Cielen, Anke; De Meester, Luc

2013-07-01

Microarrays are an ideal tool to screen for differences in gene expression of thousands of genes simultaneously. However, often commercial arrays are not available. In this study, we performed microarray analyses to evaluate patterns of gene transcription following exposure to two natural and one anthropogenic stressor. cDNA microarrays compiled of three life stage specific and three stressor-specific EST libraries, yielding 1734 different EST sequences, were used. We exposed juveniles of the water flea Daphnia magna for 48, 96 and 144 h to three stressors known to exert strong selection in natural populations of this species i.e. a sublethal concentration of the pesticide carbaryl, infective spores of the endoparasite Pasteuria ramosa, and fish predation risk mimicked by exposure to fish kairomones. A total of 148 gene fragments were differentially expressed compared to the control. Based on a PCA, the exposure treatments were separated into two main groups based on the extent of the transcriptional response: a low and a high (144 h of fish or carbaryl exposure and 96 h of parasite exposure) stress group. Firstly, we observed a general stress-related transcriptional expression profile independent of the treatment characterized by repression of transcripts involved in transcription, translation, signal transduction and energy metabolism. Secondly, we observed treatment-specific responses including signs of migration to deeper water layers in response to fish predation, structural challenge of the cuticle in response to carbaryl exposure, and disturbance of the ATP production in parasite exposure. A third important conclusion is that transcription expression patterns exhibit stress-specific changes over time. Parasite exposure shows the most differentially expressed gene fragments after 96 h. The peak of differentially expressed transcripts came only after 144 h of fish exposure, while carbaryl exposure induced a more stable number of differently expressed gene fragments over time.

Analysis and modelling of septic shock microarray data using Singular Value Decomposition.

PubMed

Allanki, Srinivas; Dixit, Madhulika; Thangaraj, Paul; Sinha, Nandan Kumar

2017-06-01

Being a high throughput technique, enormous amounts of microarray data has been generated and there arises a need for more efficient techniques of analysis, in terms of speed and accuracy. Finding the differentially expressed genes based on just fold change and p-value might not extract all the vital biological signals that occur at a lower gene expression level. Besides this, numerous mathematical models have been generated to predict the clinical outcome from microarray data, while very few, if not none, aim at predicting the vital genes that are important in a disease progression. Such models help a basic researcher narrow down and concentrate on a promising set of genes which leads to the discovery of gene-based therapies. In this article, as a first objective, we have used the lesser known and used Singular Value Decomposition (SVD) technique to build a microarray data analysis tool that works with gene expression patterns and intrinsic structure of the data in an unsupervised manner. We have re-analysed a microarray data over the clinical course of Septic shock from Cazalis et al. (2014) and have shown that our proposed analysis provides additional information compared to the conventional method. As a second objective, we developed a novel mathematical model that predicts a set of vital genes in the disease progression that works by generating samples in the continuum between health and disease, using a simple normal-distribution-based random number generator. We also verify that most of the predicted genes are indeed related to septic shock. Copyright © 2017 Elsevier Inc. All rights reserved.

Biomarkers of the Hedgehog/Smoothened pathway in healthy volunteers

PubMed Central

Kadam, Sunil K; Patel, Bharvin K R; Jones, Emma; Nguyen, Tuan S; Verma, Lalit K; Landschulz, Katherine T; Stepaniants, Sergey; Li, Bin; Brandt, John T; Brail, Leslie H

2012-01-01

The Hedgehog (Hh) pathway is involved in oncogenic transformation and tumor maintenance. The primary objective of this study was to select surrogate tissue to measure messenger ribonucleic acid (mRNA) levels of Hh pathway genes for measurement of pharmacodynamic effect. Expression of Hh pathway specific genes was measured by quantitative real time polymerase chain reaction (qRT-PCR) and global gene expression using Affymetrix U133 microarrays. Correlations were made between the expression of specific genes determined by qRT-PCR and normalized microarray data. Gene ontology analysis using microarray data for a broader set of Hh pathway genes was performed to identify additional Hh pathway-related markers in the surrogate tissue. RNA extracted from blood, hair follicle, and skin obtained from healthy subjects was analyzed by qRT-PCR for 31 genes, whereas 8 samples were analyzed for a 7-gene subset. Twelve sample sets, each with ≤500 ng total RNA derived from hair, skin, and blood, were analyzed using Affymetrix U133 microarrays. Transcripts for several Hh pathway genes were undetectable in blood using qRT-PCR. Skin was the most desirable matrix, followed by hair follicle. Whether processed by robust multiarray average or microarray suite 5 (MAS5), expression patterns of individual samples showed co-clustered signals; both normalization methods were equally effective for unsupervised analysis. The MAS5- normalized probe sets appeared better suited for supervised analysis. This work provides the basis for selection of a surrogate tissue and an expression analysis-based approach to evaluate pathway-related genes as markers of pharmacodynamic effect with novel inhibitors of the Hh pathway. PMID:22611475

A novel approach for human whole transcriptome analysis based on absolute gene expression of microarray data

PubMed Central

Bikel, Shirley; Jacobo-Albavera, Leonor; Sánchez-Muñoz, Fausto; Cornejo-Granados, Fernanda; Canizales-Quinteros, Samuel; Soberón, Xavier; Sotelo-Mundo, Rogerio R.; del Río-Navarro, Blanca E.; Mendoza-Vargas, Alfredo; Sánchez, Filiberto

2017-01-01

Background In spite of the emergence of RNA sequencing (RNA-seq), microarrays remain in widespread use for gene expression analysis in the clinic. There are over 767,000 RNA microarrays from human samples in public repositories, which are an invaluable resource for biomedical research and personalized medicine. The absolute gene expression analysis allows the transcriptome profiling of all expressed genes under a specific biological condition without the need of a reference sample. However, the background fluorescence represents a challenge to determine the absolute gene expression in microarrays. Given that the Y chromosome is absent in female subjects, we used it as a new approach for absolute gene expression analysis in which the fluorescence of the Y chromosome genes of female subjects was used as the background fluorescence for all the probes in the microarray. This fluorescence was used to establish an absolute gene expression threshold, allowing the differentiation between expressed and non-expressed genes in microarrays. Methods We extracted the RNA from 16 children leukocyte samples (nine males and seven females, ages 6–10 years). An Affymetrix Gene Chip Human Gene 1.0 ST Array was carried out for each sample and the fluorescence of 124 genes of the Y chromosome was used to calculate the absolute gene expression threshold. After that, several expressed and non-expressed genes according to our absolute gene expression threshold were compared against the expression obtained using real-time quantitative polymerase chain reaction (RT-qPCR). Results From the 124 genes of the Y chromosome, three genes (DDX3Y, TXLNG2P and EIF1AY) that displayed significant differences between sexes were used to calculate the absolute gene expression threshold. Using this threshold, we selected 13 expressed and non-expressed genes and confirmed their expression level by RT-qPCR. Then, we selected the top 5% most expressed genes and found that several KEGG pathways were significantly enriched. Interestingly, these pathways were related to the typical functions of leukocytes cells, such as antigen processing and presentation and natural killer cell mediated cytotoxicity. We also applied this method to obtain the absolute gene expression threshold in already published microarray data of liver cells, where the top 5% expressed genes showed an enrichment of typical KEGG pathways for liver cells. Our results suggest that the three selected genes of the Y chromosome can be used to calculate an absolute gene expression threshold, allowing a transcriptome profiling of microarray data without the need of an additional reference experiment. Discussion Our approach based on the establishment of a threshold for absolute gene expression analysis will allow a new way to analyze thousands of microarrays from public databases. This allows the study of different human diseases without the need of having additional samples for relative expression experiments. PMID:29230367

Microarray-based bioinformatics analysis of the combined effects of SiNPs and PbAc on cardiovascular system in zebrafish.

PubMed

Hu, Hejing; Zhang, Yannan; Shi, Yanfeng; Feng, Lin; Duan, Junchao; Sun, Zhiwei

2017-10-01

With rapid development of nanotechnology and growing environmental pollution, the combined toxic effects of SiNPs and pollutants of heavy metals like lead have received global attentions. The aim of this study was to explore the cardiovascular effects of the co-exposure of SiNPs and lead acetate (PbAc) in zebrafish using microarray and bioinformatics analysis. Although there was no other obvious cardiovascular malformation except bleeding phenotype, bradycardia, angiogenesis inhibition and declined cardiac output in zebrafish co-exposed of SiNPs and PbAc at NOAEL level, significant changes were observed in mRNA and microRNA (miRNA) expression patterns. STC-GO analysis indicated that the co-exposure might have more toxic effects on cardiovascular system than that exposure alone. Key differentially expressed genes were discerned out based on the Dynamic-gene-network, including stxbp1a, ndfip2, celf4 and gsk3b. Furthermore, several miRNAs obtained from the miRNA-Gene-Network might play crucial roles in cardiovascular disease, such as dre-miR-93, dre-miR-34a, dre-miR-181c, dre-miR-7145, dre-miR-730, dre-miR-129-5p, dre-miR-19d, dre-miR-218b, dre-miR-221. Besides, the analysis of miRNA-pathway-network indicated that the zebrafish were stimulated by the co-exposure of SiNPs and PbAc, which might cause the disturbance of calcium homeostasis and endoplasmic reticulum stress. As a result, cardiac muscle contraction might be deteriorated. In general, our data provide abundant fundamental research clues to the combined toxicity of environmental pollutants and further in-depth verifications are needed. Copyright © 2017 Elsevier Ltd. All rights reserved.

DigOut: viewing differential expression genes as outliers.

PubMed

Yu, Hui; Tu, Kang; Xie, Lu; Li, Yuan-Yuan

2010-12-01

With regards to well-replicated two-conditional microarray datasets, the selection of differentially expressed (DE) genes is a well-studied computational topic, but for multi-conditional microarray datasets with limited or no replication, the same task is not properly addressed by previous studies. This paper adopts multivariate outlier analysis to analyze replication-lacking multi-conditional microarray datasets, finding that it performs significantly better than the widely used limit fold change (LFC) model in a simulated comparative experiment. Compared with the LFC model, the multivariate outlier analysis also demonstrates improved stability against sample variations in a series of manipulated real expression datasets. The reanalysis of a real non-replicated multi-conditional expression dataset series leads to satisfactory results. In conclusion, a multivariate outlier analysis algorithm, like DigOut, is particularly useful for selecting DE genes from non-replicated multi-conditional gene expression dataset.

A comparative study of RNA-Seq and microarray data analysis on the two examples of rectal-cancer patients and Burkitt Lymphoma cells.

PubMed

Wolff, Alexander; Bayerlová, Michaela; Gaedcke, Jochen; Kube, Dieter; Beißbarth, Tim

2018-01-01

Pipeline comparisons for gene expression data are highly valuable for applied real data analyses, as they enable the selection of suitable analysis strategies for the dataset at hand. Such pipelines for RNA-Seq data should include mapping of reads, counting and differential gene expression analysis or preprocessing, normalization and differential gene expression in case of microarray analysis, in order to give a global insight into pipeline performances. Four commonly used RNA-Seq pipelines (STAR/HTSeq-Count/edgeR, STAR/RSEM/edgeR, Sailfish/edgeR, TopHat2/Cufflinks/CuffDiff)) were investigated on multiple levels (alignment and counting) and cross-compared with the microarray counterpart on the level of gene expression and gene ontology enrichment. For these comparisons we generated two matched microarray and RNA-Seq datasets: Burkitt Lymphoma cell line data and rectal cancer patient data. The overall mapping rate of STAR was 98.98% for the cell line dataset and 98.49% for the patient dataset. Tophat's overall mapping rate was 97.02% and 96.73%, respectively, while Sailfish had only an overall mapping rate of 84.81% and 54.44%. The correlation of gene expression in microarray and RNA-Seq data was moderately worse for the patient dataset (ρ = 0.67-0.69) than for the cell line dataset (ρ = 0.87-0.88). An exception were the correlation results of Cufflinks, which were substantially lower (ρ = 0.21-0.29 and 0.34-0.53). For both datasets we identified very low numbers of differentially expressed genes using the microarray platform. For RNA-Seq we checked the agreement of differentially expressed genes identified in the different pipelines and of GO-term enrichment results. In conclusion the combination of STAR aligner with HTSeq-Count followed by STAR aligner with RSEM and Sailfish generated differentially expressed genes best suited for the dataset at hand and in agreement with most of the other transcriptomics pipelines.

Prediction of regulatory gene pairs using dynamic time warping and gene ontology.

PubMed

Yang, Andy C; Hsu, Hui-Huang; Lu, Ming-Da; Tseng, Vincent S; Shih, Timothy K

2014-01-01

Selecting informative genes is the most important task for data analysis on microarray gene expression data. In this work, we aim at identifying regulatory gene pairs from microarray gene expression data. However, microarray data often contain multiple missing expression values. Missing value imputation is thus needed before further processing for regulatory gene pairs becomes possible. We develop a novel approach to first impute missing values in microarray time series data by combining k-Nearest Neighbour (KNN), Dynamic Time Warping (DTW) and Gene Ontology (GO). After missing values are imputed, we then perform gene regulation prediction based on our proposed DTW-GO distance measurement of gene pairs. Experimental results show that our approach is more accurate when compared with existing missing value imputation methods on real microarray data sets. Furthermore, our approach can also discover more regulatory gene pairs that are known in the literature than other methods.

Temperature Gradient Effect on Gas Discrimination Power of a Metal-Oxide Thin-Film Sensor Microarray

PubMed Central

Sysoev, Victor V.; Kiselev, Ilya; Frietsch, Markus; Goschnick, Joachim

2004-01-01

The paper presents results concerning the effect of spatial inhomogeneous operating temperature on the gas discrimination power of a gas-sensor microarray, with the latter based on a thin SnO2 film employed in the KAMINA electronic nose. Three different temperature distributions over the substrate are discussed: a nearly homogeneous one and two temperature gradients, equal to approx. 3.3 °C/mm and 6.7 °C/mm, applied across the sensor elements (segments) of the array. The gas discrimination power of the microarray is judged by using the Mahalanobis distance in the LDA (Linear Discrimination Analysis) coordinate system between the data clusters obtained by the response of the microarray to four target vapors: ethanol, acetone, propanol and ammonia. It is shown that the application of a temperature gradient increases the gas discrimination power of the microarray by up to 35 %.

Chondrocyte channel transcriptomics

PubMed Central

Lewis, Rebecca; May, Hannah; Mobasheri, Ali; Barrett-Jolley, Richard

2013-01-01

To date, a range of ion channels have been identified in chondrocytes using a number of different techniques, predominantly electrophysiological and/or biomolecular; each of these has its advantages and disadvantages. Here we aim to compare and contrast the data available from biophysical and microarray experiments. This letter analyses recent transcriptomics datasets from chondrocytes, accessible from the European Bioinformatics Institute (EBI). We discuss whether such bioinformatic analysis of microarray datasets can potentially accelerate identification and discovery of ion channels in chondrocytes. The ion channels which appear most frequently across these microarray datasets are discussed, along with their possible functions. We discuss whether functional or protein data exist which support the microarray data. A microarray experiment comparing gene expression in osteoarthritis and healthy cartilage is also discussed and we verify the differential expression of 2 of these genes, namely the genes encoding large calcium-activated potassium (BK) and aquaporin channels. PMID:23995703

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform.

PubMed

Salehi, Reza; Tsoi, Stephen C M; Colazo, Marcos G; Ambrose, Divakar J; Robert, Claude; Dyck, Michael K

2017-01-30

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Clustering gene expression data based on predicted differential effects of GV interaction.

PubMed

Pan, Hai-Yan; Zhu, Jun; Han, Dan-Fu

2005-02-01

Microarray has become a popular biotechnology in biological and medical research. However, systematic and stochastic variabilities in microarray data are expected and unavoidable, resulting in the problem that the raw measurements have inherent "noise" within microarray experiments. Currently, logarithmic ratios are usually analyzed by various clustering methods directly, which may introduce bias interpretation in identifying groups of genes or samples. In this paper, a statistical method based on mixed model approaches was proposed for microarray data cluster analysis. The underlying rationale of this method is to partition the observed total gene expression level into various variations caused by different factors using an ANOVA model, and to predict the differential effects of GV (gene by variety) interaction using the adjusted unbiased prediction (AUP) method. The predicted GV interaction effects can then be used as the inputs of cluster analysis. We illustrated the application of our method with a gene expression dataset and elucidated the utility of our approach using an external validation.

Comparative analysis of chrysanthemum transcriptome in response to three RNA viruses: Cucumber mosaic virus, Tomato spotted wilt virus and Potato virus X.

PubMed

Choi, Hoseong; Jo, Yeonhwa; Lian, Sen; Jo, Kyoung-Min; Chu, Hyosub; Yoon, Ju-Yeon; Choi, Seung-Kook; Kim, Kook-Hyung; Cho, Won Kyong

2015-06-01

The chrysanthemum is one of popular flowers in the world and a host for several viruses. So far, molecular interaction studies between the chrysanthemum and viruses are limited. In this study, we carried out a transcriptome analysis of chrysanthemum in response to three different viruses including Cucumber mosaic virus (CMV), Tomato spotted wilt virus (TSWV) and Potato virus X (PVX). A chrysanthemum 135K microarray derived from expressed sequence tags was successfully applied for the expression profiles of the chrysanthemum at early stage of virus infection. Finally, we identified a total of 125, 70 and 124 differentially expressed genes (DEGs) for CMV, TSWV and PVX, respectively. Many DEGs were virus specific; however, 33 DEGs were commonly regulated by three viruses. Gene ontology (GO) enrichment analysis identified a total of 132 GO terms, and of them, six GO terms related stress response and MCM complex were commonly identified for three viruses. Several genes functioning in stress response such as chitin response and ethylene mediated signaling pathway were up-regulated indicating their involvement in establishment of host immune system. In particular, TSWV infection significantly down-regulated genes related to DNA metabolic process including DNA replication, chromatin organization, histone modification and cytokinesis, and they are mostly targeted to nucleosome and MCM complex. Taken together, our comparative transcriptome analysis revealed several genes related to hormone mediated viral stress response and DNA modification. The identified chrysanthemums genes could be good candidates for further functional study associated with resistant to various plant viruses.

GenePublisher: Automated analysis of DNA microarray data.

PubMed

Knudsen, Steen; Workman, Christopher; Sicheritz-Ponten, Thomas; Friis, Carsten

2003-07-01

GenePublisher, a system for automatic analysis of data from DNA microarray experiments, has been implemented with a web interface at http://www.cbs.dtu.dk/services/GenePublisher. Raw data are uploaded to the server together with a specification of the data. The server performs normalization, statistical analysis and visualization of the data. The results are run against databases of signal transduction pathways, metabolic pathways and promoter sequences in order to extract more information. The results of the entire analysis are summarized in report form and returned to the user.

Differential gene expression in granulosa cells from polycystic ovary syndrome patients with and without insulin resistance: identification of susceptibility gene sets through network analysis.

PubMed

Kaur, Surleen; Archer, Kellie J; Devi, M Gouri; Kriplani, Alka; Strauss, Jerome F; Singh, Rita

2012-10-01

Polycystic ovary syndrome (PCOS) is a heterogeneous, genetically complex, endocrine disorder of uncertain etiology in women. Our aim was to compare the gene expression profiles in stimulated granulosa cells of PCOS women with and without insulin resistance vs. matched controls. This study included 12 normal ovulatory women (controls), 12 women with PCOS without evidence for insulin resistance (PCOS non-IR), and 16 women with insulin resistance (PCOS-IR) undergoing in vitro fertilization. Granulosa cell gene expression profiling was accomplished using Affymetrix Human Genome-U133 arrays. Differentially expressed genes were classified according to gene ontology using ingenuity pathway analysis tools. Microarray results for selected genes were confirmed by real-time quantitative PCR. A total of 211 genes were differentially expressed in PCOS non-IR and PCOS-IR granulosa cells (fold change≥1.5; P≤0.001) vs. matched controls. Diabetes mellitus and inflammation genes were significantly increased in PCOS-IR patients. Real-time quantitative PCR confirmed higher expression of NCF2 (2.13-fold), TCF7L2 (1.92-fold), and SERPINA1 (5.35-fold). Increased expression of inflammation genes ITGAX (3.68-fold) and TAB2 (1.86-fold) was confirmed in PCOS non-IR. Different cardiometabolic disease genes were differentially expressed in the two groups. Decreased expression of CAV1 (-3.58-fold) in PCOS non-IR and SPARC (-1.88-fold) in PCOS-IR was confirmed. Differential expression of genes involved in TGF-β signaling (IGF2R, increased; and HAS2, decreased), and oxidative stress (TXNIP, increased) was confirmed in both groups. Microarray analysis demonstrated differential expression of genes linked to diabetes mellitus, inflammation, cardiovascular diseases, and infertility in the granulosa cells of PCOS women with and without insulin resistance. Because these dysregulated genes are also involved in oxidative stress, lipid metabolism, and insulin signaling, we hypothesize that these genes may be involved in follicular growth arrest and metabolic disorders associated with the different phenotypes of PCOS.

Transcriptomic and Proteomic Analysis of Oenococcus oeni Adaptation to Wine Stress Conditions

PubMed Central

Margalef-Català, Mar; Araque, Isabel; Bordons, Albert; Reguant, Cristina; Bautista-Gallego, Joaquín

2016-01-01

Oenococcus oeni, the main lactic acid bacteria responsible for malolactic fermentation in wine, has to adapt to stressful conditions, such as low pH and high ethanol content. In this study, the changes in the transcriptome and the proteome of O. oeni PSU-1 during the adaptation period before MLF start have been studied. DNA microarrays were used for the transcriptomic analysis and two complementary proteomic techniques, 2-D DIGE and iTRAQ labeling were used to analyze the proteomic response. One of the most influenced functions in PSU-1 due to inoculation into wine-like medium (WLM) was translation, showing the over-expression of certain ribosomal genes and the corresponding proteins. Amino acid metabolism and transport was also altered and several peptidases were up regulated both at gene and protein level. Certain proteins involved in glutamine and glutamate metabolism showed an increased abundance revealing the key role of nitrogen uptake under stressful conditions. A strong transcriptional inhibition of carbohydrate metabolism related genes was observed. On the other hand, the transcriptional up-regulation of malate transport and citrate consumption was indicative of the use of L-malate and citrate associated to stress response and as an alternative energy source to sugar metabolism. Regarding the stress mechanisms, our results support the relevance of the thioredoxin and glutathione systems in the adaptation of O. oeni to wine related stress. Genes and proteins related to cell wall showed also significant changes indicating the relevance of the cell envelop as protective barrier to environmental stress. The differences found between transcriptomic and proteomic data suggested the relevance of post-transcriptional mechanisms and the complexity of the stress response in O. oeni adaptation. Further research should deepen into the metabolisms mostly altered due to wine conditions to elucidate the role of each mechanism in the O. oeni ability to develop MLF. PMID:27746771

Peptidoglycan microarray as a novel tool to explore protein-ligand recognition.

PubMed

Wang, Ning; Hirata, Akiyoshi; Nokihara, Kiyoshi; Fukase, Koichi; Fujimoto, Yukari

2016-11-04

Peptidoglycan is a giant bag-shaped molecule essential for bacterial cell shape and resistance to osmotic stresses. The activity of a large number of bacterial surface proteins involved in cell growth and division requires binding to this macromolecule. Recognition of peptidoglycan by immune effectors is also crucial for the establishment of the immune response against pathogens. The availability of pure and chemically defined peptidoglycan fragments is a major technical bottleneck that has precluded systematic studies of the mechanisms underpinning protein-mediated peptidoglycan recognition. Here, we report a microarray strategy suitable to carry out comprehensive studies to characterize proteins-peptidoglycan interactions. We describe a method to introduce a functional group on peptidoglycan fragments allowing their stable immobilization on amorphous carbon chip plates to minimize nonspecific binding. Such peptidoglycan microarrays were used with a model peptidoglycan binding protein-the human peptidoglycan recognition protein-S (hPGRP-S). We propose that this strategy could be implemented to carry out high-throughput analyses to study peptidoglycan binding proteins. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 422-429, 2016. © 2016 Wiley Periodicals, Inc.

Prenatal exposure to escitalopram and/or stress in rats produces limited effects on endocrine, behavioral, or gene expression measures in adult male rats

PubMed Central

Bourke, Chase H.; Stowe, Zachary N.; Neigh, Gretchen N.; Olson, Darin E.; Owens, Michael J.

2013-01-01

Stress and/or antidepressants during pregnancy have been implicated in a wide range of long-term effects in the offspring. We investigated the long-term effects of prenatal stress and/or clinically relevant antidepressant exposure on male adult offspring in a model of the pharmacotherapy of maternal depression. Female Sprague-Dawley rats were implanted with osmotic minipumps that delivered clinically relevant exposure to the antidepressant escitalopram throughout gestation. Subsequently, pregnant females were exposed on gestational days 10–20 to a chronic unpredictable mild stress paradigm. The male offspring were analyzed in adulthood. Baseline physiological measurements were largely unaltered by prenatal manipulations. Behavioral characterization of the male offspring, with or without pre-exposure to an acute stressor, did not reveal any group differences. Prenatal stress exposure resulted in a faster return towards baseline following the peak response to an acute restraint stressor, but not an airpuff startle stressor, in adulthood. Microarray analysis of the hippocampus and hypothalamus comparing all treatment groups revealed no significantly-altered transcripts. Real time PCR of the hippocampus confirmed that several transcripts in the CRFergic, serotonergic, and neural plasticity pathways were unaffected by prenatal exposures. This stress model of maternal depression and its treatment indicate that escitalopram use and/or stress during pregnancy produced no alterations in our measures of male adult behavior or the transcriptome, however prenatal stress exposure resulted in some evidence for increased glucocorticoid negative feedback following an acute restraint stress. Study design should be carefully considered before implications for human health are ascribed to prenatal exposure to stress or antidepressant medication. PMID:23906943

Expression of stress/defense-related genes in barley grown under space environment

NASA Astrophysics Data System (ADS)

Sugimoto, Manabu; Shagimardanova, Elena; Gusev, Oleg; Bingham, Gail; Levinskikh, Margarita; Sychev, Vladimir

Plants are exposed to the extreme environment in space, especially space radiation is suspected to induce oxidative stress by generating high-energy free radicals and microgravity would enhance the effect of space radiation, however, current understandings of plant growth and responses on this synergistic effect of radiation and microgravity is limited to a few experiments. In this study, expression of stress/defense-related genes in barley grown under space environment was analyzed by RT-PCR and DNA microarray experiments to understand plant responses and adaptation to space environment and to develop the space stress-tolerant plants. The seeds of barley, Hordeum vulgare L. cv. Haruna nijo, kept in the international space station (ISS) over 4 months, were germinated after 3 days of irrigation in LADA plant growth chamber onboard Russian segment of ISS and the final germination ratio was over 90 %. The height of plants was about 50 to 60 cm and flag leaf has been opened after 26 days of irrigation under 24 hr lighting, showing the similar growth to ground-grown barley. Expression levels of stress/defense-related genes in space-grown barley were compared to those in ground-grown barley by semi-quantitative RT-PCR. In 17 stress/defense-related genes that are up-regulated by oxidative stress or other abiotic stress, only catalase, pathogenesis-related protein 13, chalcone synthase, and phenylalanine ammonia-lyase genes were increased in space-grown barley. DNA microarrya analysis with the GeneChip Barley Genome Array showed the similar expression profiles of the stress/defense-related genes to those by RT-PCR experiment, suggesting that the barley germinated and grown in LADA onboard ISS is not damaged by space environment, especially oxidative stress induced by space radiation and microgravity.

Markers of inflammation and stress distinguish subsets of individuals with schizophrenia and bipolar disorder

PubMed Central

Fillman, S G; Sinclair, D; Fung, S J; Webster, M J; Shannon Weickert, C

2014-01-01

Schizophrenia and bipolar disorder share a number of common features, both symptomatically and biologically. Abnormalities in the neuroimmune and the stress-signaling pathways have been previously identified in brains of individuals with both diseases. However, the possible relationship between abnormalities in stress and neuroimmune signaling within the cortex of people with psychotic illness has not been defined. To test the hypothesis that combined alterations in brain stress responsiveness and neuroimmune/inflammatory status are characteristic of some individuals suffering from major mental illness, we examined gene expression in the Stanley Array Cohort of 35 controls, 35 individuals with schizophrenia and 34 individuals with bipolar disorder. We used levels of 8 inflammatory-related transcripts, of which SERPINA3 was significantly elevated in individuals with schizophrenia (F(2,88)=4.137, P<0.05), and 12 glucocorticoid receptor signaling (stress) pathway transcripts previously examined, to identify two clusters of individuals: a high inflammation/stress group (n=32) and a low (n=68) inflammation/stress group. The high inflammation/stress group has a significantly greater number of individuals with schizophrenia (n=15), and a trend toward having more bipolar disorder individuals (n=11), when compared with controls (n=6). Using these subgroups, we tested which microarray-assessed transcriptional changes may be associated with high inflammatory/stress groups using ingenuity analysis and found that an extended network of gene expression changes involving immune, growth factors, inhibitory signaling and cell death factors also distinguished these groups. Our work demonstrates that some of the heterogeneity in schizophrenia and bipolar disorder may be partially explained by inflammation/stress interactions, and that this biological subtype cuts across Diagnostic and Statistical Manual of Mental Disorders (DSM)-defined categories. PMID:24569695

Microarray gene-expression study in fibroblast and lymphoblastoid cell lines from antipsychotic-naïve first-episode schizophrenia patients.

PubMed

Gassó, Patricia; Mas, Sergi; Rodríguez, Natalia; Boloc, Daniel; García-Cerro, Susana; Bernardo, Miquel; Lafuente, Amalia; Parellada, Eduard

2017-12-01

Schizophrenia (SZ) is a chronic psychiatric disorder whose onset of symptoms occurs in late adolescence and early adulthood. The etiology is complex and involves important gene-environment interactions. Microarray gene-expression studies on SZ have identified alterations in several biological processes. The heterogeneity in the results can be attributed to the use of different sample types and other important confounding factors including age, illness chronicity and antipsychotic exposure. The aim of the present microarray study was to analyze, for the first time to our knowledge, differences in gene expression profiles in 18 fibroblast (FCLs) and 14 lymphoblastoid cell lines (LCLs) from antipsychotic-naïve first-episode schizophrenia (FES) patients and healthy controls. We used an analytical approach based on protein-protein interaction network construction and functional annotation analysis to identify the biological processes that are altered in SZ. Significant differences in the expression of 32 genes were found when LCLs were assessed. The network and gene set enrichment approach revealed the involvement of similar biological processes in FCLs and LCLs, including apoptosis and related biological terms such as cell cycle, autophagy, cytoskeleton organization and response to stress and stimulus. Metabolism and other processes, including signal transduction, kinase activity and phosphorylation, were also identified. These results were replicated in two independent cohorts using the same analytical approach. This provides more evidence for altered apoptotic processes in antipsychotic-naïve FES patients and other important biological functions such as cytoskeleton organization and metabolism. The convergent results obtained in both peripheral cell models support their usefulness for transcriptome studies on SZ. Copyright © 2017 Elsevier Ltd. All rights reserved.

Microarray characterization of gene expression changes in blood during acute ethanol exposure

PubMed Central

2013-01-01

Background As part of the civil aviation safety program to define the adverse effects of ethanol on flying performance, we performed a DNA microarray analysis of human whole blood samples from a five-time point study of subjects administered ethanol orally, followed by breathalyzer analysis, to monitor blood alcohol concentration (BAC) to discover significant gene expression changes in response to the ethanol exposure. Methods Subjects were administered either orange juice or orange juice with ethanol. Blood samples were taken based on BAC and total RNA was isolated from PaxGene™ blood tubes. The amplified cDNA was used in microarray and quantitative real-time polymerase chain reaction (RT-qPCR) analyses to evaluate differential gene expression. Microarray data was analyzed in a pipeline fashion to summarize and normalize and the results evaluated for relative expression across time points with multiple methods. Candidate genes showing distinctive expression patterns in response to ethanol were clustered by pattern and further analyzed for related function, pathway membership and common transcription factor binding within and across clusters. RT-qPCR was used with representative genes to confirm relative transcript levels across time to those detected in microarrays. Results Microarray analysis of samples representing 0%, 0.04%, 0.08%, return to 0.04%, and 0.02% wt/vol BAC showed that changes in gene expression could be detected across the time course. The expression changes were verified by qRT-PCR. The candidate genes of interest (GOI) identified from the microarray analysis and clustered by expression pattern across the five BAC points showed seven coordinately expressed groups. Analysis showed function-based networks, shared transcription factor binding sites and signaling pathways for members of the clusters. These include hematological functions, innate immunity and inflammation functions, metabolic functions expected of ethanol metabolism, and pancreatic and hepatic function. Five of the seven clusters showed links to the p38 MAPK pathway. Conclusions The results of this study provide a first look at changing gene expression patterns in human blood during an acute rise in blood ethanol concentration and its depletion because of metabolism and excretion, and demonstrate that it is possible to detect changes in gene expression using total RNA isolated from whole blood. The analysis approach for this study serves as a workflow to investigate the biology linked to expression changes across a time course and from these changes, to identify target genes that could serve as biomarkers linked to pilot performance. PMID:23883607

Single-Nucleotide Polymorphism-Microarray Ploidy Analysis of Paraffin-Embedded Products of Conception in Recurrent Pregnancy Loss Evaluations.

PubMed

Maslow, Bat-Sheva L; Budinetz, Tara; Sueldo, Carolina; Anspach, Erica; Engmann, Lawrence; Benadiva, Claudio; Nulsen, John C

2015-07-01

To compare the analysis of chromosome number from paraffin-embedded products of conception using single-nucleotide polymorphism (SNP) microarray with the recommended screening for the evaluation of couples presenting with recurrent pregnancy loss who do not have previous fetal cytogenetic data. We performed a retrospective cohort study including all women who presented for a new evaluation of recurrent pregnancy loss over a 2-year period (January 1, 2012, to December 31, 2013). All participants had at least two documented first-trimester losses and both the recommended screening tests and SNP microarray performed on at least one paraffin-embedded products of conception sample. Single-nucleotide polymorphism microarray identifies all 24 chromosomes (22 autosomes, X, and Y). Forty-two women with a total of 178 losses were included in the study. Paraffin-embedded products of conception from 62 losses were sent for SNP microarray. Single-nucleotide polymorphism microarray successfully diagnosed fetal chromosome number in 71% (44/62) of samples, of which 43% (19/44) were euploid and 57% (25/44) were noneuploid. Seven of 42 (17%) participants had abnormalities on recurrent pregnancy loss screening. The per-person detection rate for a cause of pregnancy loss was significantly higher in the SNP microarray (0.50; 95% confidence interval [CI] 0.36-0.64) compared with recurrent pregnancy loss evaluation (0.17; 95% CI 0.08-0.31) (P=.002). Participants with one or more euploid loss identified on paraffin-embedded products of conception were significantly more likely to have an abnormality on recurrent pregnancy loss screening than those with only noneuploid results (P=.028). The significance remained when controlling for age, number of losses, number of samples, and total pregnancies. These results suggest that SNP microarray testing of paraffin-embedded products of conception is a valuable tool for the evaluation of recurrent pregnancy loss in patients without prior fetal cytogenetic results. Recommended recurrent pregnancy loss screening was unnecessary in almost half the patients in our study. II.

eSensor: an electrochemical detection-based DNA microarray technology enabling sample-to-answer molecular diagnostics

NASA Astrophysics Data System (ADS)

Liu, Robin H.; Longiaru, Mathew

2009-05-01

DNA microarrays are becoming a widespread tool used in life science and drug screening due to its many benefits of miniaturization and integration. Microarrays permit a highly multiplexed DNA analysis. Recently, the development of new detection methods and simplified methodologies has rapidly expanded the use of microarray technologies from predominantly gene expression analysis into the arena of diagnostics. Osmetech's eSensor® is an electrochemical detection platform based on a low-to- medium density DNA hybridization array on a cost-effective printed circuit board substrate. eSensor® has been cleared by FDA for Warfarin sensitivity test and Cystic Fibrosis Carrier Detection. Other genetic-based diagnostic and infectious disease detection tests are under development. The eSensor® platform eliminates the need for an expensive laser-based optical system and fluorescent reagents. It allows one to perform hybridization and detection in a single and small instrument without any fluidic processing and handling. Furthermore, the eSensor® platform is readily adaptable to on-chip sample-to-answer genetic analyses using microfluidics technology. The eSensor® platform provides a cost-effective solution to direct sample-to-answer genetic analysis, and thus have a potential impact in the fields of point-of-care genetic analysis, environmental testing, and biological warfare agent detection.

Towards the integration, annotation and association of historical microarray experiments with RNA-seq.

PubMed

Chavan, Shweta S; Bauer, Michael A; Peterson, Erich A; Heuck, Christoph J; Johann, Donald J

2013-01-01

Transcriptome analysis by microarrays has produced important advances in biomedicine. For instance in multiple myeloma (MM), microarray approaches led to the development of an effective disease subtyping via cluster assignment, and a 70 gene risk score. Both enabled an improved molecular understanding of MM, and have provided prognostic information for the purposes of clinical management. Many researchers are now transitioning to Next Generation Sequencing (NGS) approaches and RNA-seq in particular, due to its discovery-based nature, improved sensitivity, and dynamic range. Additionally, RNA-seq allows for the analysis of gene isoforms, splice variants, and novel gene fusions. Given the voluminous amounts of historical microarray data, there is now a need to associate and integrate microarray and RNA-seq data via advanced bioinformatic approaches. Custom software was developed following a model-view-controller (MVC) approach to integrate Affymetrix probe set-IDs, and gene annotation information from a variety of sources. The tool/approach employs an assortment of strategies to integrate, cross reference, and associate microarray and RNA-seq datasets. Output from a variety of transcriptome reconstruction and quantitation tools (e.g., Cufflinks) can be directly integrated, and/or associated with Affymetrix probe set data, as well as necessary gene identifiers and/or symbols from a diversity of sources. Strategies are employed to maximize the annotation and cross referencing process. Custom gene sets (e.g., MM 70 risk score (GEP-70)) can be specified, and the tool can be directly assimilated into an RNA-seq pipeline. A novel bioinformatic approach to aid in the facilitation of both annotation and association of historic microarray data, in conjunction with richer RNA-seq data, is now assisting with the study of MM cancer biology.

Basic leucine zipper family in barley: genome-wide characterization of members and expression analysis.

PubMed

Pourabed, Ehsan; Ghane Golmohamadi, Farzan; Soleymani Monfared, Peyman; Razavi, Seyed Morteza; Shobbar, Zahra-Sadat

2015-01-01

The basic leucine zipper (bZIP) family is one of the largest and most diverse transcription factors in eukaryotes participating in many essential plant processes. We identified 141 bZIP proteins encoded by 89 genes from the Hordeum vulgare genome. HvbZIPs were classified into 11 groups based on their DNA-binding motif. Amino acid sequence alignment of the HvbZIPs basic-hinge regions revealed some highly conserved residues within each group. The leucine zipper heptads were analyzed predicting their dimerization properties. 34 conserved motifs were identified outside the bZIP domain. Phylogenetic analysis indicated that major diversification within the bZIP family predated the monocot/dicot divergence, although intra-species duplication and parallel evolution seems to be occurred afterward. Localization of HvbZIPs on the barley chromosomes revealed that different groups have been distributed on seven chromosomes of barley. Six types of intron pattern were detected within the basic-hinge regions. Most of the detected cis-elements in the promoter and UTR sequences were involved in seed development or abiotic stress response. Microarray data analysis revealed differential expression pattern of HvbZIPs in response to ABA treatment, drought, and cold stresses and during barley grain development and germination. This information would be helpful for functional characterization of bZIP transcription factors in barley.

Response of human renal tubular cells to cyclosporine and sirolimus: A toxicogenomic study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pallet, Nicolas; Rabant, Marion; Xu-Dubois, Yi-Chun

The molecular mechanisms involved in the potentially nephrotoxic response of tubular cells to immunosuppressive drugs remain poorly understood. Transcriptional profiles of human proximal tubular cells exposed to cyclosporine A (CsA), sirolimus (SRL) or their combination, were established using oligonucleotide microarrays. Hierarchical clustering of genes implicated in fibrotic processes showed a clear distinction between expression profiles with CsA and CsA + SRL treatments on the one hand and SRL treatment on the other. Functional analysis found that CsA and CsA + SRL treatments preferentially alter biological processes located at the cell membrane, such as ion transport or signal transduction, whereas SRLmore » modifies biological processes within the nucleus and related to transcriptional activity. Genome wide expression analysis suggested that CsA may induce an endoplasmic reticulum (ER) stress in tubular cells in vitro. Moreover we found that CsA exposure in vivo is associated with the upregulation of the ER stress marker BIP in kidney transplant biopsies. In conclusion, this toxicogenomic study highlights the molecular interaction networks that may contribute to the tubular response to CsA and SRL. These results may also offer a new working hypothesis for future research in the field of CsA nephrotoxicity. Further studies are needed to evaluate if ER stress detection in tubular cells in human biopsies can predict CsA nephrotoxicity.« less

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards.

PubMed

Bruland, Torunn; Anderssen, Endre; Doseth, Berit; Bergum, Hallgeir; Beisvag, Vidar; Laegreid, Astrid

2007-10-18

The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish.

Optimization of cDNA microarrays procedures using criteria that do not rely on external standards

PubMed Central

Bruland, Torunn; Anderssen, Endre; Doseth, Berit; Bergum, Hallgeir; Beisvag, Vidar; Lægreid, Astrid

2007-01-01

Background The measurement of gene expression using microarray technology is a complicated process in which a large number of factors can be varied. Due to the lack of standard calibration samples such as are used in traditional chemical analysis it may be a problem to evaluate whether changes done to the microarray procedure actually improve the identification of truly differentially expressed genes. The purpose of the present work is to report the optimization of several steps in the microarray process both in laboratory practices and in data processing using criteria that do not rely on external standards. Results We performed a cDNA microarry experiment including RNA from samples with high expected differential gene expression termed "high contrasts" (rat cell lines AR42J and NRK52E) compared to self-self hybridization, and optimized a pipeline to maximize the number of genes found to be differentially expressed in the "high contrasts" RNA samples by estimating the false discovery rate (FDR) using a null distribution obtained from the self-self experiment. The proposed high-contrast versus self-self method (HCSSM) requires only four microarrays per evaluation. The effects of blocking reagent dose, filtering, and background corrections methodologies were investigated. In our experiments a dose of 250 ng LNA (locked nucleic acid) dT blocker, no background correction and weight based filtering gave the largest number of differentially expressed genes. The choice of background correction method had a stronger impact on the estimated number of differentially expressed genes than the choice of filtering method. Cross platform microarray (Illumina) analysis was used to validate that the increase in the number of differentially expressed genes found by HCSSM was real. Conclusion The results show that HCSSM can be a useful and simple approach to optimize microarray procedures without including external standards. Our optimizing method is highly applicable to both long oligo-probe microarrays which have become commonly used for well characterized organisms such as man, mouse and rat, as well as to cDNA microarrays which are still of importance for organisms with incomplete genome sequence information such as many bacteria, plants and fish. PMID:17949480

Differential transcriptomic responses of Biomphalaria glabrata (Gastropoda, Mollusca) to bacteria and metazoan parasites, Schistosoma mansoni and Echinostoma paraensei (Digenea, Platyhelminthes)

PubMed Central

Adema, Coen M; Hanington, Patrick C.; Lun, Cheng-Man; Rosenberg, George H.; Aragon, Anthony D; Stout, Barbara A; Richard, Mara L. Lennard; Gross, Paul S.; Loker, Eric S

2009-01-01

A 70-mer oligonucleotide-based microarray (1152 features) that emphasizes stress and immune responses factors was constructed to study transcriptomic responses of the snail Biomphalaria glabrata to different immune challenges. In addition to sequences with relevant putative ID and Gene Ontology (GO) annotation, the array features non-immune factors and unknown B. glabrata ESTs for functional gene discovery. The transcription profiles of B. glabrata (3 biological replicates, each a pool of 5 snails) were recorded at 12 hours post wounding, exposure to Gram negative or Gram positive bacteria (Escherichia coli and Micrococcus luteus, respectively), or infection with compatible trematode parasites (S. mansoni or E. paraensei, 20 miracidia/snail), relative to controls, using universal reference RNA. The data were subjected to Significance Analysis for Microarrays (SAM), with a false positive rate (FPR) ≤10%. Wounding yielded a modest differential expression profile (27 up/21 down) with affected features mostly dissimilar from other treatments. Partially overlapping, yet distinct expression profiles were recorded from snails challenged with E. coli (83 up/20 down) or M. luteus (120 up/42 down), mostly showing up-regulation of defense and stress-related features. Significantly altered expression of selected immune features indicates that B. glabrata detects and responds differently to compatible trematodes. Echinostoma paraensei infection was associated mostly with down regulation of many (immune-) transcripts (42 up/68 down), whereas S. mansoni exposure yielded a preponderance of up-regulated features (140 up/23 down), with only few known immune genes affected. These observations may reflect the divergent strategies developed by trematodes during their evolution as specialized pathogens of snails to negate host defense responses. Clearly, the immune defenses of B. glabrata distinguish and respond differently to various immune challenges. PMID:19962194

Exposure to Cobalt Causes Transcriptomic and Proteomic Changes in Two Rat Liver Derived Cell Lines

PubMed Central

Permenter, Matthew G.; Dennis, William E.; Sutto, Thomas E.; Jackson, David A.; Lewis, John A.; Stallings, Jonathan D.

2013-01-01

Cobalt is a transition group metal present in trace amounts in the human diet, but in larger doses it can be acutely toxic or cause adverse health effects in chronic exposures. Its use in many industrial processes and alloys worldwide presents opportunities for occupational exposures, including military personnel. While the toxic effects of cobalt have been widely studied, the exact mechanisms of toxicity remain unclear. In order to further elucidate these mechanisms and identify potential biomarkers of exposure or effect, we exposed two rat liver-derived cell lines, H4-II-E-C3 and MH1C1, to two concentrations of cobalt chloride. We examined changes in gene expression using DNA microarrays in both cell lines and examined changes in cytoplasmic protein abundance in MH1C1 cells using mass spectrometry. We chose to closely examine differentially expressed genes and proteins changing in abundance in both cell lines in order to remove cell line specific effects. We identified enriched pathways, networks, and biological functions using commercial bioinformatic tools and manual annotation. Many of the genes, proteins, and pathways modulated by exposure to cobalt appear to be due to an induction of a hypoxic-like response and oxidative stress. Genes that may be differentially expressed due to a hypoxic-like response are involved in Hif-1α signaling, glycolysis, gluconeogenesis, and other energy metabolism related processes. Gene expression changes linked to oxidative stress are also known to be involved in the NRF2-mediated response, protein degradation, and glutathione production. Using microarray and mass spectrometry analysis, we were able to identify modulated genes and proteins, further elucidate the mechanisms of toxicity of cobalt, and identify biomarkers of exposure and effect in vitro, thus providing targets for focused in vivo studies. PMID:24386269

Changes in Global Transcriptional Profiling of Women Following Obesity Surgery Bypass.

PubMed

Pinhel, Marcela Augusta de Souza; Noronha, Natalia Yumi; Nicoletti, Carolina Ferreira; de Oliveira, Bruno Affonso Parente; Cortes-Oliveira, Cristiana; Pinhanelli, Vitor Caressato; Salgado Junior, Wilson; Machry, Ana Julia; da Silva Junior, Wilson Araújo; Souza, Dorotéia Rossi Silva; Marchini, Júlio Sérgio; Nonino, Carla Barbosa

2018-01-01

Differential gene expression in peripheral blood mononuclear cells (PBMCs) after Roux-en-Y gastric bypass (RYGB) is poorly characterized. Markers of these processes may provide a deeper understanding of the mechanisms that underlie these events. The main goal of this study was to identify changes in PBMC gene expression in women with obesity before and 6 months after RYGB-induced weight loss. The ribonucleic acid (RNA) of PBMCs from 13 obese women was analyzed before and 6 months after RYGB; the RNA of PBMCs from nine healthy women served as control. The gene expression levels were determined by microarray analysis. Significant differences in gene expression were validated by real-time quantitative polymerase chain reaction (RT-qPCR). Microarray analysis for comparison of the pre- and postoperative periods showed that 1366 genes were differentially expressed genes (DEGs). The main pathways were related to gene transcription; lipid, energy, and glycide metabolism; inflammatory and immunological response; cell differentiation; oxidative stress regulation; response to endogenous and exogenous stimuli; substrate oxidation; mTOR signaling pathway; interferon signaling; mitogen-activated protein kinases (MAPK), cAMP response element binding protein (CREB1), heat shock factor 1 (HSF1), and sterol regulatory element binding protein 1c (SREBP-1c) gene expression; adipocyte differentiation; and methylation. Six months after bariatric surgery and significant weight loss, many molecular pathways involved in obesity and metabolic diseases change. These findings are an important tool to identify potential targets for therapeutic intervention and clinical practice of nutritional genomics in obesity.

Putative DHHC-Cysteine-Rich Domain S-Acyltransferase in Plants

PubMed Central

Sun, Meihong; Liu, Shiyang; Qi, Baoxiu; Li, Xinzheng

2013-01-01

Protein S-acyltransferases (PATs) containing Asp-His-His-Cys within a Cys-rich domain (DHHC-CRD) are polytopic transmembrane proteins that are found in eukaryotic cells and mediate the S-acylation of target proteins. S-acylation is an important secondary and reversible modification that regulates the membrane association, trafficking and function of target proteins. However, little is known about the characteristics of PATs in plants. Here, we identified 804 PATs from 31 species with complete genomes. The analysis of the phylogenetic relationships suggested that all of the PATs fell into 8 groups. In addition, we analysed the phylogeny, genomic organization, chromosome localisation and expression pattern of PATs in Arabidopsis, Oryza sative, Zea mays and Glycine max. The microarray data revealed that PATs genes were expressed in different tissues and during different life stages. The preferential expression of the ZmPATs in specific tissues and the response of Zea mays to treatments with phytohormones and abiotic stress demonstrated that the PATs play roles in plant growth and development as well as in stress responses. Our data provide a useful reference for the identification and functional analysis of the members of this protein family. PMID:24155879

Phenotypic characterisation of Saccharomyces spp. yeast for tolerance to stresses encountered during fermentation of lignocellulosic residues to produce bioethanol

PubMed Central

2014-01-01

Background During industrial fermentation of lignocellulose residues to produce bioethanol, microorganisms are exposed to a number of factors that influence productivity. These include inhibitory compounds produced by the pre-treatment processes required to release constituent carbohydrates from biomass feed-stocks and during fermentation, exposure of the organisms to stressful conditions. In addition, for lignocellulosic bioethanol production, conversion of both pentose and hexose sugars is a pre-requisite for fermentative organisms for efficient and complete conversion. All these factors are important to maximise industrial efficiency, productivity and profit margins in order to make second-generation bioethanol an economically viable alternative to fossil fuels for future transport needs. Results The aim of the current study was to assess Saccharomyces yeasts for their capacity to tolerate osmotic, temperature and ethanol stresses and inhibitors that might typically be released during steam explosion of wheat straw. Phenotypic microarray analysis was used to measure tolerance as a function of growth and metabolic activity. Saccharomyces strains analysed in this study displayed natural variation to each stress condition common in bioethanol fermentations. In addition, many strains displayed tolerance to more than one stress, such as inhibitor tolerance combined with fermentation stresses. Conclusions Our results suggest that this study could identify a potential candidate strain or strains for efficient second generation bioethanol production. Knowledge of the Saccharomyces spp. strains grown in these conditions will aid the development of breeding programmes in order to generate more efficient strains for industrial fermentations. PMID:24670111

ArraySolver: an algorithm for colour-coded graphical display and Wilcoxon signed-rank statistics for comparing microarray gene expression data.

PubMed

Khan, Haseeb Ahmad

2004-01-01

The massive surge in the production of microarray data poses a great challenge for proper analysis and interpretation. In recent years numerous computational tools have been developed to extract meaningful interpretation of microarray gene expression data. However, a convenient tool for two-groups comparison of microarray data is still lacking and users have to rely on commercial statistical packages that might be costly and require special skills, in addition to extra time and effort for transferring data from one platform to other. Various statistical methods, including the t-test, analysis of variance, Pearson test and Mann-Whitney U test, have been reported for comparing microarray data, whereas the utilization of the Wilcoxon signed-rank test, which is an appropriate test for two-groups comparison of gene expression data, has largely been neglected in microarray studies. The aim of this investigation was to build an integrated tool, ArraySolver, for colour-coded graphical display and comparison of gene expression data using the Wilcoxon signed-rank test. The results of software validation showed similar outputs with ArraySolver and SPSS for large datasets. Whereas the former program appeared to be more accurate for 25 or fewer pairs (n < or = 25), suggesting its potential application in analysing molecular signatures that usually contain small numbers of genes. The main advantages of ArraySolver are easy data selection, convenient report format, accurate statistics and the familiar Excel platform.

ArraySolver: An Algorithm for Colour-Coded Graphical Display and Wilcoxon Signed-Rank Statistics for Comparing Microarray Gene Expression Data

PubMed Central

2004-01-01

The massive surge in the production of microarray data poses a great challenge for proper analysis and interpretation. In recent years numerous computational tools have been developed to extract meaningful interpretation of microarray gene expression data. However, a convenient tool for two-groups comparison of microarray data is still lacking and users have to rely on commercial statistical packages that might be costly and require special skills, in addition to extra time and effort for transferring data from one platform to other. Various statistical methods, including the t-test, analysis of variance, Pearson test and Mann–Whitney U test, have been reported for comparing microarray data, whereas the utilization of the Wilcoxon signed-rank test, which is an appropriate test for two-groups comparison of gene expression data, has largely been neglected in microarray studies. The aim of this investigation was to build an integrated tool, ArraySolver, for colour-coded graphical display and comparison of gene expression data using the Wilcoxon signed-rank test. The results of software validation showed similar outputs with ArraySolver and SPSS for large datasets. Whereas the former program appeared to be more accurate for 25 or fewer pairs (n ≤ 25), suggesting its potential application in analysing molecular signatures that usually contain small numbers of genes. The main advantages of ArraySolver are easy data selection, convenient report format, accurate statistics and the familiar Excel platform. PMID:18629036

RECOVERING FILTER-BASED MICROARRAY DATA FOR PATHWAYS ANALYSIS USING A MULTIPOINT ALIGNMENT STRATEGY

EPA Science Inventory

The use of commercial microarrays are rapidly becoming the method of choice for profiling gene expression and assessing various disease states. Research Genetics has provided a series of well defined biological and software tools to the research community for these analyses. Th...

A Transcriptomic Comparison of Two Bambara Groundnut Landraces under Dehydration Stress

PubMed Central

Khan, Faraz; Chai, Hui Hui; Ajmera, Ishan; Hodgman, Charlie; Mayes, Sean; Lu, Chungui

2017-01-01

The ability to grow crops under low-water conditions is a significant advantage in relation to global food security. Bambara groundnut is an underutilised crop grown by subsistence farmers in Africa and is known to survive in regions of water deficit. This study focuses on the analysis of the transcriptomic changes in two bambara groundnut landraces in response to dehydration stress. A cross-species hybridisation approach based on the Soybean Affymetrix GeneChip array has been employed. The differential gene expression analysis of a water-limited treatment, however, showed that the two landraces responded with almost completely different sets of genes. Hence, both landraces with very similar genotypes (as assessed by the hybridisation of genomic DNA onto the Soybean Affymetrix GeneChip) showed contrasting transcriptional behaviour in response to dehydration stress. In addition, both genotypes showed a high expression of dehydration-associated genes, even under water-sufficient conditions. Several gene regulators were identified as potentially important. Some are already known, such as WRKY40, but others may also be considered, namely PRR7, ATAUX2-11, CONSTANS-like 1, MYB60, AGL-83, and a Zinc-finger protein. These data provide a basis for drought trait research in the bambara groundnut, which will facilitate functional genomics studies. An analysis of this dataset has identified that both genotypes appear to be in a dehydration-ready state, even in the absence of dehydration stress, and may have adapted in different ways to achieve drought resistance. This will help in understanding the mechanisms underlying the ability of crops to produce viable yields under drought conditions. In addition, cross-species hybridisation to the soybean microarray has been shown to be informative for investigating the bambara groundnut transcriptome. PMID:28420201

Estimating gene function with least squares nonnegative matrix factorization.

PubMed

Wang, Guoli; Ochs, Michael F

2007-01-01

Nonnegative matrix factorization is a machine learning algorithm that has extracted information from data in a number of fields, including imaging and spectral analysis, text mining, and microarray data analysis. One limitation with the method for linking genes through microarray data in order to estimate gene function is the high variance observed in transcription levels between different genes. Least squares nonnegative matrix factorization uses estimates of the uncertainties on the mRNA levels for each gene in each condition, to guide the algorithm to a local minimum in normalized chi2, rather than a Euclidean distance or divergence between the reconstructed data and the data itself. Herein, application of this method to microarray data is demonstrated in order to predict gene function.

Microarray expression technology: from start to finish.

PubMed

Elvidge, Gareth

2006-01-01

The recent introduction of new microarray expression technologies and the further development of established platforms ensure that the researcher is presented with a range of options for performing an experiment. Whilst this has opened up the possibilities for future applications, such as exon-specific arrays, increased sample throughput and 'chromatin immunoprecipitation (ChIP) on chip' experiments, the initial decision processes and experiment planning are made more difficult. This review will give an overview of the various technologies that are available to perform a microarray expression experiment, from the initial planning stages through to the final data analysis. Both practical aspects and data analysis options will be considered. The relative advantages and disadvantages will be discussed with insights provided for future directions of the technology.

Single molecule fluorescence microscopy for ultra-sensitive RNA expression profiling

NASA Astrophysics Data System (ADS)

Hesse, Jan; Jacak, Jaroslaw; Regl, Gerhard; Eichberger, Thomas; Aberger, Fritz; Schlapak, Robert; Howorka, Stefan; Muresan, Leila; Frischauf, Anna-Maria; Schütz, Gerhard J.

2007-02-01

We developed a microarray analysis platform for ultra-sensitive RNA expression profiling of minute samples. It utilizes a novel scanning system for single molecule fluorescence detection on cm2 size samples in combination with specialized biochips, optimized for low autofluorescence and weak unspecific adsorption. 20 μg total RNA was extracted from 10 6 cells of a human keratinocyte cell line (HaCaT) and reversely transcribed in the presence of Alexa647-aha-dUTP. 1% of the resulting labeled cDNA was used for complex hybridization to a custom-made oligonucleotide microarray representing a set of 125 different genes. For low abundant genes, individual cDNA molecules hybridized to the microarray spots could be resolved. Single cDNA molecules hybridized to the chip surface appeared as diffraction limited features in the fluorescence images. The à trous wavelet method was utilized for localization and counting of the separated cDNA signals. Subsequently, the degree of labeling of the localized cDNA molecules was determined by brightness analysis for the different genes. Variations by factors up to 6 were found, which in conventional microarray analysis would result in a misrepresentation of the relative abundance of mRNAs.

Dual-color Proteomic Profiling of Complex Samples with a Microarray of 810 Cancer-related Antibodies*

PubMed Central

Schröder, Christoph; Jacob, Anette; Tonack, Sarah; Radon, Tomasz P.; Sill, Martin; Zucknick, Manuela; Rüffer, Sven; Costello, Eithne; Neoptolemos, John P.; Crnogorac-Jurcevic, Tatjana; Bauer, Andrea; Fellenberg, Kurt; Hoheisel, Jörg D.

2010-01-01

Antibody microarrays have the potential to enable comprehensive proteomic analysis of small amounts of sample material. Here, protocols are presented for the production, quality assessment, and reproducible application of antibody microarrays in a two-color mode with an array of 1,800 features, representing 810 antibodies that were directed at 741 cancer-related proteins. In addition to measures of array quality, we implemented indicators for the accuracy and significance of dual-color detection. Dual-color measurements outperform a single-color approach concerning assay reproducibility and discriminative power. In the analysis of serum samples, depletion of high-abundance proteins did not improve technical assay quality. On the contrary, depletion introduced a strong bias in protein representation. In an initial study, we demonstrated the applicability of the protocols to proteins derived from urine samples. We identified differences between urine samples from pancreatic cancer patients and healthy subjects and between sexes. This study demonstrates that biomedically relevant data can be produced. As demonstrated by the thorough quality analysis, the dual-color antibody array approach proved to be competitive with other proteomic techniques and comparable in performance to transcriptional microarray analyses. PMID:20164060

Estimating differential expression from multiple indicators

PubMed Central

Ilmjärv, Sten; Hundahl, Christian Ansgar; Reimets, Riin; Niitsoo, Margus; Kolde, Raivo; Vilo, Jaak; Vasar, Eero; Luuk, Hendrik

2014-01-01

Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR. PMID:24586062

The effect of column purification on cDNA indirect labelling for microarrays

PubMed Central

Molas, M Lia; Kiss, John Z

2007-01-01

Background The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. Results We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Conclusion Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays. PMID:17597522

The effect of column purification on cDNA indirect labelling for microarrays.

PubMed

Molas, M Lia; Kiss, John Z

2007-06-27

The success of the microarray reproducibility is dependent upon the performance of standardized procedures. Since the introduction of microarray technology for the analysis of global gene expression, reproducibility of results among different laboratories has been a major problem. Two of the main contributors to this variability are the use of different microarray platforms and different laboratory practices. In this paper, we address the latter question in terms of how variation in one of the steps of a labelling procedure affects the cDNA product prior to microarray hybridization. We used a standard procedure to label cDNA for microarray hybridization and employed different types of column chromatography for cDNA purification. After purifying labelled cDNA, we used the Agilent 2100 Bioanalyzer and agarose gel electrophoresis to assess the quality of the labelled cDNA before its hybridization onto a microarray platform. There were major differences in the cDNA profile (i.e. cDNA fragment lengths and abundance) as a result of using four different columns for purification. In addition, different columns have different efficiencies to remove rRNA contamination. This study indicates that the appropriate column to use in this type of protocol has to be experimentally determined. Finally, we present new evidence establishing the importance of testing the method of purification used during an indirect labelling procedure. Our results confirm the importance of assessing the quality of the sample in the labelling procedure prior to hybridization onto a microarray platform. Standardization of column purification systems to be used in labelling procedures will improve the reproducibility of microarray results among different laboratories. In addition, implementation of a quality control check point of the labelled samples prior to microarray hybridization will prevent hybridizing a poor quality sample to expensive micorarrays.

Functional interaction analysis of GM1-related carbohydrates and Vibrio cholerae toxins using carbohydrate microarray.

PubMed

Kim, Chang Sup; Seo, Jeong Hyun; Cha, Hyung Joon

2012-08-07

The development of analytical tools is important for understanding the infection mechanisms of pathogenic bacteria or viruses. In the present work, a functional carbohydrate microarray combined with a fluorescence immunoassay was developed to analyze the interactions of Vibrio cholerae toxin (ctx) proteins and GM1-related carbohydrates. Ctx proteins were loaded onto the surface-immobilized GM1 pentasaccharide and six related carbohydrates, and their binding affinities were detected immunologically. The analysis of the ctx-carbohydrate interactions revealed that the intrinsic selectivity of ctx was GM1 pentasaccharide ≫ GM2 tetrasaccharide > asialo GM1 tetrasaccharide ≥ GM3trisaccharide, indicating that a two-finger grip formation and the terminal monosaccharides play important roles in the ctx-GM1 interaction. In addition, whole cholera toxin (ctxAB(5)) had a stricter substrate specificity and a stronger binding affinity than only the cholera toxin B subunit (ctxB). On the basis of the quantitative analysis, the carbohydrate microarray showed the sensitivity of detection of the ctxAB(5)-GM1 interaction with a limit-of-detection (LOD) of 2 ng mL(-1) (23 pM), which is comparable to other reported high sensitivity assay tools. In addition, the carbohydrate microarray successfully detected the actual toxin directly secreted from V. cholerae, without showing cross-reactivity to other bacteria. Collectively, these results demonstrate that the functional carbohydrate microarray is suitable for analyzing toxin protein-carbohydrate interactions and can be applied as a biosensor for toxin detection.

Bacterial identification and subtyping using DNA microarray and DNA sequencing.

PubMed

Al-Khaldi, Sufian F; Mossoba, Magdi M; Allard, Marc M; Lienau, E Kurt; Brown, Eric D

2012-01-01

The era of fast and accurate discovery of biological sequence motifs in prokaryotic and eukaryotic cells is here. The co-evolution of direct genome sequencing and DNA microarray strategies not only will identify, isotype, and serotype pathogenic bacteria, but also it will aid in the discovery of new gene functions by detecting gene expressions in different diseases and environmental conditions. Microarray bacterial identification has made great advances in working with pure and mixed bacterial samples. The technological advances have moved beyond bacterial gene expression to include bacterial identification and isotyping. Application of new tools such as mid-infrared chemical imaging improves detection of hybridization in DNA microarrays. The research in this field is promising and future work will reveal the potential of infrared technology in bacterial identification. On the other hand, DNA sequencing by using 454 pyrosequencing is so cost effective that the promise of $1,000 per bacterial genome sequence is becoming a reality. Pyrosequencing technology is a simple to use technique that can produce accurate and quantitative analysis of DNA sequences with a great speed. The deposition of massive amounts of bacterial genomic information in databanks is creating fingerprint phylogenetic analysis that will ultimately replace several technologies such as Pulsed Field Gel Electrophoresis. In this chapter, we will review (1) the use of DNA microarray using fluorescence and infrared imaging detection for identification of pathogenic bacteria, and (2) use of pyrosequencing in DNA cluster analysis to fingerprint bacterial phylogenetic trees.

6.0 K microarray reveals differential transcriptomic responses in the dinoflagellate Prorocentrum minimum exposed to polychlorinated biphenyl (PCB).

PubMed

Wang, Hui; Guo, Ruoyu; Ki, Jang-Seu

2018-03-01

Endocrine disrupting chemicals (EDCs) have toxic effects on algae; however, their molecular genomic responses have not been sufficiently elucidated. Here, we evaluated genome-scaled responses of the dinoflagellate alga Prorocentrum minimum exposed to an EDC, polychlorinated biphenyl (PCB), using a 6.0 K microarray. Based on two-fold change cut-off, we identified that 609 genes (∼10.2%) responded to the PCB treatment. KEGG pathway analysis showed that differentially expressed genes (DEGs) were related to ribosomes, biosynthesis of amino acids, spliceosomes, and cellular processes. Many DEGs were involved in cell cycle progression, apoptosis, signal transduction, ion binding, and cellular transportation. In contrast, only a few genes related to photosynthesis and oxidative stress were expressed in response to PCB exposure. This was supported by that fact that there were no obvious changes in the photosynthetic efficiency and reactive oxygen species (ROS) production. These results suggest that PCB might not cause chloroplast and oxidative damage, but could lead to cell cycle arrest and apoptosis. In addition, various signal transduction and transport pathways might be disrupted in the cells, which could further contribute to cell death. These results expand the genomic understanding of the effects of EDCs on this dinoflagellate protist. Copyright © 2017 Elsevier Ltd. All rights reserved.

Image-guided genomic analysis of tissue response to laser-induced thermal stress

NASA Astrophysics Data System (ADS)

Mackanos, Mark A.; Helms, Mike; Kalish, Flora; Contag, Christopher H.

2011-05-01

The cytoprotective response to thermal injury is characterized by transcriptional activation of ``heat shock proteins'' (hsp) and proinflammatory proteins. Expression of these proteins may predict cellular survival. Microarray analyses were performed to identify spatially distinct gene expression patterns responding to thermal injury. Laser injury zones were identified by expression of a transgene reporter comprised of the 70 kD hsp gene and the firefly luciferase coding sequence. Zones included the laser spot, the surrounding region where hsp70-luc expression was increased, and a region adjacent to the surrounding region. A total of 145 genes were up-regulated in the laser irradiated region, while 69 were up-regulated in the adjacent region. At 7 hours the chemokine Cxcl3 was the highest expressed gene in the laser spot (24 fold) and adjacent region (32 fold). Chemokines were the most common up-regulated genes identified. Microarray gene expression was successfully validated using qRT- polymerase chain reaction for selected genes of interest. The early response genes are likely involved in cytoprotection and initiation of the healing response. Their regulatory elements will benefit creating the next generation reporter mice and controlling expression of therapeutic proteins. The identified genes serve as drug development targets that may prevent acute tissue damage and accelerate healing.

Epigenetic Vestiges of Early Developmental Adversity: Childhood Stress Exposure and DNA Methylation in Adolescence

ERIC Educational Resources Information Center

Essex, Marilyn J.; Boyce, W. Thomas; Hertzman, Clyde; Lam, Lucia L.; Armstrong, Jeffrey M.; Neumann, Sarah M. A.; Kobor, Michael S.

2013-01-01

Fifteen-year-old adolescents (N = 109) in a longitudinal study of child development were recruited to examine differences in DNA methylation in relation to parent reports of adversity during the adolescents' infancy and preschool periods. Microarray technology applied to 28,000 cytosine-guanine dinucleotide sites within DNA derived from buccal…

Archetypal analysis of diverse Pseudomonas aeruginosa transcriptomes reveals adaptation in cystic fibrosis airways

PubMed Central

2013-01-01

Background Analysis of global gene expression by DNA microarrays is widely used in experimental molecular biology. However, the complexity of such high-dimensional data sets makes it difficult to fully understand the underlying biological features present in the data. The aim of this study is to introduce a method for DNA microarray analysis that provides an intuitive interpretation of data through dimension reduction and pattern recognition. We present the first “Archetypal Analysis” of global gene expression. The analysis is based on microarray data from five integrated studies of Pseudomonas aeruginosa isolated from the airways of cystic fibrosis patients. Results Our analysis clustered samples into distinct groups with comprehensible characteristics since the archetypes representing the individual groups are closely related to samples present in the data set. Significant changes in gene expression between different groups identified adaptive changes of the bacteria residing in the cystic fibrosis lung. The analysis suggests a similar gene expression pattern between isolates with a high mutation rate (hypermutators) despite accumulation of different mutations for these isolates. This suggests positive selection in the cystic fibrosis lung environment, and changes in gene expression for these isolates are therefore most likely related to adaptation of the bacteria. Conclusions Archetypal analysis succeeded in identifying adaptive changes of P. aeruginosa. The combination of clustering and matrix factorization made it possible to reveal minor similarities among different groups of data, which other analytical methods failed to identify. We suggest that this analysis could be used to supplement current methods used to analyze DNA microarray data. PMID:24059747

Gene features selection for three-class disease classification via multiple orthogonal partial least square discriminant analysis and S-plot using microarray data.

PubMed

Yang, Mingxing; Li, Xiumin; Li, Zhibin; Ou, Zhimin; Liu, Ming; Liu, Suhuan; Li, Xuejun; Yang, Shuyu

2013-01-01

DNA microarray analysis is characterized by obtaining a large number of gene variables from a small number of observations. Cluster analysis is widely used to analyze DNA microarray data to make classification and diagnosis of disease. Because there are so many irrelevant and insignificant genes in a dataset, a feature selection approach must be employed in data analysis. The performance of cluster analysis of this high-throughput data depends on whether the feature selection approach chooses the most relevant genes associated with disease classes. Here we proposed a new method using multiple Orthogonal Partial Least Squares-Discriminant Analysis (mOPLS-DA) models and S-plots to select the most relevant genes to conduct three-class disease classification and prediction. We tested our method using Golub's leukemia microarray data. For three classes with subtypes, we proposed hierarchical orthogonal partial least squares-discriminant analysis (OPLS-DA) models and S-plots to select features for two main classes and their subtypes. For three classes in parallel, we employed three OPLS-DA models and S-plots to choose marker genes for each class. The power of feature selection to classify and predict three-class disease was evaluated using cluster analysis. Further, the general performance of our method was tested using four public datasets and compared with those of four other feature selection methods. The results revealed that our method effectively selected the most relevant features for disease classification and prediction, and its performance was better than that of the other methods.

Knowledge-based analysis of microarrays for the discovery of transcriptional regulation relationships

PubMed Central

2010-01-01

Background The large amount of high-throughput genomic data has facilitated the discovery of the regulatory relationships between transcription factors and their target genes. While early methods for discovery of transcriptional regulation relationships from microarray data often focused on the high-throughput experimental data alone, more recent approaches have explored the integration of external knowledge bases of gene interactions. Results In this work, we develop an algorithm that provides improved performance in the prediction of transcriptional regulatory relationships by supplementing the analysis of microarray data with a new method of integrating information from an existing knowledge base. Using a well-known dataset of yeast microarrays and the Yeast Proteome Database, a comprehensive collection of known information of yeast genes, we show that knowledge-based predictions demonstrate better sensitivity and specificity in inferring new transcriptional interactions than predictions from microarray data alone. We also show that comprehensive, direct and high-quality knowledge bases provide better prediction performance. Comparison of our results with ChIP-chip data and growth fitness data suggests that our predicted genome-wide regulatory pairs in yeast are reasonable candidates for follow-up biological verification. Conclusion High quality, comprehensive, and direct knowledge bases, when combined with appropriate bioinformatic algorithms, can significantly improve the discovery of gene regulatory relationships from high throughput gene expression data. PMID:20122245

Knowledge-based analysis of microarrays for the discovery of transcriptional regulation relationships.

PubMed

Seok, Junhee; Kaushal, Amit; Davis, Ronald W; Xiao, Wenzhong

2010-01-18

The large amount of high-throughput genomic data has facilitated the discovery of the regulatory relationships between transcription factors and their target genes. While early methods for discovery of transcriptional regulation relationships from microarray data often focused on the high-throughput experimental data alone, more recent approaches have explored the integration of external knowledge bases of gene interactions. In this work, we develop an algorithm that provides improved performance in the prediction of transcriptional regulatory relationships by supplementing the analysis of microarray data with a new method of integrating information from an existing knowledge base. Using a well-known dataset of yeast microarrays and the Yeast Proteome Database, a comprehensive collection of known information of yeast genes, we show that knowledge-based predictions demonstrate better sensitivity and specificity in inferring new transcriptional interactions than predictions from microarray data alone. We also show that comprehensive, direct and high-quality knowledge bases provide better prediction performance. Comparison of our results with ChIP-chip data and growth fitness data suggests that our predicted genome-wide regulatory pairs in yeast are reasonable candidates for follow-up biological verification. High quality, comprehensive, and direct knowledge bases, when combined with appropriate bioinformatic algorithms, can significantly improve the discovery of gene regulatory relationships from high throughput gene expression data.

Gene expression profiling of whole blood: Comparison of target preparation methods for accurate and reproducible microarray analysis

PubMed Central

Vartanian, Kristina; Slottke, Rachel; Johnstone, Timothy; Casale, Amanda; Planck, Stephen R; Choi, Dongseok; Smith, Justine R; Rosenbaum, James T; Harrington, Christina A

2009-01-01

Background Peripheral blood is an accessible and informative source of transcriptomal information for many human disease and pharmacogenomic studies. While there can be significant advantages to analyzing RNA isolated from whole blood, particularly in clinical studies, the preparation of samples for microarray analysis is complicated by the need to minimize artifacts associated with highly abundant globin RNA transcripts. The impact of globin RNA transcripts on expression profiling data can potentially be reduced by using RNA preparation and labeling methods that remove or block globin RNA during the microarray assay. We compared four different methods for preparing microarray hybridization targets from human whole blood collected in PAXGene tubes. Three of the methods utilized the Affymetrix one-cycle cDNA synthesis/in vitro transcription protocol but varied treatment of input RNA as follows: i. no treatment; ii. treatment with GLOBINclear; or iii. treatment with globin PNA oligos. In the fourth method cDNA targets were prepared with the Ovation amplification and labeling system. Results We find that microarray targets generated with labeling methods that reduce globin mRNA levels or minimize the impact of globin transcripts during hybridization detect more transcripts in the microarray assay compared with the standard Affymetrix method. Comparison of microarray results with quantitative PCR analysis of a panel of genes from the NF-kappa B pathway shows good correlation of transcript measurements produced with all four target preparation methods, although method-specific differences in overall correlation were observed. The impact of freezing blood collected in PAXGene tubes on data reproducibility was also examined. Expression profiles show little or no difference when RNA is extracted from either fresh or frozen blood samples. Conclusion RNA preparation and labeling methods designed to reduce the impact of globin mRNA transcripts can significantly improve the sensitivity of the DNA microarray expression profiling assay for whole blood samples. While blockage of globin transcripts during first strand cDNA synthesis with globin PNAs resulted in the best overall performance in this study, we conclude that selection of a protocol for expression profiling studies in blood should depend on several factors, including implementation requirements of the method and study design. RNA isolated from either freshly collected or frozen blood samples stored in PAXGene tubes can be used without altering gene expression profiles. PMID:19123946

APPLICATION OF DNA MICROARRAYS TO REPRODUCTIVE TOXICOLOGY AND THE DEVELOPMENT OF A TESTIS ARRAY

EPA Science Inventory

With the advent of sequence information for entire mammalian genomes, it is now possible to analyze gene expression and gene polymorphisms on a genomic scale. The primary tool for analysis of gene expression is the DNA microarray. We have used commercially available cDNA micro...

Microarrays for Undergraduate Classes

ERIC Educational Resources Information Center

Hancock, Dale; Nguyen, Lisa L.; Denyer, Gareth S.; Johnston, Jill M.

2006-01-01

A microarray experiment is presented that, in six laboratory sessions, takes undergraduate students from the tissue sample right through to data analysis. The model chosen, the murine erythroleukemia cell line, can be easily cultured in sufficient quantities for class use. Large changes in gene expression can be induced in these cells by…

BIOMONITORING THE TOXICOGENOMIC RESPONSE TO ENDOCRINE DISRUPTING CHEMICALS IN HUMANS, LABORATORY SPECIES AND WILDLIFE

EPA Science Inventory

With the advent of sequence information for entire eukaryotic genomes, it is now possible to analyze gene expression on a genomic scale. The primary tool for genomic analysis of gene expression is the gene microarray. We have used commercially available and custom cDNA microarray...

CLOFIBRATE-INDUCED GENE EXPRESSION CHANGES IN RAT LIVER: A CROSS-LABORATORY ANALYSIS USING MEMBRANE CDNA ARRAYS

EPA Science Inventory

Microarrays have the potential to significantly impact our ability to identify toxic hazards by the identification of mechanistically-relevant markers of toxicity. To be useful for risk assessment however, microarray data must be challenged to determine its reliability and inter...

Comparative analysis of microarray data in Arabidopsis transcriptome during compatible interactions with plant viruses

USDA-ARS?s Scientific Manuscript database

To analyze transcriptome response to virus infection, we have assembled currently available microarray data on changes in gene expression levels in compatible Arabidopsis-virus interactions. We used the mean r (Pearson’s correlation coefficient) for neighboring pairs to estimate pairwise local simil...

Gene expression profile of blood cells for the prediction of delayed cerebral ischemia after intracranial aneurysm rupture: a pilot study in humans.

PubMed

Baumann, Antoine; Devaux, Yvan; Audibert, Gérard; Zhang, Lu; Bracard, Serge; Colnat-Coulbois, Sophie; Klein, Olivier; Zannad, Faiez; Charpentier, Claire; Longrois, Dan; Mertes, Paul-Michel

2013-01-01

Delayed cerebral ischemia (DCI) is a potentially devastating complication after intracranial aneurysm rupture and its mechanisms remain poorly elucidated. Early identification of the patients prone to developing DCI after rupture may represent a major breakthrough in its prevention and treatment. The single gene approach of DCI has demonstrated interest in humans. We hypothesized that whole genome expression profile of blood cells may be useful for better comprehension and prediction of aneurysmal DCI. Over a 35-month period, 218 patients with aneurysm rupture were included in this study. DCI was defined as the occurrence of a new delayed neurological deficit occurring within 2 weeks after aneurysm rupture with evidence of ischemia either on perfusion-diffusion MRI, CT angiography or CT perfusion imaging, or with cerebral angiography. DCI patients were matched against controls based on 4 out of 5 criteria (age, sex, Fisher grade, aneurysm location and smoking status). Genome-wide expression analysis of blood cells obtained at admission was performed by microarrays. Transcriptomic analysis was performed using long oligonucleotide microarrays representing 25,000 genes. Quantitative PCR: 1 µg of total RNA extracted was reverse-transcribed, and the resulting cDNA was diluted 10-fold before performing quantitative PCR. Microarray data were first analyzed by 'Significance Analysis of Microarrays' software which includes the Benjamini correction for multiple testing. In a second step, microarray data fold change was compared using a two-tailed, paired t test. Analysis of receiver-operating characteristic (ROC) curves and the area under the ROC curves were used for prediction analysis. Logistic regression models were used to investigate the additive value of multiple biomarkers. A total of 16 patients demonstrated DCI. Significance Analysis of Microarrays software failed to retrieve significant genes, most probably because of the heterogeneity of the patients included in the microarray experiments and the small size of the DCI population sample. Standard two-tailed paired t test and C-statistic revealed significant associations between gene expression and the occurrence of DCI: in particular, the expression of neuroregulin 1 was 1.6-fold upregulated in patients with DCI (p = 0.01) and predicted DCI with an area under the ROC curve of 0.96. Logistic regression analyses revealed a significant association between neuroregulin 1 and DCI (odds ratio 1.46, 95% confidence interval 1.02-2.09, p = 0.02). This pilot study suggests that blood cells may be a reservoir of prognostic biomarkers of DCI in patients with intracranial aneurysm rupture. Despite an evident lack of power, this study elicited neuroregulin 1, a vasoreactivity-, inflammation- and angiogenesis-related gene, as a possible candidate predictor of DCI. Larger cohort studies are needed but genome-wide microarray-based studies are promising research tools for the understanding of DCI after intracranial aneurysm rupture. © 2013 S. Karger AG, Basel.

Profiling In Situ Microbial Community Structure with an Amplification Microarray

PubMed Central

Knickerbocker, Christopher; Bryant, Lexi; Golova, Julia; Wiles, Cory; Williams, Kenneth H.; Peacock, Aaron D.; Long, Philip E.

2013-01-01

The objectives of this study were to unify amplification, labeling, and microarray hybridization chemistries within a single, closed microfluidic chamber (an amplification microarray) and verify technology performance on a series of groundwater samples from an in situ field experiment designed to compare U(VI) mobility under conditions of various alkalinities (as HCO3−) during stimulated microbial activity accompanying acetate amendment. Analytical limits of detection were between 2 and 200 cell equivalents of purified DNA. Amplification microarray signatures were well correlated with 16S rRNA-targeted quantitative PCR results and hybridization microarray signatures. The succession of the microbial community was evident with and consistent between the two microarray platforms. Amplification microarray analysis of acetate-treated groundwater showed elevated levels of iron-reducing bacteria (Flexibacter, Geobacter, Rhodoferax, and Shewanella) relative to the average background profile, as expected. Identical molecular signatures were evident in the transect treated with acetate plus NaHCO3, but at much lower signal intensities and with a much more rapid decline (to nondetection). Azoarcus, Thaurea, and Methylobacterium were responsive in the acetate-only transect but not in the presence of bicarbonate. Observed differences in microbial community composition or response to bicarbonate amendment likely had an effect on measured rates of U reduction, with higher rates probable in the part of the field experiment that was amended with bicarbonate. The simplification in microarray-based work flow is a significant technological advance toward entirely closed-amplicon microarray-based tests and is generally extensible to any number of environmental monitoring applications. PMID:23160129

Bioinformatic tools for inferring functional information from plant microarray data: tools for the first steps.

PubMed

Page, Grier P; Coulibaly, Issa

2008-01-01

Microarrays are a very powerful tool for quantifying the amount of RNA in samples; however, their ability to query essentially every gene in a genome, which can number in the tens of thousands, presents analytical and interpretative problems. As a result, a variety of software and web-based tools have been developed to help with these issues. This article highlights and reviews some of the tools for the first steps in the analysis of a microarray study. We have tried for a balance between free and commercial systems. We have organized the tools by topics including image processing tools (Section 2), power analysis tools (Section 3), image analysis tools (Section 4), database tools (Section 5), databases of functional information (Section 6), annotation tools (Section 7), statistical and data mining tools (Section 8), and dissemination tools (Section 9).

Bioinformatics and Microarray Data Analysis on the Cloud.

PubMed

Calabrese, Barbara; Cannataro, Mario

2016-01-01

High-throughput platforms such as microarray, mass spectrometry, and next-generation sequencing are producing an increasing volume of omics data that needs large data storage and computing power. Cloud computing offers massive scalable computing and storage, data sharing, on-demand anytime and anywhere access to resources and applications, and thus, it may represent the key technology for facing those issues. In fact, in the recent years it has been adopted for the deployment of different bioinformatics solutions and services both in academia and in the industry. Although this, cloud computing presents several issues regarding the security and privacy of data, that are particularly important when analyzing patients data, such as in personalized medicine. This chapter reviews main academic and industrial cloud-based bioinformatics solutions; with a special focus on microarray data analysis solutions and underlines main issues and problems related to the use of such platforms for the storage and analysis of patients data.

Genome-wide identification and expression analysis of glutathione S-transferase gene family in tomato: Gaining an insight to their physiological and stress-specific roles

PubMed Central

Islam, Shiful; Rahman, Iffat Ara; Islam, Tahmina

2017-01-01

Glutathione S-transferase (GST) refers to one of the major detoxifying enzymes that plays an important role in different abiotic and biotic stress modulation pathways of plant. The present study aimed to a comprehensive genome-wide functional characterization of GST genes and proteins in tomato (Solanum lycopersicum L.). The whole genome sequence analysis revealed the presence of 90 GST genes in tomato, the largest GST gene family reported till date. Eight segmental duplicated gene pairs might contribute significantly to the expansion of SlGST gene family. Based on phylogenetic analysis of tomato, rice, and Arabidopsis GST proteins, GST family members could be further divided into ten classes. Members of each orthologous class showed high conservancy among themselves. Tau and lambda are the major classes of tomato; while tau and phi are the major classes for rice and Arabidopsis. Chromosomal localization revealed highly uneven distribution of SlGST genes in 13 different chromosomes, where chromosome 9 possessed the highest number of genes. Based on publicly available microarray data, expression analysis of 30 available SlGST genes exhibited a differential pattern in all the analyzed tissues and developmental stages. Moreover, most of the members showed highly induced expression in response to multiple biotic and abiotic stress inducers that could be harmonized with the increase in total GST enzyme activity under several stress conditions. Activity of tomato GST could be enhanced further by using some positive modulators (safeners) that have been predicted through molecular docking of SlGSTU5 and ligands. Moreover, tomato GST proteins are predicted to interact with a lot of other glutathione synthesizing and utilizing enzymes such as glutathione peroxidase, glutathione reductase, glutathione synthetase and γ-glutamyltransferase. This comprehensive genome-wide analysis and expression profiling would provide a rational platform and possibility to explore the versatile role of GST genes in crop engineering. PMID:29095889

Identification of differentially expressed genes and false discovery rate in microarray studies.

PubMed

Gusnanto, Arief; Calza, Stefano; Pawitan, Yudi

2007-04-01

To highlight the development in microarray data analysis for the identification of differentially expressed genes, particularly via control of false discovery rate. The emergence of high-throughput technology such as microarrays raises two fundamental statistical issues: multiplicity and sensitivity. We focus on the biological problem of identifying differentially expressed genes. First, multiplicity arises due to testing tens of thousands of hypotheses, rendering the standard P value meaningless. Second, known optimal single-test procedures such as the t-test perform poorly in the context of highly multiple tests. The standard approach of dealing with multiplicity is too conservative in the microarray context. The false discovery rate concept is fast becoming the key statistical assessment tool replacing the P value. We review the false discovery rate approach and argue that it is more sensible for microarray data. We also discuss some methods to take into account additional information from the microarrays to improve the false discovery rate. There is growing consensus on how to analyse microarray data using the false discovery rate framework in place of the classical P value. Further research is needed on the preprocessing of the raw data, such as the normalization step and filtering, and on finding the most sensitive test procedure.

WWOX sensitises ovarian cancer cells to paclitaxel via modulation of the ER stress response.

PubMed

Janczar, Szymon; Nautiyal, Jaya; Xiao, Yi; Curry, Edward; Sun, Mingjun; Zanini, Elisa; Paige, Adam Jw; Gabra, Hani

2017-07-27

There are clear gaps in our understanding of genes and pathways through which cancer cells facilitate survival strategies as they become chemoresistant. Paclitaxel is used in the treatment of many cancers, but development of drug resistance is common. Along with being an antimitotic agent paclitaxel also activates endoplasmic reticulum (ER) stress. Here, we examine the role of WWOX (WW domain containing oxidoreductase), a gene frequently lost in several cancers, in mediating paclitaxel response. We examine the ER stress-mediated apoptotic response to paclitaxel in WWOX-transfected epithelial ovarian cancer (EOC) cells and following siRNA knockdown of WWOX. We show that WWOX-induced apoptosis following exposure of EOC cells to paclitaxel is related to ER stress and independent of the antimitotic action of taxanes. The apoptotic response to ER stress induced by WWOX re-expression could be reversed by WWOX siRNA in EOC cells. We report that paclitaxel treatment activates both the IRE-1 and PERK kinases and that the increase in paclitaxel-mediated cell death through WWOX is dependent on active ER stress pathway. Log-rank analysis of overall survival (OS) and progression-free survival (PFS) in two prominent EOC microarray data sets (Tothill and The Cancer Genome Atlas), encompassing ~800 patients in total, confirmed clinical relevance to our findings. High WWOX mRNA expression predicted longer OS and PFS in patients treated with paclitaxel, but not in patients who were treated with only cisplatin. The association of WWOX and survival was dependent on the expression level of glucose-related protein 78 (GRP78), a key ER stress marker in paclitaxel-treated patients. We conclude that WWOX sensitises EOC to paclitaxel via ER stress-induced apoptosis, and predicts clinical outcome in patients. Thus, ER stress response mechanisms could be targeted to overcome chemoresistance in cancer.

Gene response in rice plants treated with continuous fog influenced by pH, was similar to that treated with biotic stress

PubMed Central

2014-01-01

Background Throughout Asia, including Japan, rice plants are cultivated in a wide range of areas from lowlands to highlands and are frequently exposed to fog, including acid fog. Some physiological studies have shown that acid fog can be a stress factor for plants. We analyzed the gene expression profiles of rice plants treated with artificially prepared simulated acid fog (SiAF) or simulated neutral fog (SiNF) for 1 or 7 days. Results Microarray analysis results suggested that both the SiAF and the SiNF treatments induced the expression of genes involved in the defense and stress responses in rice plants. Induction of such genes was detected in plants treated with SiAF for 1 day, and the number of induced genes increased in plants treated with SiAF for 7 days. The genes for defense and stress responses were also induced by SiNF for 7 days, although they were not induced by SiNF for 1 day. The gene expression profiles of the SiAF-treated and the SiNF-treated plants were compared to those of plants treated with other stress factors. The comparison revealed that both SiAF and SiNF treatments have similar effects to biotic stresses and ozone stress. The genes encoding NADPH oxidase and germin, which function in apoplasts, were also induced by SiAF, SiNF and biotic stresses. Conclusions These findings suggest that both the SiAF and the SiNF treatments may result in oxidative stress through the apoplastic production of reactive oxygen species. PMID:24987489

Gene profiling reveals a role for stress hormones in the molecular and behavioral response to food restriction.

PubMed

Guarnieri, Douglas J; Brayton, Catherine E; Richards, Sarah M; Maldonado-Aviles, Jaime; Trinko, Joseph R; Nelson, Jessica; Taylor, Jane R; Gourley, Shannon L; DiLeone, Ralph J

2012-02-15

Food restriction is known to enhance learning and motivation. The neural mechanisms underlying these responses likely involve alterations in gene expression in brain regions mediating the motivation to feed. Analysis of gene expression profiles in male C57BL/6J mice using whole-genome microarrays was completed in the medial prefrontal cortex, nucleus accumbens, ventral tegmental area, and the hypothalamus following a 5-day food restriction. Quantitative polymerase chain reaction was used to validate these findings and determine the time course of expression changes. Plasma levels of the stress hormone corticosterone (CORT) were measured by enzyme-linked immunosorbent assay. Expression changes were measured in adrenalectomized animals that underwent food restriction, as well as in animals receiving daily injections of CORT. Progressive ratio responding for food, a measure of motivated behavior, was assessed after CORT treatment in restricted and fed animals. Brief food restriction results in an upregulation of peripheral stress responsive genes in the mammalian brain. Time-course analysis demonstrated rapid and persistent expression changes in all four brain regions under study. Administration of CORT to nonrestricted animals was sufficient to induce a subset of the genes, and alterations in gene expression after food restriction were dependent on intact adrenal glands. CORT can increase the motivation to work for food only in the restricted state. These data demonstrate a central role for CORT in mediating both molecular and behavioral responses to food restriction. The stress hormone-induced alterations in gene expression described here may be relevant for both adaptive and pathological responses to stress. Copyright © 2012 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

Annotation of Differential Gene Expression in Small Yellow Follicles of a Broiler-Type Strain of Taiwan Country Chickens in Response to Acute Heat Stress.

PubMed

Cheng, Chuen-Yu; Tu, Wei-Lin; Wang, Shih-Han; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Chen, Shuen-Ei; Huang, San-Yuan

2015-01-01

This study investigated global gene expression in the small yellow follicles (6-8 mm diameter) of broiler-type B strain Taiwan country chickens (TCCs) in response to acute heat stress. Twelve 30-wk-old TCC hens were divided into four groups: control hens maintained at 25°C and hens subjected to 38°C acute heat stress for 2 h without recovery (H2R0), with 2-h recovery (H2R2), and with 6-h recovery (H2R6). Small yellow follicles were collected for RNA isolation and microarray analysis at the end of each time point. Results showed that 69, 51, and 76 genes were upregulated and 58, 15, 56 genes were downregulated after heat treatment of H2R0, H2R2, and H2R6, respectively, using a cutoff value of two-fold or higher. Gene ontology analysis revealed that these differentially expressed genes are associated with the biological processes of cell communication, developmental process, protein metabolic process, immune system process, and response to stimuli. Upregulation of heat shock protein 25, interleukin 6, metallopeptidase 1, and metalloproteinase 13, and downregulation of type II alpha 1 collagen, discoidin domain receptor tyrosine kinase 2, and Kruppel-like factor 2 suggested that acute heat stress induces proteolytic disintegration of the structural matrix and inflamed damage and adaptive responses of gene expression in the follicle cells. These suggestions were validated through gene expression, using quantitative real-time polymerase chain reaction. Functional annotation clarified that interleukin 6-related pathways play a critical role in regulating acute heat stress responses in the small yellow follicles of TCC hens.

Annotation of Differential Gene Expression in Small Yellow Follicles of a Broiler-Type Strain of Taiwan Country Chickens in Response to Acute Heat Stress

PubMed Central

Wang, Shih-Han; Tang, Pin-Chi; Chen, Chih-Feng; Chen, Hsin-Hsin; Lee, Yen-Pai; Chen, Shuen-Ei; Huang, San-Yuan

2015-01-01

This study investigated global gene expression in the small yellow follicles (6–8 mm diameter) of broiler-type B strain Taiwan country chickens (TCCs) in response to acute heat stress. Twelve 30-wk-old TCC hens were divided into four groups: control hens maintained at 25°C and hens subjected to 38°C acute heat stress for 2 h without recovery (H2R0), with 2-h recovery (H2R2), and with 6-h recovery (H2R6). Small yellow follicles were collected for RNA isolation and microarray analysis at the end of each time point. Results showed that 69, 51, and 76 genes were upregulated and 58, 15, 56 genes were downregulated after heat treatment of H2R0, H2R2, and H2R6, respectively, using a cutoff value of two-fold or higher. Gene ontology analysis revealed that these differentially expressed genes are associated with the biological processes of cell communication, developmental process, protein metabolic process, immune system process, and response to stimuli. Upregulation of heat shock protein 25, interleukin 6, metallopeptidase 1, and metalloproteinase 13, and downregulation of type II alpha 1 collagen, discoidin domain receptor tyrosine kinase 2, and Kruppel-like factor 2 suggested that acute heat stress induces proteolytic disintegration of the structural matrix and inflamed damage and adaptive responses of gene expression in the follicle cells. These suggestions were validated through gene expression, using quantitative real-time polymerase chain reaction. Functional annotation clarified that interleukin 6-related pathways play a critical role in regulating acute heat stress responses in the small yellow follicles of TCC hens. PMID:26587838

Transcriptional regulation of the CRK/DUF26 group of Receptor-like protein kinases by ozone and plant hormones in Arabidopsis

PubMed Central

2010-01-01

Background Plant Receptor-like/Pelle kinases (RLK) are a group of conserved signalling components that regulate developmental programs and responses to biotic and abiotic stresses. One of the largest RLK groups is formed by the Domain of Unknown Function 26 (DUF26) RLKs, also called Cysteine-rich Receptor-like Kinases (CRKs), which have been suggested to play important roles in the regulation of pathogen defence and programmed cell death. Despite the vast number of RLKs present in plants, however, only a few of them have been functionally characterized. Results We examined the transcriptional regulation of all Arabidopsis CRKs by ozone (O3), high light and pathogen/elicitor treatment - conditions known to induce the production of reactive oxygen species (ROS) in various subcellular compartments. Several CRKs were transcriptionally induced by exposure to O3 but not by light stress. O3 induces an extracellular oxidative burst, whilst light stress leads to ROS production in chloroplasts. Analysis of publicly available microarray data revealed that the transcriptional responses of the CRKs to O3 were very similar to responses to microbes or pathogen-associated molecular patterns (PAMPs). Several mutants altered in hormone biosynthesis or signalling showed changes in basal and O3-induced transcriptional responses. Conclusions Combining expression analysis from multiple treatments with mutants altered in hormone biosynthesis or signalling suggest a model in which O3 and salicylic acid (SA) activate separate signaling pathways that exhibit negative crosstalk. Although O3 is classified as an abiotic stress to plants, transcriptional profiling of CRKs showed strong similarities between the O3 and biotic stress responses. PMID:20500828

Transcriptome analysis uncovers Arabidopsis F-BOX STRESS INDUCED 1 as a regulator of jasmonic acid and abscisic acid stress gene expression.

PubMed

Gonzalez, Lauren E; Keller, Kristen; Chan, Karen X; Gessel, Megan M; Thines, Bryan C

2017-07-17

The ubiquitin 26S proteasome system (UPS) selectively degrades cellular proteins, which results in physiological changes to eukaryotic cells. F-box proteins are substrate adaptors within the UPS and are responsible for the diversity of potential protein targets. Plant genomes are enriched in F-box genes, but the vast majority of these have unknown roles. This work investigated the Arabidopsis F-box gene F-BOX STRESS INDUCED 1 (FBS1) for its effects on gene expression in order elucidate its previously unknown biological function. Using publically available Affymetrix ATH1 microarray data, we show that FBS1 is significantly co-expressed in abiotic stresses with other well-characterized stress response genes, including important stress-related transcriptional regulators. This gene suite is most highly expressed in roots under cold and salt stresses. Transcriptome analysis of fbs1-1 knock-out plants grown at a chilling temperature shows that hundreds of genes require FBS1 for appropriate expression, and that these genes are enriched in those having roles in both abiotic and biotic stress responses. Based on both this genome-wide expression data set and quantitative real-time PCR (qPCR) analysis, it is apparent that FBS1 is required for elevated expression of many jasmonic acid (JA) genes that have established roles in combatting environmental stresses, and that it also controls a subset of JA biosynthesis genes. FBS1 also significantly impacts abscisic acid (ABA) regulated genes, but this interaction is more complex, as FBS1 has both positive and negative effects on ABA-inducible and ABA-repressible gene modules. One noteworthy effect of FBS1 on ABA-related stress processes, however, is the restraint it imposes on the expression of multiple class I LIPID TRANSFER PROTEIN (LTP) gene family members that have demonstrated protective effects in water deficit-related stresses. FBS1 impacts plant stress responses by regulating hundreds of genes that respond to the plant stress hormones JA and ABA. The positive effect that FBS1 has on JA processes and the negative effect it has on at least some ABA processes indicates that it in part regulates cellular responses balanced between these two important stress hormones. More broadly then, FBS1 may aid plant cells in switching between certain biotic (JA) and abiotic (ABA) stress responses. Finally, because FBS1 regulates a subset of JA biosynthesis and response genes, we conclude that it might have a role in tuning hormone responses to particular circumstances at the transcriptional level.

Tumor Necrosis Factor-α Regulates Distinct Molecular Pathways and Gene Networks in Cultured Skeletal Muscle Cells

PubMed Central

Gupta, Sanjay K.; Dahiya, Saurabh; Lundy, Robert F.; Kumar, Ashok

2010-01-01

Background Skeletal muscle wasting is a debilitating consequence of large number of disease states and conditions. Tumor necrosis factor-α (TNF-α) is one of the most important muscle-wasting cytokine, elevated levels of which cause significant muscular abnormalities. However, the underpinning molecular mechanisms by which TNF-α causes skeletal muscle wasting are less well-understood. Methodology/Principal Findings We have used microarray, quantitative real-time PCR (QRT-PCR), Western blot, and bioinformatics tools to study the effects of TNF-α on various molecular pathways and gene networks in C2C12 cells (a mouse myoblastic cell line). Microarray analyses of C2C12 myotubes treated with TNF-α (10 ng/ml) for 18h showed differential expression of a number of genes involved in distinct molecular pathways. The genes involved in nuclear factor-kappa B (NF-kappaB) signaling, 26s proteasome pathway, Notch1 signaling, and chemokine networks are the most important ones affected by TNF-α. The expression of some of the genes in microarray dataset showed good correlation in independent QRT-PCR and Western blot assays. Analysis of TNF-treated myotubes showed that TNF-α augments the activity of both canonical and alternative NF-κB signaling pathways in myotubes. Bioinformatics analyses of microarray dataset revealed that TNF-α affects the activity of several important pathways including those involved in oxidative stress, hepatic fibrosis, mitochondrial dysfunction, cholesterol biosynthesis, and TGF-β signaling. Furthermore, TNF-α was found to affect the gene networks related to drug metabolism, cell cycle, cancer, neurological disease, organismal injury, and abnormalities in myotubes. Conclusions TNF-α regulates the expression of multiple genes involved in various toxic pathways which may be responsible for TNF-induced muscle loss in catabolic conditions. Our study suggests that TNF-α activates both canonical and alternative NF-κB signaling pathways in a time-dependent manner in skeletal muscle cells. The study provides novel insight into the mechanisms of action of TNF-α in skeletal muscle cells. PMID:20967264

Lifetime Stress Cumulatively Programs Brain Transcriptome and Impedes Stroke Recovery: Benefit of Sensory Stimulation

PubMed Central

Zucchi, Fabíola C. R.; Yao, Youli; Ilnytskyy, Yaroslav; Robbins, Jerrah C.; Soltanpour, Nasrin; Kovalchuk, Igor; Kovalchuk, Olga; Metz, Gerlinde A. S.

2014-01-01

Prenatal stress (PS) represents a critical variable affecting lifetime health trajectories, metabolic and vascular functions. Beneficial experiences may attenuate the effects of PS and its programming of health outcomes in later life. Here we investigated in a rat model (1) if PS modulates recovery following cortical ischemia in adulthood; (2) if a second hit by adult stress (AS) exaggerates stress responses and ischemic damage; and (3) if tactile stimulation (TS) attenuates the cumulative effects of PS and AS. Prenatally stressed and non-stressed adult male rats underwent focal ischemic motor cortex lesion and were tested in skilled reaching and skilled walking tasks. Two groups of rats experienced recurrent restraint stress in adulthood and one of these groups also underwent daily TS therapy. Animals that experienced both PS and AS displayed the most severe motor disabilities after lesion. By contrast, TS promoted recovery from ischemic lesion and reduced hypothalamic-pituitary-adrenal axis activity. The data also showed that cumulative effects of adverse and beneficial lifespan experiences interact with disease outcomes and brain plasticity through the modulation of gene expression. Microarray analysis of the lesion motor cortex revealed that cumulative PS and AS interact with genes related to growth factors and transcription factors, which were not affected by PS or lesion alone. TS in PS+AS animals reverted these changes, suggesting a critical role for these factors in activity-dependent motor cortical reorganization after ischemic lesion. These findings suggest that beneficial experience later in life can moderate adverse consequences of early programming to improve cerebrovascular health. PMID:24651125

An evaluation of two-channel ChIP-on-chip and DNA methylation microarray normalization strategies

PubMed Central

2012-01-01

Background The combination of chromatin immunoprecipitation with two-channel microarray technology enables genome-wide mapping of binding sites of DNA-interacting proteins (ChIP-on-chip) or sites with methylated CpG di-nucleotides (DNA methylation microarray). These powerful tools are the gateway to understanding gene transcription regulation. Since the goals of such studies, the sample preparation procedures, the microarray content and study design are all different from transcriptomics microarrays, the data pre-processing strategies traditionally applied to transcriptomics microarrays may not be appropriate. Particularly, the main challenge of the normalization of "regulation microarrays" is (i) to make the data of individual microarrays quantitatively comparable and (ii) to keep the signals of the enriched probes, representing DNA sequences from the precipitate, as distinguishable as possible from the signals of the un-enriched probes, representing DNA sequences largely absent from the precipitate. Results We compare several widely used normalization approaches (VSN, LOWESS, quantile, T-quantile, Tukey's biweight scaling, Peng's method) applied to a selection of regulation microarray datasets, ranging from DNA methylation to transcription factor binding and histone modification studies. Through comparison of the data distributions of control probes and gene promoter probes before and after normalization, and assessment of the power to identify known enriched genomic regions after normalization, we demonstrate that there are clear differences in performance between normalization procedures. Conclusion T-quantile normalization applied separately on the channels and Tukey's biweight scaling outperform other methods in terms of the conservation of enriched and un-enriched signal separation, as well as in identification of genomic regions known to be enriched. T-quantile normalization is preferable as it additionally improves comparability between microarrays. In contrast, popular normalization approaches like quantile, LOWESS, Peng's method and VSN normalization alter the data distributions of regulation microarrays to such an extent that using these approaches will impact the reliability of the downstream analysis substantially. PMID:22276688

Advantages of RNA-seq compared to RNA microarrays for transcriptome profiling of anterior cruciate ligament tears.

PubMed

Rai, Muhammad Farooq; Tycksen, Eric D; Sandell, Linda J; Brophy, Robert H

2018-01-01

Microarrays and RNA-seq are at the forefront of high throughput transcriptome analyses. Since these methodologies are based on different principles, there are concerns about the concordance of data between the two techniques. The concordance of RNA-seq and microarrays for genome-wide analysis of differential gene expression has not been rigorously assessed in clinically derived ligament tissues. To demonstrate the concordance between RNA-seq and microarrays and to assess potential benefits of RNA-seq over microarrays, we assessed differences in transcript expression in anterior cruciate ligament (ACL) tissues based on time-from-injury. ACL remnants were collected from patients with an ACL tear at the time of ACL reconstruction. RNA prepared from torn ACL remnants was subjected to Agilent microarrays (N = 24) and RNA-seq (N = 8). The correlation of biological replicates in RNA-seq and microarrays data was similar (0.98 vs. 0.97), demonstrating that each platform has high internal reproducibility. Correlations between the RNA-seq data and the individual microarrays were low, but correlations between the RNA-seq values and the geometric mean of the microarrays values were moderate. The cross-platform concordance for differentially expressed transcripts or enriched pathways was linearly correlated (r = 0.64). RNA-Seq was superior in detecting low abundance transcripts and differentiating biologically critical isoforms. Additional independent validation of transcript expression was undertaken using microfluidic PCR for selected genes. PCR data showed 100% concordance (in expression pattern) with RNA-seq and microarrays data. These findings demonstrate that RNA-seq has advantages over microarrays for transcriptome profiling of ligament tissues when available and affordable. Furthermore, these findings are likely transferable to other musculoskeletal tissues where tissue collection is challenging and cells are in low abundance. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:484-497, 2018. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

A model of binding on DNA microarrays: understanding the combined effect of probe synthesis failure, cross-hybridization, DNA fragmentation and other experimental details of affymetrix arrays

PubMed Central

2012-01-01

Background DNA microarrays are used both for research and for diagnostics. In research, Affymetrix arrays are commonly used for genome wide association studies, resequencing, and for gene expression analysis. These arrays provide large amounts of data. This data is analyzed using statistical methods that quite often discard a large portion of the information. Most of the information that is lost comes from probes that systematically fail across chips and from batch effects. The aim of this study was to develop a comprehensive model for hybridization that predicts probe intensities for Affymetrix arrays and that could provide a basis for improved microarray analysis and probe development. The first part of the model calculates probe binding affinities to all the possible targets in the hybridization solution using the Langmuir isotherm. In the second part of the model we integrate details that are specific to each experiment and contribute to the differences between hybridization in solution and on the microarray. These details include fragmentation, wash stringency, temperature, salt concentration, and scanner settings. Furthermore, the model fits probe synthesis efficiency and target concentration parameters directly to the data. All the parameters used in the model have a well-established physical origin. Results For the 302 chips that were analyzed the mean correlation between expected and observed probe intensities was 0.701 with a range of 0.88 to 0.55. All available chips were included in the analysis regardless of the data quality. Our results show that batch effects arise from differences in probe synthesis, scanner settings, wash strength, and target fragmentation. We also show that probe synthesis efficiencies for different nucleotides are not uniform. Conclusions To date this is the most complete model for binding on microarrays. This is the first model that includes both probe synthesis efficiency and hybridization kinetics/cross-hybridization. These two factors are sequence dependent and have a large impact on probe intensity. The results presented here provide novel insight into the effect of probe synthesis errors on Affymetrix microarrays; furthermore, the algorithms developed in this work provide useful tools for the analysis of cross-hybridization, probe synthesis efficiency, fragmentation, wash stringency, temperature, and salt concentration on microarray intensities. PMID:23270536

Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.

PubMed

Thormar, Hans G; Gudmundsson, Bjarki; Eiriksdottir, Freyja; Kil, Siyoen; Gunnarsson, Gudmundur H; Magnusson, Magnus Karl; Hsu, Jason C; Jonsson, Jon J

2013-04-01

The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. © 2013 American Association for Clinical Chemistry

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma

PubMed Central

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-01-01

Objective This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Methods Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Results Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification (P=0.009) or deletion (P=0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly (P=1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Conclusion Chromosomal CNVs may contribute to their transcript expression in cervical cancer. PMID:29312578

BioconductorBuntu: a Linux distribution that implements a web-based DNA microarray analysis server.

PubMed

Geeleher, Paul; Morris, Dermot; Hinde, John P; Golden, Aaron

2009-06-01

BioconductorBuntu is a custom distribution of Ubuntu Linux that automatically installs a server-side microarray processing environment, providing a user-friendly web-based GUI to many of the tools developed by the Bioconductor Project, accessible locally or across a network. System installation is via booting off a CD image or by using a Debian package provided to upgrade an existing Ubuntu installation. In its current version, several microarray analysis pipelines are supported including oligonucleotide, dual-or single-dye experiments, including post-processing with Gene Set Enrichment Analysis. BioconductorBuntu is designed to be extensible, by server-side integration of further relevant Bioconductor modules as required, facilitated by its straightforward underlying Python-based infrastructure. BioconductorBuntu offers an ideal environment for the development of processing procedures to facilitate the analysis of next-generation sequencing datasets. BioconductorBuntu is available for download under a creative commons license along with additional documentation and a tutorial from (http://bioinf.nuigalway.ie).

A benchmark for statistical microarray data analysis that preserves actual biological and technical variance.

PubMed

De Hertogh, Benoît; De Meulder, Bertrand; Berger, Fabrice; Pierre, Michael; Bareke, Eric; Gaigneaux, Anthoula; Depiereux, Eric

2010-01-11

Recent reanalysis of spike-in datasets underscored the need for new and more accurate benchmark datasets for statistical microarray analysis. We present here a fresh method using biologically-relevant data to evaluate the performance of statistical methods. Our novel method ranks the probesets from a dataset composed of publicly-available biological microarray data and extracts subset matrices with precise information/noise ratios. Our method can be used to determine the capability of different methods to better estimate variance for a given number of replicates. The mean-variance and mean-fold change relationships of the matrices revealed a closer approximation of biological reality. Performance analysis refined the results from benchmarks published previously.We show that the Shrinkage t test (close to Limma) was the best of the methods tested, except when two replicates were examined, where the Regularized t test and the Window t test performed slightly better. The R scripts used for the analysis are available at http://urbm-cluster.urbm.fundp.ac.be/~bdemeulder/.

Integrated analysis of chromosome copy number variation and gene expression in cervical carcinoma.

PubMed

Yan, Deng; Yi, Song; Chiu, Wang Chi; Qin, Liu Gui; Kin, Wong Hoi; Kwok Hung, Chung Tony; Linxiao, Han; Wai, Choy Kwong; Yi, Sui; Tao, Yang; Tao, Tang

2017-12-12

This study was conducted to explore chromosomal copy number variations (CNV) and transcript expression and to examine pathways in cervical pathogenesis using genome-wide high resolution microarrays. Genome-wide chromosomal CNVs were investigated in 6 cervical cancer cell lines by Human Genome CGH Microarray Kit (4x44K). Gene expression profiles in cervical cancer cell lines, primary cervical carcinoma and normal cervical epithelium tissues were also studied using the Whole Human Genome Microarray Kit (4x44K). Fifty common chromosomal CNVs were identified in the cervical cancer cell lines. Correlation analysis revealed that gene up-regulation or down-regulation is significantly correlated with genomic amplification ( P =0.009) or deletion ( P =0.006) events. Expression profiles were identified through cluster analysis. Gene annotation analysis pinpointed cell cycle pathways was significantly ( P =1.15E-08) affected in cervical cancer. Common CNVs were associated with cervical cancer. Chromosomal CNVs may contribute to their transcript expression in cervical cancer.

Enhancement of the Initial Rate of Ethanol Fermentation Due to Dysfunction of Yeast Stress Response Components Msn2p and/or Msn4p▿ †

PubMed Central

Watanabe, Daisuke; Wu, Hong; Noguchi, Chiemi; Zhou, Yan; Akao, Takeshi; Shimoi, Hitoshi

2011-01-01

Sake yeasts (strains of Saccharomyces cerevisiae) produce high concentrations of ethanol in sake fermentation. To investigate the molecular mechanisms underlying this brewing property, we compared gene expression of sake and laboratory yeasts in sake mash. DNA microarray and reporter gene analyses revealed defects of sake yeasts in environmental stress responses mediated by transcription factors Msn2p and/or Msn4p (Msn2/4p) and stress response elements (STRE). Furthermore, we found that dysfunction of MSN2 and/or MSN4 contributes to the higher initial rate of ethanol fermentation in both sake and laboratory yeasts. These results provide novel insights into yeast stress responses as major impediments of effective ethanol fermentation. PMID:21131516

Identifying novel glioma associated pathways based on systems biology level meta-analysis.

PubMed

Hu, Yangfan; Li, Jinquan; Yan, Wenying; Chen, Jiajia; Li, Yin; Hu, Guang; Shen, Bairong

2013-01-01

With recent advances in microarray technology, including genomics, proteomics, and metabolomics, it brings a great challenge for integrating this "-omics" data to analysis complex disease. Glioma is an extremely aggressive and lethal form of brain tumor, and thus the study of the molecule mechanism underlying glioma remains very important. To date, most studies focus on detecting the differentially expressed genes in glioma. However, the meta-analysis for pathway analysis based on multiple microarray datasets has not been systematically pursued. In this study, we therefore developed a systems biology based approach by integrating three types of omics data to identify common pathways in glioma. Firstly, the meta-analysis has been performed to study the overlapping of signatures at different levels based on the microarray gene expression data of glioma. Among these gene expression datasets, 12 pathways were found in GeneGO database that shared by four stages. Then, microRNA expression profiles and ChIP-seq data were integrated for the further pathway enrichment analysis. As a result, we suggest 5 of these pathways could be served as putative pathways in glioma. Among them, the pathway of TGF-beta-dependent induction of EMT via SMAD is of particular importance. Our results demonstrate that the meta-analysis based on systems biology level provide a more useful approach to study the molecule mechanism of complex disease. The integration of different types of omics data, including gene expression microarrays, microRNA and ChIP-seq data, suggest some common pathways correlated with glioma. These findings will offer useful potential candidates for targeted therapeutic intervention of glioma.

A single-repeat R3-MYB transcription factor MYBC1 negatively regulates freezing tolerance in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhai, Hong, E-mail: Zhai.h@hotmail.com; Bai, Xi, E-mail: baixi@neau.edu.cn; Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn

2010-04-16

We had previously identified the MYBC1 gene, which encodes a single-repeat R3-MYB protein, as a putative osmotic responding gene; however, no R3-MYB transcription factor has been reported to regulate osmotic stress tolerance. Thus, we sought to elucidate the function of MYBC1 in response to osmotic stresses. Real-time RT-PCR analysis indicated that MYBC1 expression responded to cold, dehydration, salinity and exogenous ABA at the transcript level. mybc1 mutants exhibited an increased tolerance to freezing stress, whereas 35S::MYBC1 transgenic plants exhibited decreased cold tolerance. Transcript levels of some cold-responsive genes, including CBF/DREB genes, KIN1, ADC1, ADC2 and ZAT12, though, were not alteredmore » in the mybc1 mutants or the 35S::MYBC1 transgenic plants in response to cold stress, as compared to the wild type. Microarray analysis results that are publically available were investigated and found transcript level of MYBC1 was not altered by overexpression of CBF1, CBF2, and CBF3, suggesting that MYBC1 is not down regulated by these CBF family members. Together, these results suggested that MYBC1is capable of negatively regulating the freezing tolerance of Arabidopsis in the CBF-independent pathway. In transgenic Arabidopsis carrying an MYBC1 promoter driven {beta}-glucuronidase (GUS) construct, GUS activity was observed in all tissues and was relatively stronger in the vascular tissues. Fused MYBC1 and GFP protein revealed that MYBC1 was localized exclusively in the nuclear compartment.« less

Global gene expression analysis of the heat shock response in the phytopathogen Xylella fastidiosa.

PubMed

Koide, Tie; Vêncio, Ricardo Z N; Gomes, Suely L

2006-08-01

Xylella fastidiosa is a phytopathogenic bacterium that is responsible for diseases in many economically important crops. Although different strains have been studied, little is known about X. fastidiosa stress responses. One of the better characterized stress responses in bacteria is the heat shock response, which induces the expression of specific genes to prevent protein misfolding and aggregation and to promote degradation of the irreversibly denatured polypeptides. To investigate X. fastidiosa genes involved in the heat shock response, we performed a whole-genome microarray analysis in a time course experiment. Globally, 261 genes were induced (9.7%) and 222 genes were repressed (8.3%). The expression profiles of the differentially expressed genes were grouped, and their expression patterns were validated by quantitative reverse transcription-PCR experiments. We determined the transcription start sites of six heat shock-inducible genes and analyzed their promoter regions, which allowed us to propose a putative consensus for sigma(32) promoters in Xylella and to suggest additional genes as putative members of this regulon. Besides the induction of classical heat shock protein genes, we observed the up-regulation of virulence-associated genes such as vapD and of genes for hemagglutinins, hemolysin, and xylan-degrading enzymes, which may indicate the importance of heat stress to bacterial pathogenesis. In addition, we observed the repression of genes related to fimbriae, aerobic respiration, and protein biosynthesis and the induction of genes related to the extracytoplasmic stress response and some phage-related genes, revealing the complex network of genes that work together in response to heat shock.

Differential expression and alternative splicing of rice sulphate transporter family members regulate sulphur status during plant growth, development and stress conditions.

PubMed

Kumar, Smita; Asif, Mehar Hasan; Chakrabarty, Debasis; Tripathi, Rudra Deo; Trivedi, Prabodh Kumar

2011-06-01

Sulphur, an essential nutrient required for plant growth and development, is mainly taken up by the plants as inorganic sulphate from the soil and assimilated into the sulphur reductive pathway. The uptake and transport of sulphate in plants is carried out by transporters encoded by the sulphate transporter gene family. Plant sulphate transporters have been classified with respect to their protein sequences, kinetic properties and tissue-specific localization in Arabidopsis. Though sulphate transporter genes from few other plants have also been characterized, no detailed study with respect to the structure and expression of this family from rice has been carried out. Here, we present genome-wide identification, structural and expression analyses of the rice sulphate transporter gene family. Our analysis using microarray data and MPSS database suggests that 14 rice sulphate transporters are differentially expressed during growth and development in various tissues and during biotic and abiotic stresses. Our analysis also suggests differential accumulation of splice variants of OsSultr1;1 and OsSultr4;1 transcripts during these processes. Apart from known spliced variants, we report an unusual alternative splicing of OsSultr1;1 transcript related to sulphur supply in growth medium and during stress response. Taken together, our study suggests that differential expression and alternative splicing of members of the sulphate transporter family plays an important role in regulating cellular sulphur status required for growth and development and during stress conditions. These findings significantly advance our understanding of the posttranscriptional regulatory mechanisms operating to regulate sulphur demand by the plant.

High-Throughput Quantification of SH2 Domain-Phosphopeptide Interactions with Cellulose-Peptide Conjugate Microarrays.

PubMed

Engelmann, Brett W

2017-01-01

The Src Homology 2 (SH2) domain family primarily recognizes phosphorylated tyrosine (pY) containing peptide motifs. The relative affinity preferences among competing SH2 domains for phosphopeptide ligands define "specificity space," and underpins many functional pY mediated interactions within signaling networks. The degree of promiscuity exhibited and the dynamic range of affinities supported by individual domains or phosphopeptides is best resolved by a carefully executed and controlled quantitative high-throughput experiment. Here, I describe the fabrication and application of a cellulose-peptide conjugate microarray (CPCMA) platform to the quantitative analysis of SH2 domain specificity space. Included herein are instructions for optimal experimental design with special attention paid to common sources of systematic error, phosphopeptide SPOT synthesis, microarray fabrication, analyte titrations, data capture, and analysis.

Microbial Diagnostic Microarrays for the Detection and Typing of Food- and Water-Borne (Bacterial) Pathogens

PubMed Central

Kostić, Tanja; Sessitsch, Angela

2011-01-01

Reliable and sensitive pathogen detection in clinical and environmental (including food and water) samples is of greatest importance for public health. Standard microbiological methods have several limitations and improved alternatives are needed. Most important requirements for reliable analysis include: (i) specificity; (ii) sensitivity; (iii) multiplexing potential; (iv) robustness; (v) speed; (vi) automation potential; and (vii) low cost. Microarray technology can, through its very nature, fulfill many of these requirements directly and the remaining challenges have been tackled. In this review, we attempt to compare performance characteristics of the microbial diagnostic microarrays developed for the detection and typing of food and water pathogens, and discuss limitations, points still to be addressed and issues specific for the analysis of food, water and environmental samples. PMID:27605332

The pathogenesis shared between abdominal aortic aneurysms and intracranial aneurysms: a microarray analysis.

PubMed

Wang, Wen; Li, Hao; Zhao, Zheng; Wang, Haoyuan; Zhang, Dong; Zhang, Yan; Lan, Qing; Wang, Jiangfei; Cao, Yong; Zhao, Jizong

2018-04-01

Abdominal aortic aneurysms (AAAs) and intracranial saccular aneurysms (IAs) are the most common types of aneurysms. This study was to investigate the common pathogenesis shared between these two kinds of aneurysms. We collected 12 IAs samples and 12 control arteries from the Beijing Tiantan Hospital and performed microarray analysis. In addition, we utilized the microarray datasets of IAs and AAAs from the Gene Expression Omnibus (GEO), in combination with our microarray results, to generate messenger RNA expression profiles for both AAAs and IAs in our study. Functional exploration and protein-protein interaction (PPI) analysis were performed. A total of 727 common genes were differentially expressed (404 was upregulated; 323 was downregulated) for both AAAs and IAs. The GO and pathway analyses showed that the common dysregulated genes were mainly enriched in vascular smooth muscle contraction, muscle contraction, immune response, defense response, cell activation, IL-6 signaling and chemokine signaling pathways, etc. The further protein-protein analysis identified 35 hub nodes, including TNF, IL6, MAPK13, and CCL5. These hub node genes were enriched in inflammatory response, positive regulation of IL-6 production, chemokine signaling pathway, and T/B cell receptor signaling pathway. Our study will gain new insight into the molecular mechanisms for the pathogenesis of both types of aneurysms and provide new therapeutic targets for the patients harboring AAAs and IAs.

Cloud-scale genomic signals processing classification analysis for gene expression microarray data.

PubMed

Harvey, Benjamin; Soo-Yeon Ji

2014-01-01

As microarray data available to scientists continues to increase in size and complexity, it has become overwhelmingly important to find multiple ways to bring inference though analysis of DNA/mRNA sequence data that is useful to scientists. Though there have been many attempts to elucidate the issue of bringing forth biological inference by means of wavelet preprocessing and classification, there has not been a research effort that focuses on a cloud-scale classification analysis of microarray data using Wavelet thresholding in a Cloud environment to identify significantly expressed features. This paper proposes a novel methodology that uses Wavelet based Denoising to initialize a threshold for determination of significantly expressed genes for classification. Additionally, this research was implemented and encompassed within cloud-based distributed processing environment. The utilization of Cloud computing and Wavelet thresholding was used for the classification 14 tumor classes from the Global Cancer Map (GCM). The results proved to be more accurate than using a predefined p-value for differential expression classification. This novel methodology analyzed Wavelet based threshold features of gene expression in a Cloud environment, furthermore classifying the expression of samples by analyzing gene patterns, which inform us of biological processes. Moreover, enabling researchers to face the present and forthcoming challenges that may arise in the analysis of data in functional genomics of large microarray datasets.

[Differentially expressed genes of cell signal transduction associated with benzene poisoning by cDNA microarray].

PubMed

Wang, Hong; Bi, Yongyi; Tao, Ning; Wang, Chunhong

2005-08-01

To detect the differential expression of cell signal transduction genes associated with benzene poisoning, and to explore the pathogenic mechanisms of blood system damage induced by benzene. Peripheral white blood cell gene expression profile of 7 benzene poisoning patients, including one aplastic anemia, was determined by cDNA microarray. Seven chips from normal workers were served as controls. Cluster analysis of gene expression profile was performed. Among the 4265 target genes, 176 genes associated with cell signal transduction were differentially expressed. 35 up-regulated genes including PTPRC, STAT4, IFITM1 etc were found in at least 6 pieces of microarray; 45 down-regulated genes including ARHB, PPP3CB, CDC37 etc were found in at least 5 pieces of microarray. cDNA microarray technology is an effective technique for screening the differentially expressed genes of cell signal transduction. Disorder in cell signal transduction may play certain role in the pathogenic mechanism of benzene poisoning.

A study of metaheuristic algorithms for high dimensional feature selection on microarray data

NASA Astrophysics Data System (ADS)

Dankolo, Muhammad Nasiru; Radzi, Nor Haizan Mohamed; Sallehuddin, Roselina; Mustaffa, Noorfa Haszlinna

2017-11-01

Microarray systems enable experts to examine gene profile at molecular level using machine learning algorithms. It increases the potentials of classification and diagnosis of many diseases at gene expression level. Though, numerous difficulties may affect the efficiency of machine learning algorithms which includes vast number of genes features comprised in the original data. Many of these features may be unrelated to the intended analysis. Therefore, feature selection is necessary to be performed in the data pre-processing. Many feature selection algorithms are developed and applied on microarray which including the metaheuristic optimization algorithms. This paper discusses the application of the metaheuristics algorithms for feature selection in microarray dataset. This study reveals that, the algorithms have yield an interesting result with limited resources thereby saving computational expenses of machine learning algorithms.

Microarray analysis identified Puccinia striiformis f. sp. tritici genes involved in infection and sporulation.

USDA-ARS?s Scientific Manuscript database

Puccinia striiformis f. sp. tritici (Pst) causes stripe rust, one of the most important diseases of wheat worldwide. To identify Pst genes involved in infection and sporulation, a custom oligonucleotide Genechip was made using sequences of 442 genes selected from Pst cDNA libraries. Microarray analy...

Clear cell papillary renal cell carcinoma: a chromosomal microarray analysis of two cases using a novel Molecular Inversion Probe (MIP) technology.

PubMed

Alexiev, Borislav A; Zou, Ying S

2014-12-01

Chromosomal microarray analysis using novel Molecular Inversion Probe (MIP) technology demonstrated 2,570 kb copy neutral LOH of 10q11.22 in two clear cell papillary renal cell carcinomas. In addition, one of the tumors had a big 29,784 kb deletion of 13q11-q14.2. There were two variants of unknown significance, a 2,509 kb gain of Xp22.33 and a 257 kb homozygous deletion of 8p11.22. The somatic mutation panel containing 74 mutations in nine genes did not reveal any mutations. Besides identification of submicroscopic duplications or deletions, SNP microarrays can reveal abnormal allelic imbalances including LOH and copy neutral LOH, which cannot be recognized by chromosome, FISH, and non-SNP microarray arrays. To the best of our knowledge, this is the first study demonstrating copy neutral LOH of 10q11.22 in clear cell papillary renal cell carcinomas using the new MIP SNP OncoScan FFPE Assay Kit on formalin-fixed paraffin-embedded tumor samples. Copyright © 2014 Elsevier GmbH. All rights reserved.

A Unique Procedure to Identify Cell Surface Markers Through a Spherical Self-Organizing Map Applied to DNA Microarray Analysis.

PubMed

Sugii, Yuh; Kasai, Tomonari; Ikeda, Masashi; Vaidyanath, Arun; Kumon, Kazuki; Mizutani, Akifumi; Seno, Akimasa; Tokutaka, Heizo; Kudoh, Takayuki; Seno, Masaharu

2016-01-01

To identify cell-specific markers, we designed a DNA microarray platform with oligonucleotide probes for human membrane-anchored proteins. Human glioma cell lines were analyzed using microarray and compared with normal and fetal brain tissues. For the microarray analysis, we employed a spherical self-organizing map, which is a clustering method suitable for the conversion of multidimensional data into two-dimensional data and displays the relationship on a spherical surface. Based on the gene expression profile, the cell surface characteristics were successfully mirrored onto the spherical surface, thereby distinguishing normal brain tissue from the disease model based on the strength of gene expression. The clustered glioma-specific genes were further analyzed by polymerase chain reaction procedure and immunocytochemical staining of glioma cells. Our platform and the following procedure were successfully demonstrated to categorize the genes coding for cell surface proteins that are specific to glioma cells. Our assessment demonstrates that a spherical self-organizing map is a valuable tool for distinguishing cell surface markers and can be employed in marker discovery studies for the treatment of cancer.

Systematic analysis of microarray datasets to identify Parkinson's disease‑associated pathways and genes.

PubMed

Feng, Yinling; Wang, Xuefeng

2017-03-01

In order to investigate commonly disturbed genes and pathways in various brain regions of patients with Parkinson's disease (PD), microarray datasets from previous studies were collected and systematically analyzed. Different normalization methods were applied to microarray datasets from different platforms. A strategy combining gene co‑expression networks and clinical information was adopted, using weighted gene co‑expression network analysis (WGCNA) to screen for commonly disturbed genes in different brain regions of patients with PD. Functional enrichment analysis of commonly disturbed genes was performed using the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Co‑pathway relationships were identified with Pearson's correlation coefficient tests and a hypergeometric distribution‑based test. Common genes in pathway pairs were selected out and regarded as risk genes. A total of 17 microarray datasets from 7 platforms were retained for further analysis. Five gene coexpression modules were identified, containing 9,745, 736, 233, 101 and 93 genes, respectively. One module was significantly correlated with PD samples and thus the 736 genes it contained were considered to be candidate PD‑associated genes. Functional enrichment analysis demonstrated that these genes were implicated in oxidative phosphorylation and PD. A total of 44 pathway pairs and 52 risk genes were revealed, and a risk gene pathway relationship network was constructed. Eight modules were identified and were revealed to be associated with PD, cancers and metabolism. A number of disturbed pathways and risk genes were unveiled in PD, and these findings may help advance understanding of PD pathogenesis.

Gene set analysis approaches for RNA-seq data: performance evaluation and application guideline

PubMed Central

Rahmatallah, Yasir; Emmert-Streib, Frank

2016-01-01

Transcriptome sequencing (RNA-seq) is gradually replacing microarrays for high-throughput studies of gene expression. The main challenge of analyzing microarray data is not in finding differentially expressed genes, but in gaining insights into the biological processes underlying phenotypic differences. To interpret experimental results from microarrays, gene set analysis (GSA) has become the method of choice, in particular because it incorporates pre-existing biological knowledge (in a form of functionally related gene sets) into the analysis. Here we provide a brief review of several statistically different GSA approaches (competitive and self-contained) that can be adapted from microarrays practice as well as those specifically designed for RNA-seq. We evaluate their performance (in terms of Type I error rate, power, robustness to the sample size and heterogeneity, as well as the sensitivity to different types of selection biases) on simulated and real RNA-seq data. Not surprisingly, the performance of various GSA approaches depends only on the statistical hypothesis they test and does not depend on whether the test was developed for microarrays or RNA-seq data. Interestingly, we found that competitive methods have lower power as well as robustness to the samples heterogeneity than self-contained methods, leading to poor results reproducibility. We also found that the power of unsupervised competitive methods depends on the balance between up- and down-regulated genes in tested gene sets. These properties of competitive methods have been overlooked before. Our evaluation provides a concise guideline for selecting GSA approaches, best performing under particular experimental settings in the context of RNA-seq. PMID:26342128

Comparison of RNA-seq and microarray-based models for clinical endpoint prediction.

PubMed

Zhang, Wenqian; Yu, Ying; Hertwig, Falk; Thierry-Mieg, Jean; Zhang, Wenwei; Thierry-Mieg, Danielle; Wang, Jian; Furlanello, Cesare; Devanarayan, Viswanath; Cheng, Jie; Deng, Youping; Hero, Barbara; Hong, Huixiao; Jia, Meiwen; Li, Li; Lin, Simon M; Nikolsky, Yuri; Oberthuer, André; Qing, Tao; Su, Zhenqiang; Volland, Ruth; Wang, Charles; Wang, May D; Ai, Junmei; Albanese, Davide; Asgharzadeh, Shahab; Avigad, Smadar; Bao, Wenjun; Bessarabova, Marina; Brilliant, Murray H; Brors, Benedikt; Chierici, Marco; Chu, Tzu-Ming; Zhang, Jibin; Grundy, Richard G; He, Min Max; Hebbring, Scott; Kaufman, Howard L; Lababidi, Samir; Lancashire, Lee J; Li, Yan; Lu, Xin X; Luo, Heng; Ma, Xiwen; Ning, Baitang; Noguera, Rosa; Peifer, Martin; Phan, John H; Roels, Frederik; Rosswog, Carolina; Shao, Susan; Shen, Jie; Theissen, Jessica; Tonini, Gian Paolo; Vandesompele, Jo; Wu, Po-Yen; Xiao, Wenzhong; Xu, Joshua; Xu, Weihong; Xuan, Jiekun; Yang, Yong; Ye, Zhan; Dong, Zirui; Zhang, Ke K; Yin, Ye; Zhao, Chen; Zheng, Yuanting; Wolfinger, Russell D; Shi, Tieliu; Malkas, Linda H; Berthold, Frank; Wang, Jun; Tong, Weida; Shi, Leming; Peng, Zhiyu; Fischer, Matthias

2015-06-25

Gene expression profiling is being widely applied in cancer research to identify biomarkers for clinical endpoint prediction. Since RNA-seq provides a powerful tool for transcriptome-based applications beyond the limitations of microarrays, we sought to systematically evaluate the performance of RNA-seq-based and microarray-based classifiers in this MAQC-III/SEQC study for clinical endpoint prediction using neuroblastoma as a model. We generate gene expression profiles from 498 primary neuroblastomas using both RNA-seq and 44 k microarrays. Characterization of the neuroblastoma transcriptome by RNA-seq reveals that more than 48,000 genes and 200,000 transcripts are being expressed in this malignancy. We also find that RNA-seq provides much more detailed information on specific transcript expression patterns in clinico-genetic neuroblastoma subgroups than microarrays. To systematically compare the power of RNA-seq and microarray-based models in predicting clinical endpoints, we divide the cohort randomly into training and validation sets and develop 360 predictive models on six clinical endpoints of varying predictability. Evaluation of factors potentially affecting model performances reveals that prediction accuracies are most strongly influenced by the nature of the clinical endpoint, whereas technological platforms (RNA-seq vs. microarrays), RNA-seq data analysis pipelines, and feature levels (gene vs. transcript vs. exon-junction level) do not significantly affect performances of the models. We demonstrate that RNA-seq outperforms microarrays in determining the transcriptomic characteristics of cancer, while RNA-seq and microarray-based models perform similarly in clinical endpoint prediction. Our findings may be valuable to guide future studies on the development of gene expression-based predictive models and their implementation in clinical practice.

Transcriptome Pathway Analysis of Pathological and Physiological Aldosterone-Producing Human Tissues.

PubMed

Zhou, Junhua; Lam, Brian; Neogi, Sudeshna G; Yeo, Giles S H; Azizan, Elena A B; Brown, Morris J

2016-12-01

Primary aldosteronism is present in ≈10% of hypertensives. We previously performed a microarray assay on aldosterone-producing adenomas and their paired zona glomerulosa and fasciculata. Confirmation of top genes validated the study design and functional experiments of zona glomerulosa selective genes established the role of the encoded proteins in aldosterone regulation. In this study, we further analyzed our microarray data using AmiGO 2 for gene ontology enrichment and Ingenuity Pathway Analysis to identify potential biological processes and canonical pathways involved in pathological and physiological aldosterone regulation. Genes differentially regulated in aldosterone-producing adenoma and zona glomerulosa were associated with steroid metabolic processes gene ontology terms. Terms related to the Wnt signaling pathway were enriched in zona glomerulosa only. Ingenuity Pathway Analysis showed "NRF2-mediated oxidative stress response pathway" and "LPS (lipopolysaccharide)/IL-1 (interleukin-1)-mediated inhibition of RXR (retinoid X receptor) function" were affected in both aldosterone-producing adenoma and zona glomerulosa with associated genes having up to 21- and 8-fold differences, respectively. Comparing KCNJ5-mutant aldosterone-producing adenoma, zona glomerulosa, and zona fasciculata samples with wild-type samples, 138, 56, and 59 genes were differentially expressed, respectively (fold-change >2; P<0.05). ACSS3, encoding the enzyme that synthesizes acetyl-CoA, was the top gene upregulated in KCNJ5-mutant aldosterone-producing adenoma compared with wild-type. NEFM, a gene highly upregulated in zona glomerulosa, was upregulated in KCNJ5 wild-type aldosterone-producing adenomas. NR4A2, the transcription factor for aldosterone synthase, was highly expressed in zona fasciculata adjacent to a KCNJ5-mutant aldosterone-producing adenoma. Further interrogation of these genes and pathways could potentially provide further insights into the pathology of primary aldosteronism. © 2016 The Authors.

Roles of the Sodium-Translocating NADH:Quinone Oxidoreductase (Na+-NQR) on Vibrio cholerae Metabolism, Motility and Osmotic Stress Resistance

PubMed Central

Minato, Yusuke; Halang, Petra; Quinn, Matthew J.; Faulkner, Wyatt J.; Aagesen, Alisha M.; Steuber, Julia; Stevens, Jan F.; Häse, Claudia C.

2014-01-01

The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology. PMID:24811312

A comprehensive sensitivity analysis of microarray breast cancer classification under feature variability

PubMed Central

2009-01-01

Background Large discrepancies in signature composition and outcome concordance have been observed between different microarray breast cancer expression profiling studies. This is often ascribed to differences in array platform as well as biological variability. We conjecture that other reasons for the observed discrepancies are the measurement error associated with each feature and the choice of preprocessing method. Microarray data are known to be subject to technical variation and the confidence intervals around individual point estimates of expression levels can be wide. Furthermore, the estimated expression values also vary depending on the selected preprocessing scheme. In microarray breast cancer classification studies, however, these two forms of feature variability are almost always ignored and hence their exact role is unclear. Results We have performed a comprehensive sensitivity analysis of microarray breast cancer classification under the two types of feature variability mentioned above. We used data from six state of the art preprocessing methods, using a compendium consisting of eight diferent datasets, involving 1131 hybridizations, containing data from both one and two-color array technology. For a wide range of classifiers, we performed a joint study on performance, concordance and stability. In the stability analysis we explicitly tested classifiers for their noise tolerance by using perturbed expression profiles that are based on uncertainty information directly related to the preprocessing methods. Our results indicate that signature composition is strongly influenced by feature variability, even if the array platform and the stratification of patient samples are identical. In addition, we show that there is often a high level of discordance between individual class assignments for signatures constructed on data coming from different preprocessing schemes, even if the actual signature composition is identical. Conclusion Feature variability can have a strong impact on breast cancer signature composition, as well as the classification of individual patient samples. We therefore strongly recommend that feature variability is considered in analyzing data from microarray breast cancer expression profiling experiments. PMID:19941644

Mutation analysis of 272 Spanish families affected by autosomal recessive retinitis pigmentosa using a genotyping microarray.

PubMed

Ávila-Fernández, Almudena; Cantalapiedra, Diego; Aller, Elena; Vallespín, Elena; Aguirre-Lambán, Jana; Blanco-Kelly, Fiona; Corton, M; Riveiro-Álvarez, Rosa; Allikmets, Rando; Trujillo-Tiebas, María José; Millán, José M; Cremers, Frans P M; Ayuso, Carmen

2010-12-03

Retinitis pigmentosa (RP) is a genetically heterogeneous disorder characterized by progressive loss of vision. The aim of this study was to identify the causative mutations in 272 Spanish families using a genotyping microarray. 272 unrelated Spanish families, 107 with autosomal recessive RP (arRP) and 165 with sporadic RP (sRP), were studied using the APEX genotyping microarray. The families were also classified by clinical criteria: 86 juveniles and 186 typical RP families. Haplotype and sequence analysis were performed to identify the second mutated allele. At least one-gene variant was found in 14% and 16% of the juvenile and typical RP groups respectively. Further study identified four new mutations, providing both causative changes in 11% of the families. Retinol Dehydrogenase 12 (RDH12) was the most frequently mutated gene in the juvenile RP group, and Usher Syndrome 2A (USH2A) and Ceramide Kinase-Like (CERKL) were the most frequently mutated genes in the typical RP group. The only variant found in CERKL was p.Arg257Stop, the most frequent mutation. The genotyping microarray combined with segregation and sequence analysis allowed us to identify the causative mutations in 11% of the families. Due to the low number of characterized families, this approach should be used in tandem with other techniques.

Curation of microarray oligonucleotides and corresponding ESTs/cDNAs used for gene expression analysis in zebra finches.

PubMed

Lovell, Peter V; Huizinga, Nicole A; Getachew, Abel; Mees, Brianna; Friedrich, Samantha R; Wirthlin, Morgan; Mello, Claudio V

2018-05-18

Zebra finches are a major model organism for investigating mechanisms of vocal learning, a trait that enables spoken language in humans. The development of cDNA collections with expressed sequence tags (ESTs) and microarrays has allowed for extensive molecular characterizations of circuitry underlying vocal learning and production. However, poor database curation can lead to errors in transcriptome and bioinformatics analyses, limiting the impact of these resources. Here we used genomic alignments and synteny analysis for orthology verification to curate and reannotate ~ 35% of the oligonucleotides and corresponding ESTs/cDNAs that make-up Agilent microarrays for gene expression analysis in finches. We found that: (1) 5475 out of 43,084 oligos (a) failed to align to the zebra finch genome, (b) aligned to multiple loci, or (c) aligned to Chr_un only, and thus need to be flagged until a better genome assembly is available, or (d) reflect cloning artifacts; (2) Out of 9635 valid oligos examined further, 3120 were incorrectly named, including 1533 with no known orthologs; and (3) 2635 oligos required name update. The resulting curated dataset provides a reference for correcting gene identification errors in previous finch microarrays studies, and avoiding such errors in future studies.

A Model-Based Joint Identification of Differentially Expressed Genes and Phenotype-Associated Genes

PubMed Central

Seo, Minseok; Shin, Su-kyung; Kwon, Eun-Young; Kim, Sung-Eun; Bae, Yun-Jung; Lee, Seungyeoun; Sung, Mi-Kyung; Choi, Myung-Sook; Park, Taesung

2016-01-01

Over the last decade, many analytical methods and tools have been developed for microarray data. The detection of differentially expressed genes (DEGs) among different treatment groups is often a primary purpose of microarray data analysis. In addition, association studies investigating the relationship between genes and a phenotype of interest such as survival time are also popular in microarray data analysis. Phenotype association analysis provides a list of phenotype-associated genes (PAGs). However, it is sometimes necessary to identify genes that are both DEGs and PAGs. We consider the joint identification of DEGs and PAGs in microarray data analyses. The first approach we used was a naïve approach that detects DEGs and PAGs separately and then identifies the genes in an intersection of the list of PAGs and DEGs. The second approach we considered was a hierarchical approach that detects DEGs first and then chooses PAGs from among the DEGs or vice versa. In this study, we propose a new model-based approach for the joint identification of DEGs and PAGs. Unlike the previous two-step approaches, the proposed method identifies genes simultaneously that are DEGs and PAGs. This method uses standard regression models but adopts different null hypothesis from ordinary regression models, which allows us to perform joint identification in one-step. The proposed model-based methods were evaluated using experimental data and simulation studies. The proposed methods were used to analyze a microarray experiment in which the main interest lies in detecting genes that are both DEGs and PAGs, where DEGs are identified between two diet groups and PAGs are associated with four phenotypes reflecting the expression of leptin, adiponectin, insulin-like growth factor 1, and insulin. Model-based approaches provided a larger number of genes, which are both DEGs and PAGs, than other methods. Simulation studies showed that they have more power than other methods. Through analysis of data from experimental microarrays and simulation studies, the proposed model-based approach was shown to provide a more powerful result than the naïve approach and the hierarchical approach. Since our approach is model-based, it is very flexible and can easily handle different types of covariates. PMID:26964035

Strong Magnetic Field Induced Changes of Gene Expression in Arabidopsis

NASA Astrophysics Data System (ADS)

Paul, A.-L.; Ferl, R. J.; Klingenberg, B.; Brooks, J. S.; Morgan, A. N.; Yowtak, J.; Meisel, M. W.

2005-07-01

We review our studies of the biological impact of magnetic field strengths of up to 30 T on transgenic arabidopsis plants engineered with a stress response gene consisting of the alcohol dehydrogenase (Adh) gene promoter driving the β-glucuronidase (GUS) gene reporter. Field strengths in excess of 15 T induce expression of the Adh/GUS transgene in the roots and leaves. Microarray analyses indicate that such field strengths have a far reaching effect on the genome. Wide spread induction of stress-related genes and transcription factors, and a depression of genes associated with cell wall metabolism are prominent examples.

The water-water cycle is essential for chloroplast protection in the absence of stress.

PubMed

Rizhsky, Ludmila; Liang, Hongjian; Mittler, Ron

2003-10-03

Maintaining electron flow through the photosynthetic apparatus, even in the absence of a sufficient amount of NADP+ as an electron acceptor, is essential for chloroplast protection from photooxidative stress. At least two different pathways are thought to participate in this process, i.e. cyclic electron flow and the water-water cycle. Although the function of the water-water cycle was inferred from a number of biochemical and physiological studies, genetic evidence for the function of this cycle is very limited. Here we show that knockdown Arabidopsis plants with suppressed expression of the key water-water cycle enzyme, thylakoid-attached copper/zinc superoxide dismutase (KD-SOD), are suppressed in their growth and development. Chloroplast size, chlorophyll content, and photosynthetic activity were also reduced in KD-SOD plants. Microarray analysis of KD-SOD plants, grown under controlled conditions, revealed changes in transcript expression consistent with an acclimation response to light stress. Although a number of transcripts involved in the defense of plants from oxidative stress were induced in KD-SOD plants, and seedlings of KD-SOD plants were more tolerant to oxidative stress, these mechanisms were unable to compensate for the suppression of the water-water cycle in mature leaves. Thus, the localization of copper/zinc superoxide dismutase at the vicinity of photosystem I may be essential for its function. Our studies provide genetic evidence for the importance of the water-water cycle in protecting the photosynthetic apparatus of higher plants from photooxidative damage.