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1

Selection of reference genes for qPCR in hairy root cultures of peanut  

PubMed Central

Background Hairy root cultures produced via Agrobacterium rhizogenes-mediated transformation have emerged as practical biological models to elucidate the biosynthesis of specialized metabolites. To effectively understand the expression patterns of the genes involved in the metabolic pathways of these compounds, reference genes need to be systematically validated under specific experimental conditions as established by the MIQE (Minimum Information for Publication of Quantitative Real-Time PCR Experiments) guidelines. In the present report we describe the first validation of reference genes for RT-qPCR in hairy root cultures of peanut which produce stilbenoids upon elicitor treatments. Results A total of 21 candidate reference genes were evaluated. Nineteen genes were selected based on previous qPCR studies in plants and two were from the T-DNAs transferred from A. rhizogenes. Nucleotide sequences of peanut candidate genes were obtained using their homologous sequences in Arabidopsis. To identify the suitable primers, calibration curves were obtained for each candidate reference gene. After data analysis, 12 candidate genes meeting standard efficiency criteria were selected. The expression stability of these genes was analyzed using geNorm and NormFinder algorithms and a ranking was established based on expression stability of the genes. Candidate reference gene expression was shown to have less variation in methyl jasmonate (MeJA) treated root cultures than those treated with sodium acetate (NaOAc). Conclusions This work constitutes the first effort to validate reference genes for RT-qPCR in hairy roots. While these genes were selected under conditions of NaOAc and MeJA treatment, we anticipate these genes to provide good targets for reference genes for hairy roots under a variety of stress conditions. The lead reference genes were a gene encoding for a TATA box binding protein (TBP2) and a gene encoding a ribosomal protein (RPL8C). A commonly used reference gene GAPDH showed low stability of expression suggesting that its use may lead to inaccurate gene expression profiles when used for data normalization in stress-stimulated hairy roots. Likewise the A. rhizogenes transgene rolC showed less expression stability than GAPDH. This study proposes that a minimum of two reference genes should be used for a normalization procedure in gene expression profiling using elicited hairy roots. PMID:21985172

2011-01-01

2

Selection of reference genes for gene expression studies in porcine skeletal muscle using SYBR green qPCR.  

PubMed

Quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA. Internal controls such as reference genes are used to normalize mRNA levels between different samples for an exact comparison of gene transcription level. However, the expression levels of these reference genes may vary between cell types, developmental stages, species and experimental conditions, thus proper normalization strategy is an important precondition for reliable conclusions. In this study, we explored 10 commonly used reference genes in porcine skeletal muscle using SYBR green qPCR. We used both geNorm and NormFinder to analyze the expression stability and found that PPIA, HPRT and eEF-1? were suitable internal controls for porcine skeletal muscle. However, PPIA, HPRT and SDHA were suitable for skeletal muscle of western pigs while PPIA, eEF-1? and HPRT for indigenous Chinese pigs. Normalized qPCR data of ROCK2 were compared with microarray data to evaluate our developed set of reference genes. PMID:20887758

Feng, Xiaoting; Xiong, Yuanzhu; Qian, Hui; Lei, Minggang; Xu, Dequan; Ren, Zhuqing

2010-11-01

3

Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis  

Microsoft Academic Search

The selection and validation of reference genes constitute a key point for gene expression analysis based on qPCR, requiring\\u000a efficient normalization approaches. In this work, the expression profiles of eight genes were evaluated to identify novel\\u000a reference genes for transcriptional studies associated to the senescence process in sunflower. Three alternative strategies\\u000a were applied for the evaluation of gene expression stability

Paula FernandezJulio; Julio A. Di Rienzo; Sebastián Moschen; Guillermo A. A. Dosio; Luis A. N. Aguirrezábal; H. Esteban Hopp; Norma Paniego; Ruth A. Heinz

2011-01-01

4

Selection of reliable reference genes for qPCR studies on chondroprotective action  

PubMed Central

Background Chondroprotective agents (CPA) such as glucosamine, curcumin and diacerein represent potential remedies for the management of osteoarthritis and several studies have been performed on their effects in-vitro and in-vivo. For the investigation of chondroprotective action on chondrocyte gene expression, quantitative real-time RT-PCR is the method of choice. However, validation of applied normalization strategies represents a crucial and sometimes neglected step in the analysis process. Therefore, the present study aimed to determine the expression stability of common reference genes (ACTB, Beta actin; GAPDH, Glyceraldehyde-3-phosphate; B2M, Beta-2-microglobulin; HPRT1, Hypoxanthine phosphoribosyl-transferase I; SDHA, Succinate dehydrogenase complex, subunit A; YWHAZ, Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) under the influence of glucosamine, curcumin and diacerein in the IL-1?-stimulated C-28/I2 chondrocyte model, using the geNorm software tool. Results CPA treatment of C-28/I2 chondrocytes significantly affected the expression level of many reference genes (p < 0.05). According to their expression stability, geNorm analysis revealed rankings of the 3 most stable genes (from most stable to least stable) as follows: GAPDH, B2M and SDHA in glucosamine treated samples and HPRT1, GAPDH and B2M in curcumin or diacerein treated samples. Interestingly, ACTB was one of the most variably expressed genes throughout all experiments. Conclusion Our study points out the problem of relying on commonly used reference genes without an accurate validation process. For normalization purposes in gene profiling studies on glucosamine action, the genes GAPDH, B2M and SDHA are recommended as single reference genes depending on the expression level of the target gene or more favourably in combination. For experiments with curcumin and diacerein the use of HPRT1, GAPDH and B2M should be considered. PMID:17324259

Toegel, Stefan; Huang, Wenwen; Piana, Claudia; Unger, Frank M; Wirth, Michael; Goldring, Mary B; Gabor, Franz; Viernstein, Helmut

2007-01-01

5

Selection of Suitable Reference Genes for qPCR Normalization under Abiotic Stresses in Oenanthe javanica (BI.) DC  

PubMed Central

Accurate normalization of gene expression data is an absolute prerequisite to obtain reliable results in qPCR analysis. Oenanthe javanica, an aquatic perennial herb, belongs to the Oenanthe genus in Apiaceae family, with known medicinal properties. In the current study, O. javanica was subjected to hormone stimuli (gibberellin, salicylic acid, methyl jasmonate, and abscisic acid) and abiotic stresses (heat, cold, salt, and drought), and the expression of nine candidate reference genes (eIF-4?, ACT7, TIP41, GAPDH, SAND, EF-1?, PP2A, TBP, and TUB) was evaluated. Stability of the genes was assessed using geNorm, NormFinder and BestKeeper. All the genes presented distinct expression profiles under the experimental conditions analyzed. Under abiotic stress conditions, ACT7 and PP2A genes displayed the maximum stability; PP2A and SAND were the most stable genes under hormone stimuli. Even though PP2A gene was most stable across all the samples, individual analysis revealed changes in expression profile. To further validate the suitability of the reference genes identified in this study, the expression level of M6PR gene under salt treatment was studied. Based on our data, we propose that it is essential to normalize the target gene expression with specific reference genes under different experimental conditions for most accurate results. To our knowledge, this is the first systematic analysis for reference genes under abiotic stress and hormone stimuli conditions in O. javanica. This will be beneficial for future studies on O. javanica and other plants in Apiaceae family at molecular level. PMID:24651080

Li, Meng-Yao; Ma, Jing; Tan, Guo-Fei; Xiong, Ai-Sheng

2014-01-01

6

Evaluation of Reference Genes for RT qPCR Analyses of Structure-Specific and Hormone Regulated Gene Expression in Physcomitrella patens Gametophytes  

PubMed Central

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

7

Evaluation of reference genes for RT qPCR analyses of structure-specific and hormone regulated gene expression in Physcomitrella patens gametophytes.  

PubMed

The use of the moss Physcomitrella patens as a model system to study plant development and physiology is rapidly expanding. The strategic position of P. patens within the green lineage between algae and vascular plants, the high efficiency with which transgenes are incorporated by homologous recombination, advantages associated with the haploid gametophyte representing the dominant phase of the P. patens life cycle, the simple structure of protonemata, leafy shoots and rhizoids that constitute the haploid gametophyte, as well as a readily accessible high-quality genome sequence make this moss a very attractive experimental system. The investigation of the genetic and hormonal control of P. patens development heavily depends on the analysis of gene expression patterns by real time quantitative PCR (RT qPCR). This technique requires well characterized sets of reference genes, which display minimal expression level variations under all analyzed conditions, for data normalization. Sets of suitable reference genes have been described for most widely used model systems including e.g. Arabidopsis thaliana, but not for P. patens. Here, we present a RT qPCR based comparison of transcript levels of 12 selected candidate reference genes in a range of gametophytic P. patens structures at different developmental stages, and in P. patens protonemata treated with hormones or hormone transport inhibitors. Analysis of these RT qPCR data using GeNorm and NormFinder software resulted in the identification of sets of P. patens reference genes suitable for gene expression analysis under all tested conditions, and suggested that the two best reference genes are sufficient for effective data normalization under each of these conditions. PMID:23951063

Le Bail, Aude; Scholz, Sebastian; Kost, Benedikt

2013-01-01

8

Quantifying mRNA and MicroRNA with qPCR in Cervical Carcinogenesis: A Validation of Reference Genes to Ensure Accurate Data  

PubMed Central

A number of recent studies have catalogued global gene expression patterns in a panel of normal, tumoral cervical tissues so that potential biomarkers can be identified. The qPCR has been one of the most widely used technologies for detecting these potential biomarkers. However, few studies have investigated a correct strategy for the normalization of data in qPCR assays for cervical tissues. The aim of this study was to validate reference genes in cervical tissues to ensure accurate quantification of mRNA and miRNA levels in cervical carcinogenesis. For this purpose, some issues for obtaining reliable qPCR data were evaluated such as the following: geNorm analysis with a set of samples which meet all of the cervical tissue conditions (Normal + CIN1 + CIN2 + CIN3 + Cancer); the use of individual Ct values versus pooled Ct values; and the use of a single (or multiple) reference genes to quantify mRNA and miRNA expression levels. Two different data sets were put on the geNorm to assess the expression stability of the candidate reference genes: the first dataset comprised the quantities of the individual Ct values; and the second dataset comprised the quantities of the pooled Ct values. Moreover, in this study, all the candidate reference genes were analyzed as a single “normalizer”. The normalization strategies were assessed by measuring p16INK4a and miR-203 transcripts in qPCR assays. We found that the use of pooled Ct values, can lead to a misinterpretation of the results, which suggests that the maintenance of inter-individual variability is a key factor in ensuring the reliability of the qPCR data. In addition, it should be stressed that a proper validation of the suitability of the reference genes is required for each experimental setting, since the indiscriminate use of a reference gene can also lead to discrepant results. PMID:25365304

Leitao, Maria da Conceicao Gomes; Coimbra, Eliane Campos; de Lima, Rita de Cassia Pereira; Guimaraes, Marilea de Lima; Heraclio, Sandra de Andrade; Silva Neto, Jacinto da Costa; de Freitas, Antonio Carlos

2014-01-01

9

Validation of Potential Reference Genes for qPCR in Maize across Abiotic Stresses, Hormone Treatments, and Tissue Types  

PubMed Central

The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1?), tubulin beta (?-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1?, ?-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types. PMID:24810581

Lan, Hai; Gao, Shibin; Liu, Hailan; Liu, Jian; Cao, Moju; Pan, Guangtang; Rong, Tingzhao; Zhang, Suzhi

2014-01-01

10

Effect of irradiation on the expression of DNA repair genes studied in human fibroblasts by real-time qPCR using three methods of reference gene validation.  

PubMed

The aim of this study was to determine the effects of ionizing radiation on gene expression by using for a first time a qPCR platform specifically established for the detection of 94 DNA repair genes but also to test the robustness of these results by using three analytical methods (global pattern recognition, ??Cq/Normfinder and ??Cq/Genorm). Study was focused on these genes because DNA repair is known primarily to determine the radiation response. Six strains of normal human fibroblasts were exposed to 2 Gy, and changes in gene expression were analyzed 24 h thereafter. A significant change in gene expression was found for only few genes, but the genes detected were mostly different for the three analytical methods used. For GPR, a significant change was found for four genes, in contrast to the eight or nine genes when applying ??Cq/Genorm or ??Cq/Normfinder, respectively. When using all three methods, a significant change in expression was only seen for GADD45A and PCNA. These data demonstrate that (1) the genes identified to show an altered expression upon irradiation strongly depend on the analytical method applied, and that (2) overall GADD45A and PCNA appear to play a central role in this response, while no significant change is induced for any of the other DNA repair genes tested. PMID:23884658

Reuther, Sebastian; Reiter, Martina; Raabe, Annette; Dikomey, Ekkehard

2013-11-01

11

Selecting control genes for RT-QPCR using public microarray data  

PubMed Central

Background Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different samples uses one to several control genes (e.g. housekeeping genes), from which a baseline reference level is constructed. Thus, the choice of the control genes is of utmost importance, yet there is not a generally accepted standard technique for screening a large number of candidates and identifying the best ones. Results We propose a novel approach for scoring and ranking candidate genes for their suitability as control genes. Our approach relies on publicly available microarray data and allows the combination of multiple data sets originating from different platforms and/or representing different pathologies. The use of microarray data allows the screening of tens of thousands of genes, producing very comprehensive lists of candidates. We also provide two lists of candidate control genes: one which is breast cancer-specific and one with more general applicability. Two genes from the breast cancer list which had not been previously used as control genes are identified and validated by RT-QPCR. Open source R functions are available at Conclusion We proposed a new method for identifying candidate control genes for RT-QPCR which was able to rank thousands of genes according to some predefined suitability criteria and we applied it to the case of breast cancer. We also empirically showed that translating the results from microarray to PCR platform was achievable. PMID:19187545

Popovici, Vlad; Goldstein, Darlene R; Antonov, Janine; Jaggi, Rolf; Delorenzi, Mauro; Wirapati, Pratyaksha

2009-01-01

12

Methods for qPCR gene expression profiling applied to 1440 lymphoblastoid single cells  

PubMed Central

The stochastic nature of generating eukaryotic transcripts challenges conventional methods for obtaining and analyzing single-cell gene expression data. In order to address the inherent noise, detailed methods are described on how to collect data on multiple genes in a large number of single cells using microfluidic arrays. As part of a study exploring the effect of genotype on Wnt pathway activation, data were collected for 96 qPCR assays on 1440 lymphoblastoid cells. The description of methods includes preliminary data processing steps. The methods used in the collection and analysis of single-cell qPCR data are contrasted with those used in conventional qPCR. PMID:23079396

Livak, Kenneth J.; Wills, Quin F.; Tipping, Alex J.; Datta, Krishnalekha; Mittal, Rowena; Goldson, Andrew J.; Sexton, Darren W.; Holmes, Chris C.

2013-01-01

13

Selecting control genes for RT-QPCR using public microarray data  

Microsoft Academic Search

BACKGROUND: Gene expression analysis has emerged as a major biological research area, with real-time quantitative reverse transcription PCR (RT-QPCR) being one of the most accurate and widely used techniques for expression profiling of selected genes. In order to obtain results that are comparable across assays, a stable normalization strategy is required. In general, the normalization of PCR measurements between different

Vlad Popovici; Darlene R. Goldstein; Janine Antonov; Rolf Jaggi; Mauro Delorenzi; Pratyaksha Wirapati

2009-01-01

14

Novel reference genes for quantifying transcriptional responses of Escherichia coli to protein overexpression by quantitative PCR  

E-print Network

Background: Accurate interpretation of quantitative PCR (qPCR) data requires normalization using constitutively expressed reference genes. Ribosomal RNA is often used as a reference gene for transcriptional studies in E. ...

Zhou, Kang

15

Next-generation qPCR for the high-throughput measurement of gene expression in multiple leukocyte subsets.  

PubMed

Clinical studies of gene expression are increasingly using the whole blood, peripheral blood mononuclear cells, and leukocyte subsets involved in the innate and adaptive immune responses. However, the small amount of RNA available in the clinical setting is a limitation for commonly used methods such as quantitative polymerase chain reactions (qPCR) and microarrays. Our aim was to design 96 gene assays to simultaneously measure gene expression in the whole blood and seven leukocyte subsets using a new-generation qPCR method--high-throughput nanofluidic reverse transcription qPCR (HT RT-qPCR). The leukocyte subset purity was 94% to 98% for seven subsets and was less for the ?? T-cell receptor subset (80%). The HT RT-qPCR replicate sample measurements were highly reproducible (r = 0.997, p < 2.2 × 10(-16)), and the ??Ct values from HT RT-qPCR correlated significantly with those from qPCR. The control genes were differentially expressed across the eight leukocyte subsets in the control subjects (p = 1.3 × 10(-5), analysis of variance). Two analytical methods, absolute and relative, gave concordant results and were significantly correlated (p = 1.9 × 10(-9)). HT RT-qPCR permits the rapid, reproducible, and quantitative measurement of multiple transcripts using minimal sample amounts. The protocol described yielded leukocyte subsets of high purity and identified two analytic methods for use. PMID:23690294

Adamski, Mateusz G; Li, Yan; Wagner, Erin; Yu, Hua; Seales-Bailey, Chloe; Soper, Steven A; Murphy, Michael; Baird, Alison E

2013-10-01

16

Selection and validation of reference genes for transcript normalization in gene expression studies in Catharanthus roseus.  

PubMed

Quantitative Real-Time PCR (qPCR), a sensitive and commonly used technique for gene expression analysis, requires stably expressed reference genes for normalization of gene expression. Up to now, only one reference gene for qPCR analysis, corresponding to 40S Ribosomal protein S9 (RPS9), was available for the medicinal plant Catharanthus roseus, the only source of the commercial anticancer drugs vinblastine and vincristine. Here, we screened for additional reference genes for this plant species by mining C. roseus RNA-Seq data for orthologs of 22 genes known to be stably expressed in Arabidopsis thaliana and qualified as superior reference genes for this model plant species. Based on this, eight candidate C. roseus reference genes were identified and, together with RPS9, evaluated by performing qPCR on a series of different C. roseus explants and tissue cultures. NormFinder, geNorm and BestKeeper analyses of the resulting qPCR data revealed that the orthologs of At2g28390 (SAND family protein, SAND), At2g32170 (N2227-like family protein, N2227) and At4g26410 (Expressed protein, EXP) had the highest expression stability across the different C. roseus samples and are superior as reference genes as compared to the traditionally used RPS9. Analysis of publicly available C. roseus RNA-Seq data confirmed the expression stability of SAND and N2227, underscoring their value as reference genes for C. roseus qPCR analysis. PMID:25058454

Pollier, Jacob; Vanden Bossche, Robin; Rischer, Heiko; Goossens, Alain

2014-10-01

17

QuantStudioTM services at OHSU --qPCR gene expression  

E-print Network

RNA profiling #12;The all-in-one qPCR instrument Combining flexible throughput capabilities with a streamlined workflow, the QuantStudioTM 12K Flex system takes you from targeted discovery through confirmation,000datapointsinan8-hourday ·Simplehigh-throughputOpenArray® workflow

Chapman, Michael S.

18

Reference Gene Selection for Quantitative PCR Studies in Sheep Neutrophils  

PubMed Central

Reference genes are essential for studying mRNA expression with quantitative PCR (qPCR). We investigated 11 potential neutrophil reference genes (RPL19, GAPDH, ACTB, B2M, HPRT, G6PD, TFRC, PGK1, YWHAZ, SDHA and GYPC) for sheep under disease conditions of foot rot (FR) and with or without Se supplementation. Initial screening was based on gene expression level (<28 Cq cycles) and variability (SD < 1.5 Cq cycles) and excluded TFRC, GYPC and HPRT from further analysis. Expression stability of the remaining genes was evaluated using four software programs: geNorm, NormFinder, BestKeeper and the comparative delta Cq method. The neutrophil reference genes, G6PD, YWHAZ, GAPDH, RPL19 and SDHA, consistently ranked among the top five most stable genes under these experimental conditions. The SDHA gene expression was not stable in FR-diseased sheep receiving Se treatment and, thus, cannot be recommended as a reference gene. The commonly used genes, PGK1, ACTB and B2M, were not reliable reference genes, underscoring the need to validate neutrophil reference genes under different experimental conditions. Multiple references genes rather than a single gene may provide more robust and reliable results. The best pair of reference genes was SDHA/G6PD in healthy sheep and GADPH/YWHAZ in FR-diseased sheep. PMID:23722658

Vorachek, William R.; Hugejiletu; Bobe, Gerd; Hall, Jean A.

2013-01-01

19

Superior Cross-Species Reference Genes: A Blueberry Case Study  

PubMed Central

The advent of affordable Next Generation Sequencing technologies has had major impact on studies of many crop species, where access to genomic technologies and genome-scale data sets has been extremely limited until now. The recent development of genomic resources in blueberry will enable the application of high throughput gene expression approaches that should relatively quickly increase our understanding of blueberry physiology. These studies, however, require a highly accurate and robust workflow and make necessary the identification of reference genes with high expression stability for correct target gene normalization. To create a set of superior reference genes for blueberry expression analyses, we mined a publicly available transcriptome data set from blueberry for orthologs to a set of Arabidopsis genes that showed the most stable expression in a developmental series. In total, the expression stability of 13 putative reference genes was evaluated by qPCR and a set of new references with high stability values across a developmental series in fruits and floral buds of blueberry were identified. We also demonstrated the need to use at least two, preferably three, reference genes to avoid inconsistencies in results, even when superior reference genes are used. The new references identified here provide a valuable resource for accurate normalization of gene expression in Vaccinium spp. and may be useful for other members of the Ericaceae family as well. PMID:24058469

Die, Jose V.; Rowland, Lisa J.

2013-01-01

20

Evaluation and Validation of Reference Genes for Normalization of Quantitative Real-Time PCR Based Gene Expression Studies in Peanut  

PubMed Central

The quantitative real-time PCR (qPCR) based techniques have become essential for gene expression studies and high-throughput molecular characterization of transgenic events. Normalizing to reference gene in relative quantification make results from qPCR more reliable when compared to absolute quantification, but requires robust reference genes. Since, ideal reference gene should be species specific, no single internal control gene is universal for use as a reference gene across various plant developmental stages and diverse growth conditions. Here, we present validation studies of multiple stably expressed reference genes in cultivated peanut with minimal variations in temporal and spatial expression when subjected to various biotic and abiotic stresses. Stability in the expression of eight candidate reference genes including ADH3, ACT11, ATPsyn, CYP2, ELF1B, G6PD, LEC and UBC1 was compared in diverse peanut plant samples. The samples were categorized into distinct experimental sets to check the suitability of candidate genes for accurate and reliable normalization of gene expression using qPCR. Stability in expression of the references genes in eight sets of samples was determined by geNorm and NormFinder methods. While three candidate reference genes including ADH3, G6PD and ELF1B were identified to be stably expressed across experiments, LEC was observed to be the least stable, and hence must be avoided for gene expression studies in peanut. Inclusion of the former two genes gave sufficiently reliable results; nonetheless, the addition of the third reference gene ELF1B may be potentially better in a diverse set of tissue samples of peanut. PMID:24167633

Cindhuri, Katamreddy Sri; Sharma, Kiran K.

2013-01-01

21

Optimizing a qPCR Gene Expression Quantification Assay for S. epidermidis Biofilms: A Comparison between Commercial Kits and a Customized Protocol  

PubMed Central

Staphylococcus epidermidis biofilm-related infections are a current concern within the medical community due to their high incidence and prevalence, particularly in patients with indwelling medical devices. Biofilm gene expression analysis by quantitative real-time PCR (qPCR) has been increasingly used to understand the role of biofilm formation in the pathogenesis of S. epidermidis infections. However, depending on the RNA extraction procedure, and cDNA synthesis and qPCR master mixes used, gene expression quantification can be suboptimal. We recently showed that some RNA extraction kits are not suitable for S. epidermidis biofilms, due to sample composition, in particular the presence of the extracellular matrix. In this work, we describe a custom RNA extraction assay followed by the evaluation of gene expression using different commercial reverse transcriptase kits and qPCR master mixes. Our custom RNA extraction assay was able to produce good quality RNA with reproducible gene expression quantification, reducing the time and the costs associated. We also tested the effect of reducing cDNA and qPCR reaction volumes and, in most of the cases tested, no significant differences were found. Finally, we titered the SYBR Green I concentrations in standard PCR master mixes and compared the normalized expression of the genes icaA, bhp, aap, psm?1 and agrB using 4 distinct biofilm forming S. epidermidis strains to the results obtained with commercially available kits. The overall results demonstrated that despite some statistically, but not biologically significant differences observed, the customized qPCR protocol resulted in the same gene expression trend presented by the commercially available kits used. PMID:22629403

Franca, Angela; Freitas, Ana I.; Henriques, Ana F.; Cerca, Nuno

2012-01-01

22

Monitoring foreign gene incorporation into the plastome of Chlamydomonas reinhardtii by multiplex qPCR.  

PubMed

The genetic material of the Chlamydomonas reinhardtii chloroplast can be easily manipulated and creation of transgenic plastomes is of interest for both photosynthetic research and for biofuel and biomass production. Because multiple copies of the chloroplast genome are present, it is important to understand whether, following the introduction of a foreign gene, the resulting transgenic plastome is homoplasmic or heteroplasmic. By quantitative PCR together with a simple DNA extraction procedure and a series of DNA oligonucleotides the following protocol will determine the extent of foreign gene incorporation into a host chloroplast plastome. This approach is used to follow the degree of heteroplasmy following biolistic transformation of several transgenic strains. The approach used is quick, simple to set up, and gives an accurate quantitation of foreign genes within of the chloroplast plastome. Possible future uses of the technique are discussed. PMID:23649166

Johnson, Eric A

2013-05-01

23

Evaluation of the 23S rRNA gene as target for qPCR based quantification of Frankia in soils.  

PubMed

The 23S rRNA gene was evaluated as target for the development of Sybr Green-based quantitative PCR (qPCR) for the analysis of nitrogen-fixing members of the genus Frankia or subgroups of these in soil. A qPCR with a primer combination targeting all nitrogen-fixing frankiae (clusters 1, 2 and 3) resulted in numbers similar to those obtained with a previously developed qPCR using nifH gene sequences, both with respect to introduced and indigenous Frankia populations. Primer combinations more specifically targeting three subgroups of the Alnus host infection group (cluster 1) or members of the Elaeagnus host infection group (cluster 3) were specific for introduced strains of the target group, with numbers corresponding to those obtained by quantification of nitrogen-fixing frankiae with both the 23S rRNA and nifH genes as target. Method verification on indigenous Frankia populations in soils, i.e. in depth profiles from four sites at an Alnus glutinosa stand, revealed declining numbers in the depth profiles, with similar abundance of all nitrogen-fixing frankiae independent of 23S rRNA or nifH gene targets, and corresponding numbers of one group of frankiae of the Alnus host infection only, with no detections of frankiae representing the Elaeagnus, Casuarina, or a second subgroup of the Alnus host infection groups. PMID:24315016

Samant, Suvidha; Amann, Rudolf I; Hahn, Dittmar

2014-05-01

24

Gapdh gene expression is modulated by inflammatory arthritis and is not suitable for qPCR normalization.  

PubMed

Gene expression studies are fundamental for the understanding of complex diseases, providing new insights into the pathogenic process and new tools for diagnostic and patient stratification. Gene profiling studies by real-time PCR require the use of reference genes for normalization and an appropriate validation is essential for accurate results. We performed a comprehensive assessment of six common housekeeping genes in the K/BxN serum-induced arthritis model in mice. Classical statistics and NormFinder analyses pointed out Gapdh as the less stable and therefore unsuitable as a reference control. Gapdh was considerably down-regulated in arthritic joints and therefore produced an overestimation of transcriptional changes. Hptr, B2m, and Rpl13a showed the most constant expression. Collectively our data advise against the use of Gapdh in gene expression studies in the acute phase of the K/BxN model and adds a cautionary note on the need to validate the reference genes for reliable, comparable, and reproducible results. PMID:24493325

Montero-Melendez, Trinidad; Perretti, Mauro

2014-08-01

25

Evaluation of reference genes for quantitative real-time PCR in the brain, pituitary, and gonads of songbirds.  

PubMed

Quantitative real-time PCR (qPCR) is becoming a popular tool for the quantification of gene expression in the brain and endocrine tissues of songbirds. Accurate analysis of qPCR data relies on the selection of appropriate reference genes for normalization, yet few papers on songbirds contain evidence of reference gene validation. Here, we evaluated the expression of ten potential reference genes (18S, ACTB, GAPDH, HMBS, HPRT, PPIA, RPL4, RPL32, TFRC, and UBC) in brain, pituitary, ovary, and testis in two species of songbirds: zebra finch and white-throated sparrow. We used two algorithms, geNorm and NormFinder, to assess the stability of these reference genes in our samples. We found that the suitability of some of the most popular reference genes for target gene normalization in mammals, such as 18S, depended highly on tissue type. Thus, they are not the best choices for brain and gonad in these songbirds. In contrast, we identified alternative genes, such as HPRT, RPL4 and PPIA, that were highly stable in brain, pituitary, and gonad in these species. Our results suggest that the validation of reference genes in mammals does not necessarily extrapolate to other taxonomic groups. For researchers wishing to identify and evaluate suitable reference genes for qPCR in songbirds, our results should serve as a starting point and should help increase the power and utility of songbird models in behavioral neuroendocrinology. PMID:24780145

Zinzow-Kramer, Wendy M; Horton, Brent M; Maney, Donna L

2014-07-01

26

Reliable Reference Genes for Normalization of Gene Expression in Cucumber Grown under Different Nitrogen Nutrition  

PubMed Central

In plants, nitrogen is the most important nutritional factor limiting the yield of cultivated crops. Since nitrogen is essential for synthesis of nucleotides, amino acids and proteins, studies on gene expression in plants cultivated under different nitrogen availability require particularly careful selection of suitable reference genes which are not affected by nitrogen limitation. Therefore, the objective of this study was to select the most reliable reference genes for qPCR analysis of target cucumber genes under varying nitrogen source and availability. Among twelve candidate cucumber genes used in this study, five are highly homologous to the commonly used internal controls, whereas seven novel candidates were previously identified through the query of the cucumber genome. The expression of putative reference genes and the target CsNRT1.1 gene was analyzed in roots, stems and leaves of cucumbers grown under nitrogen deprivation, varying nitrate availability or different sources of nitrogen (glutamate, glutamine or NH3). The stability of candidate genes expression significantly varied depending on the tissue type and nitrogen supply. However, in most of the outputs genes encoding CACS, TIP41, F-box protein and EF? proved to be the most suitable for normalization of CsNRT1.1 expression. In addition, our results suggest the inclusion of 3 or 4 references to obtain highly reliable results of target genes expression in all cucumber organs under nitrogen-related stress. PMID:24058446

Warzybok, Anna; Migocka, Magdalena

2013-01-01

27

Diversity and Distribution of Marine Synechococcus: Multiple Gene Phylogenies for Consensus Classification and Development of qPCR Assays for Sensitive Measurement of Clades in the Ocean.  

PubMed

Marine Synechococcus is a globally significant genus of cyanobacteria that is comprised of multiple genetic lineages or clades. These clades are thought to represent ecologically distinct units, or ecotypes. Because multiple clades often co-occur together in the oceans, Synechococcus are ideal microbes to explore how closely related bacterial taxa within the same functional guild of organisms co-exist and partition marine habitats. Here we sequenced multiple gene loci from cultured strains to confirm the congruency of clade classifications between the 16S-23S rDNA internally transcribed spacer (ITS), 16S rDNA, narB, ntcA, and rpoC1 loci commonly used in Synechococcus diversity studies. We designed quantitative PCR (qPCR) assays that target the ITS for 10 Synechococcus clades, including four clades, XV, XVI, CRD1, and CRD2, not covered by previous assays employing other loci. Our new qPCR assays are very sensitive and specific, detecting down to tens of cells per ml. Application of these qPCR assays to field samples from the northwest Atlantic showed clear shifts in Synechococcus community composition across a coastal to open-ocean transect. Consistent with previous studies, clades I and IV dominated cold, coastal Synechococcus communities. Clades II and X were abundant at the two warmer, off-shore stations, and at all stations multiple Synechococcus clades co-occurred. qPCR assays developed here provide valuable tools to further explore the dynamics of microbial community structure and the mechanisms of co-existence. PMID:22723796

Ahlgren, Nathan A; Rocap, Gabrielle

2012-01-01

28

Diversity and Distribution of Marine Synechococcus: Multiple Gene Phylogenies for Consensus Classification and Development of qPCR Assays for Sensitive Measurement of Clades in the Ocean  

PubMed Central

Marine Synechococcus is a globally significant genus of cyanobacteria that is comprised of multiple genetic lineages or clades. These clades are thought to represent ecologically distinct units, or ecotypes. Because multiple clades often co-occur together in the oceans, Synechococcus are ideal microbes to explore how closely related bacterial taxa within the same functional guild of organisms co-exist and partition marine habitats. Here we sequenced multiple gene loci from cultured strains to confirm the congruency of clade classifications between the 16S–23S rDNA internally transcribed spacer (ITS), 16S rDNA, narB, ntcA, and rpoC1 loci commonly used in Synechococcus diversity studies. We designed quantitative PCR (qPCR) assays that target the ITS for 10 Synechococcus clades, including four clades, XV, XVI, CRD1, and CRD2, not covered by previous assays employing other loci. Our new qPCR assays are very sensitive and specific, detecting down to tens of cells per ml. Application of these qPCR assays to field samples from the northwest Atlantic showed clear shifts in Synechococcus community composition across a coastal to open-ocean transect. Consistent with previous studies, clades I and IV dominated cold, coastal Synechococcus communities. Clades II and X were abundant at the two warmer, off-shore stations, and at all stations multiple Synechococcus clades co-occurred. qPCR assays developed here provide valuable tools to further explore the dynamics of microbial community structure and the mechanisms of co-existence. PMID:22723796

Ahlgren, Nathan A.; Rocap, Gabrielle

2012-01-01

29

Defining suitable reference genes for RT-qPCR analysis on human sertoli cells after 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) exposure.  

PubMed

Quantitative real-time RT-PCR (qPCR) has proven to be a valuable molecular technique to quantify gene expression. There are few studies in the literature that describe suitable reference genes to normalize gene expression data. Studies of transcriptionally disruptive toxins, like tetrachlorodibenzo-p-dioxin (TCDD), require careful consideration of reference genes. The present study was designed to validate potential reference genes in human Sertoli cells after exposure to TCDD. 32 candidate reference genes were analyzed to determine their applicability. geNorm and NormFinder softwares were used to obtain an estimation of the expression stability of the 32 genes and to identify the most suitable genes for qPCR data normalization. PMID:25078986

Ribeiro, Mariana Antunes; Dos Reis, Mariana Bisarro; de Moraes, Leonardo Nazário; Briton-Jones, Christine; Rainho, Cláudia Aparecida; Scarano, Wellerson Rodrigo

2014-11-01

30

Validation of reference genes for quantitative real-time PCR during Chinese wolfberry fruit development.  

PubMed

Lycium barbarum L., a woody bush that grows in Eurasia and North Africa, is an ornamental and medicinal plant. Its fruits have been used for centuries in China as a traditional herbal medicine and as a valuable nourishing tonic. There has been no report describing the selection of reference genes for stringent normalization for quantitative PCR (qPCR) in L. barbarum. The present study identified reliable reference genes for normalization of qPCR data in L. barbarum during fruit development from among eight candidate genes (GAPDH, TEF G, EF 1a, UBQ, TUB a, SAMS, EF2 and Hsp80) using the geNorm and NormFinder statistical algorithms. The results showed that the best-ranked references genes differed across the samples. A combination of GAPDH and EF1a would be appropriate as a reference panel for normalizing gene expression data across fruit developmental stages. A combination of EF 1a and SAMS would be appropriate as a reference panel for normalizing gene expression data at the stage A tested, whereas the combination of TUB a, and TEF G was the most suitable for stage B. EF2 and Hsp80 exhibited the most stable expression under stage C and stage D. NormFinder ranking of reference gene candidates was slightly different from that determined by geNorm. These results provide guidelines for the selection of reference genes under different development stages and also represent a foundation for more accurate and widespread use of qRT-PCR in L. barbarum gene analysis. PMID:23811043

Wang, Lijuan; Wang, Yancai; Zhou, Ping

2013-09-01

31

Identification of Appropriate Reference Genes for Human Mesenchymal Cells during Expansion and Differentiation  

PubMed Central

Background Quantitative real time polymerase chain reaction (qPCR) is an extremely powerful technique for monitoring gene expression. The quantity of the messenger ribonucleic acids (mRNA) of interest should be normalized using a reference gene, in order to avoid unreliable results originated by the obtained RNA quality and quantity, manipulation errors and inhibitory contaminants. A reference gene is any gene that is stably and consistently expressed under the conditions being studied. Completely false data can be generated if a reference gene is not chosen adequately. Results In the present study, we compared expression levels of five putative reference genes (HPRT1, ACTB, GAPDH, RPL13A and B2M) in primary cultures of four different human cells: mesenchymal stromal cells obtained from bone marrow, adipose tissue or umbilical cord Wharto?s Jelly, and dermal fibroblasts, under different expansion and differentiation conditions. We observed that reference genes are not the same for different cells under the same culture conditions. Conclusion Most stable reference genes under our experimental conditions were: RPL13A for adipose tissue- and Wharto?s Jelly-derived mesenchymal stromal cells, and HPRT1 for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts. ACTB was the most unstable gene when evaluating adipose tissue- and Wharto?s Jelly-derived mesenchymal stromal cells, whilst GAPDH and B2M were the most unstable genes for bone marrow-derived mesenchymal stromal cells and dermal fibroblasts, respectively. PMID:24023904

Amable, Paola Romina; Teixeira, Marcus Vinicius Telles; Carias, Rosana Bizon Vieira; Granjeiro, Jose Mauro; Borojevic, Radovan

2013-01-01

32

A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli.  

PubMed

Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements have combined the high-throughput performance of the microarray with the specificity and sensitivity of real-time qPCR to make large-scale screening efforts both time- and cost-effective. This study identified and evaluated a panel of 28 genetic markers including known virulence and regulatory genes, O-antigen genes, and select prophage regions of O157 and non-O157 EHEC that can be used with high-throughput PCR to virulotype, serotype, and preliminarily subtype large numbers of isolates. The PCR assays for the target genes were shown to be robust using multiple extraction methods and PCR platforms. Preliminary quantitative PCR showed that an EHEC concentration of 10(4) CFU/ml or lower could be detected, with a linear range of detection over five to six orders of magnitude. The panel of 28 target genes has the potential to become an integral tool in outbreak, environmental, and genetic investigations of EHEC. PMID:21925264

Gonzales, Tina K; Kulow, Megan; Park, Dong-Jin; Kaspar, Charles W; Anklam, Kelly S; Pertzborn, Kelly M; Kerrish, Kristen D; Ivanek, Renata; Döpfer, Dörte

2011-01-01

33

Validation of Suitable Reference Genes for Expression Normalization in Echinococcus spp. Larval Stages  

PubMed Central

In recent years, a significant amount of sequence data (both genomic and transcriptomic) for Echinococcus spp. has been published, thereby facilitating the analysis of genes expressed during a specific stage or involved in parasite development. To perform a suitable gene expression quantification analysis, the use of validated reference genes is strongly recommended. Thus, the aim of this work was to identify suitable reference genes to allow reliable expression normalization for genes of interest in Echinococcus granulosus sensu stricto (s.s.) (G1) and Echinococcus ortleppi upon induction of the early pre-adult development. Untreated protoscoleces (PS) and pepsin-treated protoscoleces (PSP) from E. granulosus s.s. (G1) and E. ortleppi metacestode were used. The gene expression stability of eleven candidate reference genes (?TUB, NDUFV2, RPL13, TBP, CYP-1, RPII, EF-1?, ?ACT-1, GAPDH, ETIF4A-III and MAPK3) was assessed using geNorm, Normfinder, and RefFinder. Our qPCR data showed a good correlation with the recently published RNA-seq data. Regarding expression stability, EF-1? and TBP were the most stable genes for both species. Interestingly, ?ACT-1 (the most commonly used reference gene), and GAPDH and ETIF4A-III (previously identified as housekeeping genes) did not behave stably in our assay conditions. We propose the use of EF-1? as a reference gene for studies involving gene expression analysis in both PS and PSP experimental conditions for E. granulosus s.s. and E. ortleppi. To demonstrate its applicability, EF-1? was used as a normalizer gene in the relative quantification of transcripts from genes coding for antigen B subunits. The same EF-1? reference gene may be used in studies with other Echinococcus sensu lato species. This report validates suitable reference genes for species of class Cestoda, phylum Platyhelminthes, thus providing a foundation for further validation in other epidemiologically important cestode species, such as those from the Taenia genus. PMID:25014071

Espinola, Sergio Martin; Ferreira, Henrique Bunselmeyer; Zaha, Arnaldo

2014-01-01

34

Reference Gene Identification for Reverse Transcription-Quantitative Polymerase Chain Reaction Analysis in an Ischemic Wound-Healing Model  

PubMed Central

Reference genes are often used in RT-quantitative PCR (qPCR) analysis to normalize gene expression levels to a gene that is expressed stably across study groups. They ultimately serve as a control in RT-qPCR analysis, producing more accurate interpretation of results. Whereas many reference genes have been used in various wound-healing studies, the most stable reference gene for ischemic wound-healing analysis has yet to be identified. The goal of this study was to determine systematically the most stable reference gene for studying gene expression in a rat ischemic wound-healing model using RT-qPCR. Twelve commonly used reference genes were analyzed using RT-qPCR and geNorm data analysis to determine stability across normal and ischemic skin tissue. It was ultimately determined that Ubiquitin C (UBC) and ?-2 Microglobulin (B2M) are the most stably conserved reference genes across normal and ischemic skin tissue. UBC and B2M represent reliable reference genes for RT-qPCR studies in the rat ischemic wound model and are unaffected by sustained tissue ischemia. The geometric mean of these two stable genes provides an accurate normalization factor. These results provide insight on dependence of reference-gene stability on experimental parameters and the importance of such reference-gene investigations. PMID:24294111

Ruedrich, Elizabeth D.; Henzel, Mary K.; Hausman, Bryan S.; Bogie, Kath M.

2013-01-01

35

QPCR: Application for real-time PCR data management and analysis  

PubMed Central

Background Since its introduction quantitative real-time polymerase chain reaction (qPCR) has become the standard method for quantification of gene expression. Its high sensitivity, large dynamic range, and accuracy led to the development of numerous applications with an increasing number of samples to be analyzed. Data analysis consists of a number of steps, which have to be carried out in several different applications. Currently, no single tool is available which incorporates storage, management, and multiple methods covering the complete analysis pipeline. Results QPCR is a versatile web-based Java application that allows to store, manage, and analyze data from relative quantification qPCR experiments. It comprises a parser to import generated data from qPCR instruments and includes a variety of analysis methods to calculate cycle-threshold and amplification efficiency values. The analysis pipeline includes technical and biological replicate handling, incorporation of sample or gene specific efficiency, normalization using single or multiple reference genes, inter-run calibration, and fold change calculation. Moreover, the application supports assessment of error propagation throughout all analysis steps and allows conducting statistical tests on biological replicates. Results can be visualized in customizable charts and exported for further investigation. Conclusion We have developed a web-based system designed to enhance and facilitate the analysis of qPCR experiments. It covers the complete analysis workflow combining parsing, analysis, and generation of charts into one single application. The system is freely available at PMID:19712446

Pabinger, Stephan; Thallinger, Gerhard G; Snajder, Rene; Eichhorn, Heiko; Rader, Robert; Trajanoski, Zlatko

2009-01-01

36

Identification of two putative reference genes from grapevine suitable for gene expression analysis in berry and related tissues derived from RNA-Seq data  

PubMed Central

Background Data normalization is a key step in gene expression analysis by qPCR. Endogenous control genes are used to estimate variations and experimental errors occurring during sample preparation and expression measurements. However, the transcription level of the most commonly used reference genes can vary considerably in samples obtained from different individuals, tissues, developmental stages and under variable physiological conditions, resulting in a misinterpretation of the performance of the target gene(s). This issue has been scarcely approached in woody species such as grapevine. Results A statistical criterion was applied to select a sub-set of 19 candidate reference genes from a total of 242 non-differentially expressed (NDE) genes derived from a RNA-Seq experiment comprising ca. 500 million reads obtained from 14 table-grape genotypes sampled at four phenological stages. From the 19 candidate reference genes, VvAIG1 (AvrRpt2-induced gene) and VvTCPB (T-complex 1 beta-like protein) were found to be the most stable ones after comparing the complete set of genotypes and phenological stages studied. This result was further validated by qPCR and geNorm analyses. Conclusions Based on the evidence presented in this work, we propose to use the grapevine genes VvAIG1 or VvTCPB or both as a reference tool to normalize RNA expression in qPCR assays or other quantitative method intended to measure gene expression in berries and other tissues of this fruit crop, sampled at different developmental stages and physiological conditions. PMID:24330674

2013-01-01

37

Selection of proper reference genes for the cyanobacterium Synechococcus PCC 7002 using real-time quantitative PCR.  

PubMed

Synechococcus sp. PCC 7002 is known to be tolerant to most of the environmental factors in natural habitats of Cyanobacteria. Gene expression can be easily studied in this cyanobacterium, as its complete genome sequence is available. These properties make Synechococcus sp. PCC 7002 an appropriate model organism for biotechnological applications. To study the gene expression in Cyanobacteria, real-time quantitative PCR (qPCR) can be used, but as this is a highly sensitive method, data standardization is indicated between samples. The most commonly used strategy is normalization against internal reference genes. Synechococcus sp. PCC 7002 has not yet been evaluated for the best reference genes. In this work, six candidate genes were analyzed for this purpose. Cyanobacterial cultures were exposed to several stress conditions, and three different algorithms were used for ranking the reference genes: geNorm, NormFinder, and BestKeeper. Moreover, gene expression stability value M and single-control normalization error E were calculated. Our data provided a list of reference genes that can be used in qPCR experiments in Synechococcus sp. PCC 7002. PMID:25115691

Szekeres, Edina; Sicora, Cosmin; Drago?, Nicolae; Drug?, Bogdan

2014-10-01

38

Validation of Reference Genes for Quantitative Expression Analysis by Real-Time RT-PCR in Four Lepidopteran Insects  

PubMed Central

Quantitative real-time polymerase chain reaction (qPCR) is an efficient and widely used technique to monitor gene expression. Housekeeping genes (HKGs) are often empirically selected as the reference genes for data normalization. However, the suitability of HKGs used as the reference genes has been seldom validated. Here, six HKGs were chosen (actin A3, actin A1, GAPDH, G3PDH, E2F, rp49) in four lepidopteran insects Bombyx mori L. (Lepidoptera: Bombycidae), Plutella xylostella L. (Plutellidae), Chilo suppressalis Walker (Crambidae), and Spodoptera exigua Hübner (Noctuidae) to study their expression stability. The algorithms of geNorm, NormFinder, stability index, and ?Ct analysis were used to evaluate these HKGs. Across different developmental stages, actin A1 was the most stable in P. xylostella and C. suppressalis, but it was the least stable in B. mori and S. exigua. Rp49 and GAPDH were the most stable in B. mori and S. exigua, respectively. In different tissues, GAPDH, E2F, and Rp49 were the most stable in B. mori, S. exigua, and C. suppressalis, respectively. The relative abundances of Siwi genes estimated by 2-??Ct method were tested with different HKGs as the reference gene, proving the importance of internal controls in qPCR data analysis. The results not only presented a list of suitable reference genes in four lepidopteran insects, but also proved that the expression stabilities of HKGs were different among evolutionarily close species. There was no single universal reference gene that could be used in all situations. It is indispensable to validate the expression of HKGs before using them as the internal control in qPCR. PMID:22938136

Teng, Xiaolu; Zhang, Zan; He, Guiling; Yang, Liwen; Li, Fei

2012-01-01

39

Validation of Reference Genes for Real-Time PCR of Reproductive System in the Black Tiger Shrimp  

PubMed Central

Gene expression of reproductive system of the black tiger shrimp (Peneaus monodon) has been widely studied to address poor maturation problem in captivity. However, a systematic evaluation of reference genes in quantitative real-time PCR (qPCR) for P. monodon reproductive organs is lacking. In this study, the stability of four potential reference genes (18s rRNA, GAPDH, ?-actin, and EF1-?) was examined in the reproductive tissues in various conditions using bioinformatic tools: NormFinder and geNorm. For NormFinder, EF1-? and GAPDH ranked first and second as the most stable genes in testis groups whereas GAPDH and EF1-? were for ovaries from wild-caught broodstock and domesticated groups. EF1-? and ?-actin ranked first and second for the eyestalk ablated ovaries. For geNorm, EF1-? and GAPDH had the best stability in all testis and ovaries from domesticated groups whereas EF1-? and ?-actin were the best for ovaries from wild-caught and eyestalk ablated groups. Moreover, the expression levels of two well-known reproductive genes, Dmc1 and Vitellogenin, were used to validate these reference genes. When normalized to EF1-?, the expected expression patterns were obtained in all cases. Therefore, this work suggests that EF1-? is more versatile as reference genes in qPCR analysis for reproductive system in P. monodon. PMID:23285145

Leelatanawit, Rungnapa; Klanchui, Amornpan; Uawisetwathana, Umaporn; Karoonuthaisiri, Nitsara

2012-01-01

40

Selection and Expression Profiles of Reference Genes in Mouse Preimplantation Embryos of Different Ploidies at Various Developmental Stages  

PubMed Central

Real-time reverse transcription quantitative polymerase chain reaction (qPCR) has become the most frequently used system for studies of gene expression. Manystudies have provided reliable evidence that the transcription levels of reference genes are not constant at different developmental stages and in different experimental conditions. However, suitable reference genes which are stably expressed in polyploid preimplantation embryos of different developmental stages have not yet been identified. Therefore, it is critical to verify candidate reference genes to analyze gene expression accurately in both diploid and polyploid embryos. We examined the expression levels of 12 candidate reference genes in preimplantation embryos of four different ploidies at six developmental stages. Stability analysis of the reference genes was performed by four independent software programs, and the stability of three genes was evaluated by comparison with the Oct4 expression level during preimplantation development in diploid embryos. The expression levels of most genes in the polyploid embryos were higher than that in the diploid embryos, but the increasing degree were disproportionate with the ploidies. There were no significant difference in reference gene expressions among embryos of different ploidies when they reached the morula stage, and the expression level remained flat until the blastocyst stage. Ubc, Ppia, and Pgk1 were the three most stable reference genes in diploid and polyploid embryos. PMID:24927500

Gu, Yanli; Shen, Xinghui; Zhou, Dongjie; Wang, Zhendong; Zhang, Na; Shan, Zhiyan; Jin, Lianhong; Lei, Lei

2014-01-01

41

Reference Gene Selection for Quantitative Real-time PCR Normalization in Caragana intermedia under Different Abiotic Stress Conditions  

PubMed Central

Quantitative real-time reverse transcription polymerase chain reaction (qPCR), a sensitive technique for gene expression analysis, depends on the stability of the reference genes used for data normalization. Caragana intermedia, a native desert shrub with strong drought-resistance, sand-fixing capacity and high forage value that is widespread in the desert land of west and northwest China, has not been investigated regarding the identification of reference genes suitable for the normalization of qPCR data. In this study, 10 candidate reference genes were analyzed in C. intermedia subjected to different abiotic (osmotic, salt, cold and heat) stresses, in two distinct plant organs (roots and leaves). The expression stability of these genes was assessed using geNorm, NormFinder and BestKeeper algorithms. The best-ranked reference genes differed across the different sets of samples, but UNK2, PP2A and SAND were the most stable across all tested samples. UNK2 and SAND would be appropriate for normalizing gene expression data for salt-treated roots, whereas the combination of UNK2, SAND and EF-1? would be appropriate for salt-treated leaves. UNK1, UNK2 and PP2A would be appropriate for PEG-treated (osmotic) roots, whereas the combination of TIP41 and PP2A was the most suitable for PEG-treated leaves. SAND, PP2A and TIP41 exhibited the most stable expression in heat-treated leaves. In cold-treated leaves, SAND and EF-1? were the most stably expressed. To further validate the suitability of the reference genes identified in this study, the expression levels of DREB1 and DREB2 (homologs of AtDREB1 and AtDREB2) were studied in parallel. This study is the first systematic analysis for the selection of superior reference genes for qPCR in C. intermedia under different abiotic stress conditions, and will benefit future studies on gene expression in C. intermedia and other species of the leguminous genus Caragana. PMID:23301042

Zhu, Jianfeng; Zhang, Lifeng; Li, Wanfeng; Han, Suying; Yang, Wenhua; Qi, Liwang

2013-01-01

42

Identification of cell-specific patterns of reference gene stability in quantitative reverse-transcriptase polymerase chain reaction studies of embryonic, placental and neural stem models of prenatal ethanol exposure.  

PubMed

Identification of the transcriptional networks disrupted by prenatal ethanol exposure remains a core requirement to better understanding the molecular mechanisms of alcohol-induced teratogenesis. In this regard, quantitative reverse-transcriptase polymerase chain reaction (qPCR) has emerged as an essential technique in our efforts to characterize alterations in gene expression brought on by exposure to alcohol. However, many publications continue to report the utilization of inappropriate methods of qPCR normalization, and for many in vitro models, no consistent set of empirically tested normalization controls have been identified. In the present study, we sought to identify a group of candidate reference genes for use within studies of alcohol exposed embryonic, placental, and neurosphere stem cells under both conditions maintaining stemness as well as throughout in vitro differentiation. To this end, we surveyed the recent literature and compiled a short list of fourteen candidate genes commonly used as normalization controls in qPCR studies of gene expression. This list included: Actb, B2m, Gapdh, Gusb, H2afz, Hk2, Hmbs, Hprt, Mrpl1, Pgk1, Ppia, Sdha, Tbp, and Ywhaz. From these studies, we find no single candidate gene was consistently refractory to the influence of alcohol nor completely stable throughout in vitro differentiation. Accordingly, we propose normalizing qPCR measurements to the geometric mean C(T) values obtained for three independent reference mRNAs as a reliable method to accurately interpret qPCR data and assess alterations in gene expression within alcohol treated cultures. Highlighting the importance of careful and empirical reference gene selection, the commonly used reference gene Actb was often amongst the least stable candidate genes tested. In fact, it would not serve as a valid normalization control in many cases. Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens. PMID:23317542

Carnahan, Mindy N; Veazey, Kylee J; Muller, Daria; Tingling, Joseph D; Miranda, Rajesh C; Golding, Michael C

2013-03-01

43

Combining qPCR and functional gene microarrays to directly link changes in the expression of the nirS gene to denitrification rates in aquatic sediment mesocosms  

NASA Astrophysics Data System (ADS)

Molecular methods for the investigation of biogeochemical processes, including denitrification, are being developed at an astonishing rate, but it remains difficult to use the molecular information to understand the regulation and variation in biogeochemical transformation rates. By combining information on gene abundance and expression for nirS, a key gene in denitrification, with quantitative modeling of nitrogen fluxes, we can begin to understand the scales on which genetic signals vary in space and time, and how they relate to biogeochemical function. We used quantitative PCR, a functional gene microarray, and biogeochemical modeling to assess how denitrifier community composition (evaluated by DNA and cDNA of the nirS gene) changed over time in estuarine sediment mesocosms. Sediments and water were collected from coastal Massachusetts and maintained in replicated 20 L mesocosm experiments for 45 days. Sediments were collected for microbial analysis at weekly intervals throughout the experiment. Concentrations of all major nitrogen species were measured daily and used to derive rates of nitrification and denitrification from a Monte Carlo-based nonnegative least-squares analysis of finite difference equations. Denitrification rates peaked between day 18 and day 22, slightly after the peaks in nitrite concentration that were generated from oxidization of remineralized ammonium. In most mesocosms the peak in denitrification rates coincided with the peak in nirS gene abundance (DNA). Peaks in the expression of the nirS gene (cDNA), however, did not always correlate with peaks in the denitrification rates. The nirS microarray contained 39 archetype probes, three of which accounted for more than 60% of the DNA hybridization signal. Two of these clades also dominated the hybridization signal in cDNA, indicating that those organisms that are actively expressing nirS are not always the dominant members of the community. Fifteen of the 39 probes accounted for less than 1% of the hybridization signal, but the number of probes that accounted for more than 1% of the DNA hybridization signal decreased during the mesocosm experiments while the number of probes with significant cDNA hybridization signals increased through the experiment. This suggests that as nitrate supply became more available a greater diversity of denitrifiers were able to upregulate their gene expression and actively engage in denitrification. Among the clades that represented more than 60% of the hybridization signal in either the DNA or the cDNA, none are available in pure culture so nothing is known of their ecology or physiology. This approach provides a powerful way to examine the fine-scale connections between nirS gene abundance and expression and the biogeochemical fluxes that result.

Bowen, J. L.; Babbin, A. R.; Ward, B. B.

2010-12-01

44

A new reliable reference gene UBA52 for quantitative real-time polymerase chain reaction studies in pyloric cecal tissues of the starfish Asterias rubens.  

PubMed

The starfish Asterias rubens is one of the most abundant echinoderm species in the White, Barents, North, and Baltic Seas. This species is an important component of marine ecosystems and a model object for certain biological studies, in particular those requiring quantitative estimation of gene expression. As a rule, expression at the transcriptional level is estimated by real-time qPCR using the ??Ct method, which allows the comparison of the copy number of target gene transcripts in samples with unknown mRNA/cDNA concentration. Application of this method requires normalization of the results relative to genes with stable expression levels (reference genes). The identification of reference genes is still a challenging task since data of this kind are missing for certain taxa, whereas the use of "standard" endogenous control genes without additional tests might lead to erroneous conclusions. We performed a preliminary analysis of the expression of many housekeeping genes in the pyloric ceca of A. rubens by high-throughput sequencing under normal and heat shock conditions. For one of them, the ubiquitin gene UBA52, low variation of expression (not greater than 2-fold) was shown using real-time qPCR. Tissues of pyloric ceca of normal adults and underyearlings and of adults after heat shock were used. The data obtained suggest that the UBA52 gene may be used as reference for normalization of gene expression at the mRNA level in the starfish A. rubens and probably in closely related species. PMID:24938608

Sadritdinova, A F; Dmitriev, A A; Snezhkina, A V; Belenikin, M S; Krasnov, G S; Manylov, O G; Kudryavtsev, A A; Melnikova, N V; Speranskaya, A S; Darii, M V; Lakunina, V A; Uroshlev, L A; Smurov, A O; Stepanov, O A; Kudryavtseva, A V

2014-01-01

45

Use of Maximum Likelihood-Mixed Models to select stable reference genes: a case of heat stress response in sheep  

PubMed Central

Background Reference genes with stable expression are required to normalize expression differences of target genes in qPCR experiments. Several procedures and companion software have been proposed to find the most stable genes. Model based procedures are attractive because they provide a solid statistical framework. NormFinder, a widely used software, uses a model based method. The pairwise comparison procedure implemented in GeNorm is a simpler procedure but one of the most extensively used. In the present work a statistical approach based in Maximum Likelihood estimation under mixed models was tested and compared with NormFinder and geNorm softwares. Sixteen candidate genes were tested in whole blood samples from control and heat stressed sheep. Results A model including gene and treatment as fixed effects, sample (animal), gene by treatment, gene by sample and treatment by sample interactions as random effects with heteroskedastic residual variance in gene by treatment levels was selected using goodness of fit and predictive ability criteria among a variety of models. Mean Square Error obtained under the selected model was used as indicator of gene expression stability. Genes top and bottom ranked by the three approaches were similar; however, notable differences for the best pair of genes selected for each method and the remaining genes of the rankings were shown. Differences among the expression values of normalized targets for each statistical approach were also found. Conclusions Optimal statistical properties of Maximum Likelihood estimation joined to mixed model flexibility allow for more accurate estimation of expression stability of genes under many different situations. Accurate selection of reference genes has a direct impact over the normalized expression values of a given target gene. This may be critical when the aim of the study is to compare expression rate differences among samples under different environmental conditions, tissues, cell types or genotypes. To select reference genes not only statistical but also functional and biological criteria should be considered. Under the method here proposed SDHA/MDH1 have arisen as the best set of reference genes to be used in qPCR assays to study heat shock in ovine blood samples. PMID:21849053

2011-01-01

46

Identification of Endogenous Reference Genes for the Analysis of microRNA Expression in the Hippocampus of the Pilocarpine-Induced Model of Mesial Temporal Lobe Epilepsy  

PubMed Central

Real-time quantitative RT-PCR (qPCR) is one of the most powerful techniques for analyzing miRNA expression because of its sensitivity and specificity. However, in this type of analysis, a suitable normalizer is required to ensure that gene expression is unaffected by the experimental condition. To the best of our knowledge, there are no reported studies that performed a detailed identification and validation of suitable reference genes for miRNA qPCR during the epileptogenic process. Here, using a pilocarpine (PILO) model of mesial temporal lobe epilepsy (MTLE), we investigated five potential reference genes, performing a stability expression analysis using geNorm and NormFinder softwares. As a validation strategy, we used each one of the candidate reference genes to measure PILO-induced changes in microRNA-146a levels, a gene whose expression pattern variation in the PILO injected model is known. Our results indicated U6SnRNA and SnoRNA as the most stable candidate reference genes. By geNorm analysis, the normalization factor should preferably contain at least two of the best candidate reference genes (snoRNA and U6SnRNA). In fact, when normalized using the best combination of reference genes, microRNA-146a transcripts were found to be significantly increased in chronic stage, which is consistent with the pattern reported in different models. Conversely, when reference genes were individually employed for normalization, we failed to detect up-regulation of the microRNA-146a gene in the hippocampus of epileptic rats. The data presented here support that the combination of snoRNA and U6SnRNA was the minimum necessary for an accurate normalization of gene expression at the different stages of epileptogenesis that we tested. PMID:24964029

de Araujo, Mykaella Andrade; Marques, Thalita Ewellyn Batista Sales; Taniele-Silva, Jamile; Souza, Fernanda Maria de Araujo; de Andrade, Tiago Gomes; Garcia-Cairasco, Norberto; Paco-Larson, Maria Luisa; Gitai, Daniel Leite Goes

2014-01-01

47

Validation of reference genes for gene expression analysis in olive (Olea europaea) mesocarp tissue by quantitative real-time RT-PCR  

PubMed Central

Background Gene expression analysis using quantitative reverse transcription PCR (qRT-PCR) is a robust method wherein the expression levels of target genes are normalised using internal control genes, known as reference genes, to derive changes in gene expression levels. Although reference genes have recently been suggested for olive tissues, combined/independent analysis on different cultivars has not yet been tested. Therefore, an assessment of reference genes was required to validate the recent findings and select stably expressed genes across different olive cultivars. Results A total of eight candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), serine/threonine-protein phosphatase catalytic subunit (PP2A), elongation factor 1 alpha (EF1-alpha), polyubiquitin (OUB2), aquaporin tonoplast intrinsic protein (TIP2), tubulin alpha (TUBA), 60S ribosomal protein L18-3 (60S RBP L18-3) and polypyrimidine tract-binding protein homolog 3 (PTB)] were chosen based on their stability in olive tissues as well as in other plants. Expression stability was examined by qRT-PCR across 12 biological samples, representing mesocarp tissues at various developmental stages in three different olive cultivars, Barnea, Frantoio and Picual, independently and together during the 2009 season with two software programs, GeNorm and BestKeeper. Both software packages identified GAPDH, EF1-alpha and PP2A as the three most stable reference genes across the three cultivars and in the cultivar, Barnea. GAPDH, EF1-alpha and 60S RBP L18-3 were found to be most stable reference genes in the cultivar Frantoio while 60S RBP L18-3, OUB2 and PP2A were found to be most stable reference genes in the cultivar Picual. Conclusions The analyses of expression stability of reference genes using qRT-PCR revealed that GAPDH, EF1-alpha, PP2A, 60S RBP L18-3 and OUB2 are suitable reference genes for expression analysis in developing Olea europaea mesocarp tissues, displaying the highest level of expression stability across three different olive cultivars, Barnea, Frantoio and Picual, however the combination of the three most stable reference genes do vary amongst individual cultivars. This study will provide guidance to other researchers to select reference genes for normalization against target genes by qPCR across tissues obtained from the mesocarp region of the olive fruit in the cultivars, Barnea, Frantoio and Picual. PMID:24884716

2014-01-01

48

Evaluation of internal reference genes for quantitative expression analysis by real-time reverse transcription-PCR in somatic cells from goat milk.  

PubMed

Reverse transcription (RT) quantitative real-time PCR (qPCR) is the most accurate and easy-to-perform technique to measure the expression level of a selected gene of interest by quantifying mRNA transcripts. The use of reference genes is commonly accepted as the most reliable approach to normalize RT-qPCR data and reduce possible errors generated in the quantification of gene expression. The optimal number and choice of reference genes are experimentally validated for specific tissues or cell types and experimental designs. To date, data on qPCR normalization in goats are scarce and the most suitable reference genes in this species have been identified for only a limited number of tissues. The aim of this study was to determine an optimal combination of stably expressed reference genes in caprine milk somatic cells (MSC) from healthy and infected mammary glands. For the purpose, we performed RT-qPCR for 10 commonly used reference genes from various functional classes and then determined their expression level in MSC from goats intramammary challenged with Staphylococcus aureus and in MSC from healthy controls, with a view to select genes whose stability would be unaffected under infection conditions. The geNorm and NormFinder algorithms were used for validating the reference genes. Furthermore, to demonstrate the importance of normalization of gene expression with appropriate reference genes, we tested the effect of using a combination of the least stable genes for expression analysis evaluation. On the basis of our evaluation, we recommend the use of a panel of reference genes that should include G6PD, YWHAZ, and ACTB for caprine MSC gene expression profiling. The expression of the 2 genes of interest, pentraxin-related protein (PTX3) and secreted phosphoprotein 1 (SPP1), was evaluated by RT-qPCR in all samples collected pre- and postinfection, and the recommended reference genes were used to normalize the data. Our study provides a validated panel of optimal reference genes for the identification of genes differentially expressed by qRT-PCR in caprine MSC. Moreover, we provided a set of intron-spanning primer sequences that could be suitable for gene expression experiments using SYBR Green chemistry on other caprine tissues and cells. PMID:24119819

Modesto, P; Peletto, S; Pisoni, G; Cremonesi, P; Castiglioni, B; Colussi, S; Caramelli, M; Bronzo, V; Moroni, P; Acutis, P L

2013-12-01

49

A method for quantitative analysis of standard and high-throughput qPCR expression data based on input sample quantity.  

PubMed

Over the past decade rapid advances have occurred in the understanding of RNA expression and its regulation. Quantitative polymerase chain reactions (qPCR) have become the gold standard for quantifying gene expression. Microfluidic next generation, high throughput qPCR now permits the detection of transcript copy number in thousands of reactions simultaneously, dramatically increasing the sensitivity over standard qPCR. Here we present a gene expression analysis method applicable to both standard polymerase chain reactions (qPCR) and high throughput qPCR. This technique is adjusted to the input sample quantity (e.g., the number of cells) and is independent of control gene expression. It is efficiency-corrected and with the use of a universal reference sample (commercial complementary DNA (cDNA)) permits the normalization of results between different batches and between different instruments--regardless of potential differences in transcript amplification efficiency. Modifications of the input quantity method include (1) the achievement of absolute quantification and (2) a non-efficiency corrected analysis. When compared to other commonly used algorithms the input quantity method proved to be valid. This method is of particular value for clinical studies of whole blood and circulating leukocytes where cell counts are readily available. PMID:25090612

Adamski, Mateusz G; Gumann, Patryk; Baird, Alison E

2014-01-01

50

Evaluation of Reference Genes for Accurate Normalization of Gene Expression for Real Time-Quantitative PCR in Pyrus pyrifolia Using Different Tissue Samples and Seasonal Conditions  

PubMed Central

We have evaluated suitable reference genes for real time (RT)-quantitative PCR (qPCR) analysis in Japanese pear (Pyrus pyrifolia). We tested most frequently used genes in the literature such as ?-Tubulin, Histone H3, Actin, Elongation factor-1?, Glyceraldehyde-3-phosphate dehydrogenase, together with newly added genes Annexin, SAND and TIP41. A total of 17 primer combinations for these eight genes were evaluated using cDNAs synthesized from 16 tissue samples from four groups, namely: flower bud, flower organ, fruit flesh and fruit skin. Gene expression stabilities were analyzed using geNorm and NormFinder software packages or by ?Ct method. geNorm analysis indicated three best performing genes as being sufficient for reliable normalization of RT-qPCR data. Suitable reference genes were different among sample groups, suggesting the importance of validation of gene expression stability of reference genes in the samples of interest. Ranking of stability was basically similar between geNorm and NormFinder, suggesting usefulness of these programs based on different algorithms. ?Ct method suggested somewhat different results in some groups such as flower organ or fruit skin; though the overall results were in good correlation with geNorm or NormFinder. Gene expression of two cold-inducible genes PpCBF2 and PpCBF4 were quantified using the three most and the three least stable reference genes suggested by geNorm. Although normalized quantities were different between them, the relative quantities within a group of samples were similar even when the least stable reference genes were used. Our data suggested that using the geometric mean value of three reference genes for normalization is quite a reliable approach to evaluating gene expression by RT-qPCR. We propose that the initial evaluation of gene expression stability by ?Ct method, and subsequent evaluation by geNorm or NormFinder for limited number of superior gene candidates will be a practical way of finding out reliable reference genes. PMID:24466117

Saito, Takanori; Moriguchi, Takaya

2014-01-01

51

Careful Selection of Reference Genes Is Required for Reliable Performance of RT-qPCR in Human Normal and Cancer Cell Lines  

PubMed Central

Reverse Transcription - quantitative Polymerase Chain Reaction (RT-qPCR) is a standard technique in most laboratories. The selection of reference genes is essential for data normalization and the selection of suitable reference genes remains critical. Our aim was to 1) review the literature since implementation of the MIQE guidelines in order to identify the degree of acceptance; 2) compare various algorithms in their expression stability; 3) identify a set of suitable and most reliable reference genes for a variety of human cancer cell lines. A PubMed database review was performed and publications since 2009 were selected. Twelve putative reference genes were profiled in normal and various cancer cell lines (n?=?25) using 2-step RT-qPCR. Investigated reference genes were ranked according to their expression stability by five algorithms (geNorm, Normfinder, BestKeeper, comparative ?Ct, and RefFinder). Our review revealed 37 publications, with two thirds patient samples and one third cell lines. qPCR efficiency was given in 68.4% of all publications, but only 28.9% of all studies provided RNA/cDNA amount and standard curves. GeNorm and Normfinder algorithms were used in 60.5% in combination. In our selection of 25 cancer cell lines, we identified HSPCB, RRN18S, and RPS13 as the most stable expressed reference genes. In the subset of ovarian cancer cell lines, the reference genes were PPIA, RPS13 and SDHA, clearly demonstrating the necessity to select genes depending on the research focus. Moreover, a cohort of at least three suitable reference genes needs to be established in advance to the experiments, according to the guidelines. For establishing a set of reference genes for gene normalization we recommend the use of ideally three reference genes selected by at least three stability algorithms. The unfortunate lack of compliance to the MIQE guidelines reflects that these need to be further established in the research community. PMID:23554992

Jacob, Francis; Guertler, Rea; Naim, Stephanie; Nixdorf, Sheri; Fedier, Andre; Hacker, Neville F.; Heinzelmann-Schwarz, Viola

2013-01-01

52

Critical appraisal of quantitative PCR results in colorectal cancer research: can we rely on published qPCR results?  

PubMed

The use of real-time quantitative polymerase chain reaction (qPCR) in cancer research has become ubiquitous. The relative simplicity of qPCR experiments, which deliver fast and cost-effective results, means that each year an increasing number of papers utilizing this technique are being published. But how reliable are the published results? Since the validity of gene expression data is greatly dependent on appropriate normalisation to compensate for sample-to-sample and run-to-run variation, we have evaluated the adequacy of normalisation procedures in qPCR-based experiments. Consequently, we assessed all colorectal cancer publications that made use of qPCR from 2006 until August 2013 for the number of reference genes used and whether they had been validated. Using even these minimal evaluation criteria, the validity of only three percent (6/179) of the publications can be adequately assessed. We describe common errors, and conclude that the current state of reporting on qPCR in colorectal cancer research is disquieting. Extrapolated to the study of cancer in general, it is clear that the majority of studies using qPCR cannot be reliably assessed and that at best, the results of these studies may or may not be valid and at worst, pervasive incorrect normalisation is resulting in the wholesale publication of incorrect conclusions. This survey demonstrates that the existence of guidelines, such as MIQE, is necessary but not sufficient to address this problem and suggests that the scientific community should examine its responsibility and be aware of the implications of these findings for current and future research. PMID:24423493

Dijkstra, J R; van Kempen, L C; Nagtegaal, I D; Bustin, S A

2014-06-01

53

Reference genes for normalizing transcription in diploid and tetraploid Arabidopsis  

PubMed Central

Published transcription data from a set of 19 diploid Arabidopsis thaliana and 5 tetraploid (3 allo- and 2 auto- tetraploid) Arabidopsis accessions were re-analysed to identify reliable reference genes for normalization purposes. Five conventional and 16 novel reference genes previously derived from microarray data covering a wide range of abundance in absolute expression levels in diploid A. thaliana Col-0 were employed. Transcript abundance was well conserved for all 21 potential reference genes in the diploid A. thaliana accessions, with geNorm and NormFinder analysis indicating that AT5G46630, AT1G13320, AT4G26410, AT5G60390 and AT5G08290 were the most stable. However, conservation was less good among the tetraploid accessions, with the transcription of seven of the 21 genes being undetectable in all allotetraploids. The most stable gene was AT5G46630, while AT1G13440 was the unstable one. Hence, the choice of reference gene(s) for A. thaliana is quite wide, but with respect to the analysis of transcriptomic data derived from the tetraploids, it is probably necessary to select more than one reference gene. PMID:25345678

Wang, Haibin; Wang, Jingjing; Jiang, Jiafu; Chen, Sumei; Guan, Zhiyong; Liao, Yuan; Chen, Fadi

2014-01-01

54

Selection of Reference Genes for Quantitative Real-Time PCR Normalization in Panax ginseng at Different Stages of Growth and in Different Organs  

PubMed Central

Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1? were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng. PMID:25393243

Liu, Jing; Wang, Qun; Sun, Minying; Zhu, Linlin; Yang, Michael; Zhao, Yu

2014-01-01

55

The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses.  

PubMed

Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses. PMID:25391499

Guo, Jinlong; Ling, Hui; Wu, Qibin; Xu, Liping; Que, Youxiong

2014-01-01

56

The choice of reference genes for assessing gene expression in sugarcane under salinity and drought stresses  

PubMed Central

Sugarcane (Saccharum spp. hybrids) is a world-wide cash crop for sugar and biofuel in tropical and subtropical regions and suffers serious losses in cane yield and sugar content under salinity and drought stresses. Although real-time quantitative PCR has a numerous advantage in the expression quantification of stress-related genes for the elaboration of the corresponding molecular mechanism in sugarcane, the variation happened across the process of gene expression quantification should be normalized and monitored by introducing one or several reference genes. To validate suitable reference genes or gene sets for sugarcane gene expression normalization, 13 candidate reference genes have been tested across 12 NaCl- and PEG-treated sugarcane samples for four sugarcane genotypes using four commonly used systematic statistical algorithms termed geNorm, BestKeeper, NormFinder and the deltaCt method. The results demonstrated that glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and eukaryotic elongation factor 1-alpha (eEF-1a) were identified as suitable reference genes for gene expression normalization under salinity/drought-treatment in sugarcane. Moreover, the expression analyses of SuSK and 6PGDH further validated that a combination of clathrin adaptor complex (CAC) and cullin (CUL) as reference should be better for gene expression normalization. These results can facilitate the future research on gene expression in sugarcane under salinity and drought stresses. PMID:25391499

Guo, Jinlong; Ling, Hui; Wu, Qibin; Xu, Liping; Que, Youxiong

2014-01-01

57

Reference-based gene model prediction on DNA contigs  

SciTech Connect

This paper presents an algorithm for constructing multiple gene models on a set of contigs of a large genomic clone. The algorithm first uses pattern recognition-based methods to locate exons or partial exons in each contig, and then applies protein homology or EST information from the databases, as reference models, to parse the predicted exons into gene models. In the phase of gene model construction, the algorithm uses a unified framework for genes ranging from situation with homologous proteins/ESTs to no homologous protein/EST in the database. By exploiting protein homology or EST information, the algorithm is able to (1) parse exons into multiple gene models over a set of DNA contigs (possibly unoriented and unordered); (2) remove falsely predicted exons; and (3) identify and locate exons missed by the initial exon prediction. 16 refs., 3 figs.

Xu, Ying; Uberbacher, E.C. [Oak Ridge National Lab., TN (United States)

1997-12-01

58

Reference-based gene model prediction on DNA contigs  

SciTech Connect

This paper presents an algorithm for constructing multiple gene models on a set of contigs of a large genomic clone. The algorithm first uses pattern recognition-based methods to locate exons or partial exons in each contig, and then applies protein homology or EST information from the databases, as reference models, to parse the predicted exons into gene models. In the phase of gene model construction, the algorithm uses a unified framework for genes ranging from situation with homologous proteins/ESTs to no homologous protein/EST in the database. By exploiting protein homology or EST information, the algorithm is able to (1) parse exons into multiple gene models over a set of DNA contigs (possibly unoriented and unordered); (2) remove falsely predicted exons; and (3) identify and locate exons missed by the initial exon prediction.

Xu, Y.; Uberbacher, E.C. [Oak Ridge National Lab., TN (United States). Computer Sciences and Mathematics Div.

1997-01-01

59

Reference genes for gene expression studies in wheat flag leaves grown under different farming conditions  

PubMed Central

Background Internal control genes with highly uniform expression throughout the experimental conditions are required for accurate gene expression analysis as no universal reference genes exists. In this study, the expression stability of 24 candidate genes from Triticum aestivum cv. Cubus flag leaves grown under organic and conventional farming systems was evaluated in two locations in order to select suitable genes that can be used for normalization of real-time quantitative reverse-transcription PCR (RT-qPCR) reactions. The genes were selected among the most common used reference genes as well as genes encoding proteins involved in several metabolic pathways. Findings Individual genes displayed different expression rates across all samples assayed. Applying geNorm, a set of three potential reference genes were suitable for normalization of RT-qPCR reactions in winter wheat flag leaves cv. Cubus: TaFNRII (ferredoxin-NADP(H) oxidoreductase; AJ457980.1), ACT2 (actin 2; TC234027), and rrn26 (a putative homologue to RNA 26S gene; AL827977.1). In addition of these three genes that were also top-ranked by NormFinder, two extra genes: CYP18-2 (Cyclophilin A, AY456122.1) and TaWIN1 (14-3-3 like protein, AB042193) were most consistently stably expressed. Furthermore, we showed that TaFNRII, ACT2, and CYP18-2 are suitable for gene expression normalization in other two winter wheat varieties (Tommi and Centenaire) grown under three treatments (organic, conventional and no nitrogen) and a different environment than the one tested with cv. Cubus. Conclusions This study provides a new set of reference genes which should improve the accuracy of gene expression analyses when using wheat flag leaves as those related to the improvement of nitrogen use efficiency for cereal production. PMID:21951810

2011-01-01

60

A qPCR Assay to Detect and Quantify Shiga Toxin-Producing E. coli (STEC) in Cattle and on Farms: A Potential Predictive Tool for STEC Culture-Positive Farms  

PubMed Central

Shiga toxin-producing E. coli (STEC), of various serogroups harboring the intimin gene, form a serious threat to human health. They are asymptomatically carried by cattle. In this study, a quantitative real-time PCR (qPCR) method was developed as a molecular method to detect and quantify Shiga toxin genes stx1 and stx2 and the intimin gene eae. Subsequently, 59 fecal samples from six farms were tested using qPCR and a culture method as a reference. Three farms had contaminated animals as demonstrated by the culture method. Culture-positive farms showed moderate significantly higher stx prevalences than culture-negative farms (p = 0.05). This is the first study which showed preliminary results that qPCR can predict STEC farm contamination, with a specificity of 77% and a sensitivity of 83%, as compared with the culture method. Furthermore, the presence or quantity of stx genes in feces was not correlated to the isolation of STEC from the individual animal. Quantitative data thus did not add value to the results. Finally, the detection of both stx and eae genes within the same fecal sample or farm using qPCR was not correlated with the isolation of an eae-harboring STEC strain from the respective sample or farm using the culture method. PMID:24681714

Verstraete, Karen; Van Coillie, Els; Werbrouck, Hadewig; Van Weyenberg, Stephanie; Herman, Lieve; Del-Favero, Jurgen; De Rijk, Peter; De Zutter, Lieven; Joris, Maria-Adelheid; Heyndrickx, Marc; De Reu, Koen

2014-01-01

61

Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis  

PubMed Central

Background Reference genes are used as internal standards to normalize mRNA abundance in quantitative real-time PCR and thereby allow a direct comparison between samples. So far most of these expression studies used human or classical laboratory model species whereas studies on non-model organism under in-situ conditions are quite rare. However, only studies in free-ranging populations can reveal the effects of natural selection on the expression levels of functional important genes. In order to test the feasibility of gene expression studies in wildlife samples we transferred and validated potential reference genes that were developed for lab mice (Mus musculus) to samples of wild yellow-necked mice, Apodemus flavicollis. The stability and suitability of eight potential reference genes was accessed by the programs BestKeeper, NormFinder and geNorm. Findings Although the three programs used different algorithms the ranking order of reference genes was significantly concordant and geNorm differed in only one, NormFinder in two positions compared to BestKeeper. The genes ordered by their mean rank from the most to the least stable gene were: Rps18, Sdha, Canx, Actg1, Pgk1, Ubc, Rpl13a and Actb. Analyses of the normalization factor revealed best results when the five most stable genes were included for normalization. Discussion We established a SYBR green qPCR assay for liver samples of wild A. flavicollis and conclude that five genes should be used for appropriate normalization. Our study provides the basis to investigate differential expression of genes under selection under natural selection conditions in liver samples of A. flavicollis. This approach might also be applicable to other non-model organisms. PMID:20030847

2009-01-01

62

Reliable reference gene selection for Cordyceps militaris gene expression studies under different developmental stages and media.  

PubMed

Cordyceps militaris is considered a model organism for the study of Cordyceps species, which are highly prized in traditional Chinese medicine. Gene expression analysis has become more popular and important in studies of this fungus. Reference gene validation under different experimental conditions is crucial for RT-qPCR analysis. In this study, eight candidate reference genes, actin, cox5, gpd, rpb1, tef1, try, tub, and ubi, were selected and their expression stability was evaluated in C. militaris samples using four algorithms, genorm, normfinder, bestkeeper, and the comparative ?Ct method. Three sets of samples, five different developmental stages cultured in wheat medium and pupae, and all the samples pool were included. The results showed that rpb1 was the best reference gene during all developmental stages examined, while the most common reference genes, actin and tub, were not suitable internal controls. Cox5 also performed poorly and was less stable in our analysis. The ranks of ubi and gpd were inconsistent in different sample sets by different methods. Our results provide guidelines for reference gene selection at different developmental stages and also represent a foundation for more accurate and widespread use of RT-qPCR in C. militaris gene expression analysis. PMID:24953133

Lian, Tiantian; Yang, Tao; Liu, Guijun; Sun, Junde; Dong, Caihong

2014-07-01

63

Evaluation of reference genes for gene expression analysis using quantitative RT-PCR in Azospirillum brasilense.  

PubMed

Azospirillum brasilense is a nitrogen fixing bacterium that has been shown to have various beneficial effects on plant growth and yield. Under normal conditions A. brasilense exists in a motile flagellated form, which, under starvation or stress conditions, can undergo differentiation into an encapsulated, cyst-like form. Quantitative RT-PCR can be used to analyse changes in gene expression during this differentiation process. The accuracy of quantification of mRNA levels by qRT-PCR relies on the normalisation of data against stably expressed reference genes. No suitable set of reference genes has yet been described for A. brasilense. Here we evaluated the expression of ten candidate reference genes (16S rRNA, gapB, glyA, gyrA, proC, pykA, recA, recF, rpoD, and tpiA) in wild-type and mutant A. brasilense strains under different culture conditions, including conditions that induce differentiation. Analysis with the software programs BestKeeper, NormFinder and GeNorm indicated that gyrA, glyA and recA are the most stably expressed reference genes in A. brasilense. The results also suggested that the use of two reference genes (gyrA and glyA) is sufficient for effective normalisation of qRT-PCR data. PMID:24841066

McMillan, Mary; Pereg, Lily

2014-01-01

64

Validation of reference genes for quantitative measurement of immune gene expression in shrimp.  

PubMed

To accurately measure the relative expression of a target gene, mRNA expression data is routinely normalized with reference to an internal control gene. We examined the transcriptional stability of four internal control genes, beta-actin, glyceraldehyde-3 phosphate dehydrogenase (GAPDH), elongation factor1-alpha (EF1-alpha), and 18S ribosomal RNA (18S rRNA) while measuring the mRNA expression of a gene encoding a pattern recognition protein, lipopolysaccharide and glucan binding protein (LGBP) gene, in healthy and white spot syndrome virus (WSSV) infected shrimp (Penaeus stylirostris) before and after (4, 8, 16 and 32 h) challenge using real-time RT-PCR. Here, we describe a method to rank the internal control genes based on a linear regression analysis. This analysis enables us to analyze the multivariate data set, e.g. time course study samples with control and treatment groups. Using the linear regression analysis and the WSSV-challenged time course samples, GAPDH was found to be the most stable internal control gene followed by the genes EF1-alpha, 18S rRNA and beta-actin. Using the program geNorm, GAPDH was also found to be the most stable gene followed by the genes EF1-alpha, beta-actin and 18S rRNA. Using the program NormFinder, the ranking of the internal control genes were in the order of EF1-alpha>GAPDH>18S rRNA>beta-actin. The ability to compare the healthy and WSSV infected samples in parallel by the regression analysis makes this method a very useful approach while identifying the optimal reference gene for gene expression analysis. PMID:19297025

Dhar, Arun K; Bowers, Robert M; Licon, Kate S; Veazey, Gregory; Read, Betsy

2009-05-01

65

Identification of Suitable Reference Genes for Gene Expression Normalization in qRT-PCR Analysis in Watermelon  

PubMed Central

Watermelon is one of the major Cucurbitaceae crops and the recent availability of genome sequence greatly facilitates the fundamental researches on it. Quantitative real-time reverse transcriptase PCR (qRT–PCR) is the preferred method for gene expression analyses, and using validated reference genes for normalization is crucial to ensure the accuracy of this method. However, a systematic validation of reference genes has not been conducted on watermelon. In this study, transcripts of 15 candidate reference genes were quantified in watermelon using qRT–PCR, and the stability of these genes was compared using geNorm and NormFinder. geNorm identified ClTUA and ClACT, ClEF1? and ClACT, and ClCAC and ClTUA as the best pairs of reference genes in watermelon organs and tissues under normal growth conditions, abiotic stress, and biotic stress, respectively. NormFinder identified ClYLS8, ClUBCP, and ClCAC as the best single reference genes under the above experimental conditions, respectively. ClYLS8 and ClPP2A were identified as the best reference genes across all samples. Two to nine reference genes were required for more reliable normalization depending on the experimental conditions. The widely used watermelon reference gene 18SrRNA was less stable than the other reference genes under the experimental conditions. Catalase family genes were identified in watermelon genome, and used to validate the reliability of the identified reference genes. ClCAT1and ClCAT2 were induced and upregulated in the first 24 h, whereas ClCAT3 was downregulated in the leaves under low temperature stress. However, the expression levels of these genes were significantly overestimated and misinterpreted when 18SrRNA was used as a reference gene. These results provide a good starting point for reference gene selection in qRT–PCR analyses involving watermelon. PMID:24587403

Gao, Lingyun; Zhao, Shuang; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

66

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.)  

PubMed Central

Background Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs/tissues examined. It is therefore important to identify the best reference genes to use in each biological system before using qRT-PCR to investigate differential gene expression. In this paper we evaluate different candidate HKGs for developmental transcriptomic studies in the economically-important flax fiber- and oil-crop (Linum usitatissimum L). Results Specific primers were designed in order to quantify the expression levels of 20 different potential housekeeping genes in flax roots, internal- and external-stem tissues, leaves and flowers at different developmental stages. After calculations of PCR efficiencies, 13 HKGs were retained and their expression stabilities evaluated by the computer algorithms geNorm and NormFinder. According to geNorm, 2 Transcriptional Elongation Factors (TEFs) and 1 Ubiquitin gene are necessary for normalizing gene expression when all studied samples are considered. However, only 2 TEFs are required for normalizing expression in stem tissues. In contrast, NormFinder identified glyceraldehyde-3-phosphate dehydrogenase (GADPH) as the most stably expressed gene when all samples were grouped together, as well as when samples were classed into different sub-groups. qRT-PCR was then used to investigate the relative expression levels of two splice variants of the flax LuMYB1 gene (homologue of AtMYB59). LuMYB1-1 and LuMYB1-2 were highly expressed in the internal stem tissues as compared to outer stem tissues and other samples. This result was confirmed with both geNorm-designated- and NormFinder-designated-reference genes. Conclusions The use of 2 different statistical algorithms results in the identification of different combinations of flax HKGs for expression data normalization. Despite such differences, the use of geNorm-designated- and NormFinder-designated-reference genes enabled us to accurately compare the expression levels of a flax MYB gene in different organs and tissues. Our identification and validation of suitable flax HKGs will facilitate future developmental transcriptomic studies in this economically-important plant. PMID:20403198

2010-01-01

67

Identification of Suitable Reference Genes for gene Expression Studies by qRT-PCR in the Blister Beetle Mylabris cichorii  

PubMed Central

The blister beetle Mylabris cichorii L. (Coleoptera: Meloidae) is a traditional medicinal insect recorded in the Chinese Pharmacopoeia. It synthesizes cantharidin, which kills cancer cells efficiently. Only males produce large amounts of cantharidin. Reference genes are required as endogenous controls for the analysis of differential gene expression in M. cichorii. Our study chose 10 genes as candidate reference genes. The stability of expression of these genes was analyzed by quantitative PCR and determined with two algorithms, geNorm and Normfinder. We recommend UBE3A and RPL22e as suitable reference genes in females and UBE3A, TAF5, and RPL22e in males. PMID:25368050

Wang, Yu; Wang, Zhong-Kang; Huang, Yi; Liao, Yu-Feng; Yin, You-Ping

2014-01-01

68

Real-Time PCR (qPCR) Primer Design Using Free Online Software  

ERIC Educational Resources Information Center

Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

Thornton, Brenda; Basu, Chhandak

2011-01-01

69

Microarray-based uncovering reference genes for quantitative real time PCR in grapevine under abiotic stress  

PubMed Central

Background Quantitative real time polymerase chain reaction is becoming the primary tool for detecting mRNA and transcription data analysis as it shows to have advantages over other more commonly used techniques. Nevertheless, it also presents a few shortcomings, with the most import being the need for data normalisation, usually with a reference gene. Therefore the choice of the reference gene(s) is of great importance for correct data analysis. Microarray data, when available, can be of great assistance when choosing reference genes. Grapevine was submitted to water stress and heat stress as well as a combination of both to test the stability of the possible reference genes. Results Using the analysis of microarray data available for grapevine, six possible reference genes were selected for RT-qPCR validation: PADCP, ubiq, TIF, TIF-GTP, VH1-IK, aladin-related. Two additional genes that are commonly used as reference genes were included: act and L2. The stability of those genes was tested in leaves of grapevine in both field plants and in greenhouse plants under water or heat stress or a combination of both. Gene stability was analyzed with the softwares GeNorm, NormFinder and the ?Cq method resulting in several combinations of reference genes suitable for data normalisation. In order to assess the best combination, the reference genes were tested in putative stress marker genes (PCO, Galsynt, BKCoAS and HSP17) also chosen from the same microarray, in water stress, heat stress and the combination of both. Conclusions Each method selected different gene combinations (PADCP?+?act, TIF?+?TIF-GTP and ubiq?+?act). However, as none of the combinations diverged significantly from the others used to normalize the expression of the putative stress marker genes, then any combination is suitable for data normalisation under the conditions tested. Here we prove the accuracy of choosing grapevine reference genes for RT-qPCR through a microarray analysis. PMID:22564373

2012-01-01

70

Validation of Reference Housekeeping Genes for Gene Expression Studies in Western Corn Rootworm (Diabrotica virgifera virgifera)  

PubMed Central

Quantitative Real-time PCR (qRT-PCR) is a powerful technique to investigate comparative gene expression. In general, normalization of results using a highly stable housekeeping gene (HKG) as an internal control is recommended and necessary. However, there are several reports suggesting that regulation of some HKGs is affected by different conditions. The western corn rootworm (WCR), Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), is a serious pest of corn in the United States and Europe. The expression profile of target genes related to insecticide exposure, resistance, and RNA interference has become an important experimental technique for study of western corn rootworms; however, lack of information on reliable HKGs under different conditions makes the interpretation of qRT-PCR results difficult. In this study, four distinct algorithms (Genorm, NormFinder, BestKeeper and delta-CT) and five candidate HKGs to genes of reference (?-actin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ?-tubulin; RPS9, ribosomal protein S9; EF1a, elongation factor-1?) were evaluated to determine the most reliable HKG under different experimental conditions including exposure to dsRNA and Bt toxins and among different tissues and developmental stages. Although all the HKGs tested exhibited relatively stable expression among the different treatments, some differences were noted. Among the five candidate reference genes evaluated, ?-actin exhibited highly stable expression among different life stages. RPS9 exhibited the most similar pattern of expression among dsRNA treatments, and both experiments indicated that EF1a was the second most stable gene. EF1a was also the most stable for Bt exposure and among different tissues. These results will enable researchers to use more accurate and reliable normalization of qRT-PCR data in WCR experiments. PMID:25356627

Barros Rodrigues, Thais; Khajuria, Chitvan; Wang, Haichuan; Matz, Natalie; Cunha Cardoso, Danielle; Valicente, Fernando Hercos; Zhou, Xuguo; Siegfried, Blair

2014-01-01

71

Validation of reference genes for gene expression analysis in Valsa mali var. mali using real-time quantitative PCR.  

PubMed

Valsa mali var. mali (Vmm), is the predominant species of apple valsa canker in China. Modern analysis of genes involved in virulence or pathogenicity usually implicate gene expression analysis most often performed using real-time quantitative polymerase chain reaction (RT-qPCR). However, for relative gene expression analysis pertinent reference genes have to be validated before using them as internal reference. This has not been reported for Vmm, so far. Therefore, eight commonly used housekeeping genes (ACT, CYP, EF1-?, G6PDH, GAPDH, L13, TUB, and UBQ) were cloned and evaluated for their expression stability by geNorm and NormFinder. Overall, all of the candidate reference genes were found to be suitable for gene expression analysis. After analysis of 10 samples from different strains and abiotic stress treatments, G6PDH appeared to be the most suitable reference gene, whereas GAPDH was the least suitable. Moreover, taking G6PDH combined with L13 or CYP as reference genes, improved the reliability of RT-qPCR significantly. The influence of the reference system on expression data was demonstrated by analyzing Vmmpg-1 encoding an endo-polygalacturonase gene. Pectinases are considered key pathogenicity factors for this fungus. In order to better understand the role of pectinases in pathogenicity of Vmm, RT-qPCR was used for expression analysis. Our results may provide a guideline for future studies on gene expression of V. mali var. mali by using RT-qPCR. PMID:23508400

Yin, Zhiyuan; Ke, Xiwang; Huang, Dingxuan; Gao, Xiaoning; Voegele, Ralf T; Kang, Zhensheng; Huang, Lili

2013-09-01

72

Reference: Biol. Bull. 196: 359-362. (June 1999) Horizontal Gene Transfer: Pitfalls and Promises  

E-print Network

Reference: Biol. Bull. 196: 359-362. (June 1999) Horizontal Gene Transfer: Pitfalls and Promises J). The invocation of horizontal gene transfer events is often regarded as a last-ditch attempt by systematists phylogenies are due to actual events in evolution (horizontal gene transfer or gene duplications [see 2

Gogarten, J. Peter

73

Reference gene selection for gene expression analysis of oocytes collected from dairy cattle and buffaloes during winter and summer.  

PubMed

Oocytes from dairy cattle and buffaloes have severely compromised developmental competence during summer. While analysis of gene expression is a powerful technique for understanding the factors affecting developmental hindrance in oocytes, analysis by real-time reverse transcription PCR (RT-PCR) relies on the correct normalization by reference genes showing stable expression. Furthermore, several studies have found that genes commonly used as reference standards do not behave as expected depending on cell type and experimental design. Hence, it is recommended to evaluate expression stability of candidate reference genes for a specific experimental condition before employing them as internal controls. In acknowledgment of the importance of seasonal effects on oocyte gene expression, the aim of this study was to evaluate the stability of expression levels of ten well-known reference genes (ACTB, GAPDH, GUSB, HIST1H2AG, HPRT1, PPIA, RPL15, SDHA, TBP and YWHAZ) using oocytes collected from different categories of dairy cattle and buffaloes during winter and summer. A normalization factor was provided for cattle (RPL15, PPIA and GUSB) and buffaloes (YWHAZ, GUSB and GAPDH) based on the expression of the three most stable reference genes in each species. Normalization of non-reference target genes by these reference genes was shown to be considerably different from normalization by less stable reference genes, further highlighting the need for careful selection of internal controls. Therefore, due to the high variability of reference genes among experimental groups, we conclude that data normalized by internal controls can be misleading and should be compared to not normalized data or to data normalized by an external control in order to better interpret the biological relevance of gene expression analysis. PMID:24676354

Macabelli, Carolina Habermann; Ferreira, Roberta Machado; Gimenes, Lindsay Unno; de Carvalho, Nelcio Antonio Tonizza; Soares, Júlia Gleyci; Ayres, Henderson; Ferraz, Márcio Leão; Watanabe, Yeda Fumie; Watanabe, Osnir Yoshime; Sangalli, Juliano Rodrigues; Smith, Lawrence Charles; Baruselli, Pietro Sampaio; Meirelles, Flávio Vieira; Chiaratti, Marcos Roberto

2014-01-01

74

Identifying reference genes with stable expression from high throughput sequence data  

PubMed Central

Genes that are constitutively expressed across multiple environmental stimuli are crucial to quantifying differentially expressed genes, particularly when employing quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) assays. However, the identification of these potential reference genes in non-model organisms is challenging and is often guided by expression patterns in distantly related organisms. Here, transcriptome datasets from the diatom Thalassiosira pseudonana grown under replete, phosphorus-limited, iron-limited, and phosphorus and iron co-limited nutrient regimes were analyzed through literature-based searches for homologous reference genes, k-means clustering, and analysis of sequence counts (ASC) to identify putative reference genes. A total of 9759 genes were identified and screened for stable expression. Literature-based searches surveyed 18 generally accepted reference genes, revealing 101 homologs in T. pseudonana with variable expression and a wide range of mean tags per million. k-means analysis parsed the whole transcriptome into 15 clusters. The two most stable clusters contained 709 genes, but still had distinct patterns in expression. ASC analyses identified 179 genes that were stably expressed (posterior probability < 0.1 for 1.25 fold change). Genes known to have a stable expression pattern across the test treatments, like actin, were identified in this pool of 179 candidate genes. ASC can be employed on data without biological replicates and was more robust than the k-means approach in isolating genes with stable expression. The intersection of the genes identified through ASC with commonly used reference genes from the literature suggests that actin and ubiquitin ligase may be useful reference genes for T. pseudonana and potentially other diatoms. With the wealth of transcriptome sequence data becoming available, ASC can be easily applied to transcriptome datasets from other phytoplankton to identify reference genes. PMID:23162540

Alexander, Harriet; Jenkins, Bethany D.; Rynearson, Tatiana A.; Saito, Mak A.; Mercier, Melissa L.; Dyhrman, Sonya T.

2012-01-01

75

Exploring Valid Reference Genes for Quantitative Real-time PCR Analysis in Plutella xylostella (Lepidoptera: Plutellidae)  

PubMed Central

Abstract: Quantitative real-time PCR (qRT-PCR), a primary tool in gene expression analysis, requires an appropriate normalization strategy to control for variation among samples. The best option is to compare the mRNA level of a target gene with that of reference gene(s) whose expression level is stable across various experimental conditions. In this study, expression profiles of eight candidate reference genes from the diamondback moth, Plutella xylostella, were evaluated under diverse experimental conditions. RefFinder, a web-based analysis tool, integrates four major computational programs including geNorm, Normfinder, BestKeeper, and the comparative ?Ct method to comprehensively rank the tested candidate genes. Elongation factor 1 (EF1) was the most suited reference gene for the biotic factors (development stage, tissue, and strain). In contrast, although appropriate reference gene(s) do exist for several abiotic factors (temperature, photoperiod, insecticide, and mechanical injury), we were not able to identify a single universal reference gene. Nevertheless, a suite of candidate reference genes were specifically recommended for selected experimental conditions. Our finding is the first step toward establishing a standardized qRT-PCR analysis of this agriculturally important insect pest. PMID:23983612

Fu, Wei; Xie, Wen; Zhang, Zhuo; Wang, Shaoli; Wu, Qingjun; Liu, Yong; Zhou, Xiaomao; Zhou, Xuguo; Zhang, Youjun

2013-01-01

76

Selection of reliable reference genes for gene expression study in nasopharyngeal carcinoma  

PubMed Central

Aim: To construct a system for selecting reference genes (RGs) and to select the most optimal RGs for gene expression studies in nasopharyngeal carcinoma (NPC). Methods: The total RNAs from 20 NPC samples were each labeled with Cy5-dUTP. To create a common control, the total RNA from 15 nasopharyngeal phlogistic (NP) tissues was mixed and labeled via reverse transcription with Cy3-dUTP. cDNA microarrays containing 14 112 genes were then performed. A mathematical approach was constructed to screen stably expressed genes from the microarray data. Using this method, three genes (YARS, EIF3S7, and PFDN1) were selected as candidate RGs. Furthermore, 7 commonly used RGs (HPRT1, GAPDH, TBP, ACTB, B2M, G6PDH, and HBB) were selected as additional potential RGs. Real-time PCR was used to detect these 10 candidate genes' expression levels and the geNorm program was used to find the optimal RGs for NPC studies. Results: On the basis of the 10 candidate genes' expression stability level, geNorm analysis identified the optimal single RG (YARS or HPRT1) and the most suitable set of RGs (HPRT1, YARS, and EIF3S7) for NPC gene expression studies. In addition, this analysis determined that B2M, G6PDH, and HBB were not appropriate for use as RGs. Interestingly, ACTB was the least stable RG in our study, even though previous studies had indicated that it was one of the most stable RGs. Three novel candidate genes (YARS, EIF3S7, and PFDN1), which were selected from microarray data, were all identified as suitable RGs for NPC research. A RG-selecting system was then constructed, which combines microarray data analysis, a literature screen, real-time PCR, and bioinformatic analysis. Conclusion: We construct a RG-selecting system that helps find the optimal RGs. This process, applied to NPC research, determined the single RG (YARS or HPRT1) and the set of RGs (HPRT1, YARS, and EIF3S7) that are the most suitable internal controls. PMID:21052085

Guo, Yi; Chen, Jia-xin; Yang, Shu; Fu, Xu-ping; Zhang, Zheng; Chen, Ke-he; Huang, Yan; Li, Yao; Xie, Yi; Mao, Yu-min

2010-01-01

77

Expression Profiling in Bemisia tabaci under Insecticide Treatment: Indicating the Necessity for Custom Reference Gene Selection  

PubMed Central

Finding a suitable reference gene is the key for qRT-PCR analysis. However, none of the reference gene discovered thus far can be utilized universally under various biotic and abiotic experimental conditions. In this study, we further examine the stability of candidate reference genes under a single abiotic factor, insecticide treatment. After being exposed to eight commercially available insecticides, which belong to five different classes, the expression profiles of eight housekeeping genes in the sweetpotato whitefly, Bemisia tabaci, one of the most invasive and destructive pests in the world, were investigated using qRT-PCR analysis. In summary, elongation factor 1? (EF1?), ?-tubulin (TUB1?) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified as the most stable reference genes under the insecticide treatment. The initial assessment of candidate reference genes was further validated with the expression of two target genes, a P450 (Cyp6cm1) and a glutathione S-transferase (GST). However, ranking of reference genes varied substantially among intra- and inter-classes of insecticides. These combined data strongly suggested the necessity of conducting custom reference gene selection designed for each and every experimental condition, even when examining the same abiotic or biotic factor. PMID:24498122

Zhou, Xuguo; Gao, Xiwu

2014-01-01

78

Identification of novel reference genes based on MeSH categories.  

PubMed

Transcriptome experiments are performed to assess protein abundance through mRNA expression analysis. Expression levels of genes vary depending on the experimental conditions and the cell response. Transcriptome data must be diverse and yet comparable in reference to stably expressed genes, even if they are generated from different experiments on the same biological context from various laboratories. In this study, expression patterns of 9090 microarray samples grouped into 381 NCBI-GEO datasets were investigated to identify novel candidate reference genes using randomizations and Receiver Operating Characteristic (ROC) curves. The analysis demonstrated that cell type specific reference gene sets display less variability than a united set for all tissues. Therefore, constitutively and stably expressed, origin specific novel reference gene sets were identified based on their coefficient of variation and percentage of occurrence in all GEO datasets, which were classified using Medical Subject Headings (MeSH). A large number of MeSH grouped reference gene lists are presented as novel tissue specific reference gene lists. The most commonly observed 17 genes in these sets were compared for their expression in 8 hepatocellular, 5 breast and 3 colon carcinoma cells by RT-qPCR to verify tissue specificity. Indeed, commonly used housekeeping genes GAPDH, Actin and EEF2 had tissue specific variations, whereas several ribosomal genes were among the most stably expressed genes in vitro. Our results confirm that two or more reference genes should be used in combination for differential expression analysis of large-scale data obtained from microarray or next generation sequencing studies. Therefore context dependent reference gene sets, as presented in this study, are required for normalization of expression data from diverse technological backgrounds. PMID:24682035

Ersahin, Tulin; Carkacioglu, Levent; Can, Tolga; Konu, Ozlen; Atalay, Volkan; Cetin-Atalay, Rengul

2014-01-01

79

Identification of Novel Reference Genes Based on MeSH Categories  

PubMed Central

Transcriptome experiments are performed to assess protein abundance through mRNA expression analysis. Expression levels of genes vary depending on the experimental conditions and the cell response. Transcriptome data must be diverse and yet comparable in reference to stably expressed genes, even if they are generated from different experiments on the same biological context from various laboratories. In this study, expression patterns of 9090 microarray samples grouped into 381 NCBI-GEO datasets were investigated to identify novel candidate reference genes using randomizations and Receiver Operating Characteristic (ROC) curves. The analysis demonstrated that cell type specific reference gene sets display less variability than a united set for all tissues. Therefore, constitutively and stably expressed, origin specific novel reference gene sets were identified based on their coefficient of variation and percentage of occurrence in all GEO datasets, which were classified using Medical Subject Headings (MeSH). A large number of MeSH grouped reference gene lists are presented as novel tissue specific reference gene lists. The most commonly observed 17 genes in these sets were compared for their expression in 8 hepatocellular, 5 breast and 3 colon carcinoma cells by RT-qPCR to verify tissue specificity. Indeed, commonly used housekeeping genes GAPDH, Actin and EEF2 had tissue specific variations, whereas several ribosomal genes were among the most stably expressed genes in vitro. Our results confirm that two or more reference genes should be used in combination for differential expression analysis of large-scale data obtained from microarray or next generation sequencing studies. Therefore context dependent reference gene sets, as presented in this study, are required for normalization of expression data from diverse technological backgrounds. PMID:24682035

Can, Tolga; Konu, Ozlen; Atalay, Volkan; Cetin-Atalay, Rengul

2014-01-01

80

Identification and validation of reference genes for quantitative RT-PCR normalization in wheat  

PubMed Central

Background Usually the reference genes used in gene expression analysis have been chosen for their known or suspected housekeeping roles, however the variation observed in most of them hinders their effective use. The assessed lack of validated reference genes emphasizes the importance of a systematic study for their identification. For selecting candidate reference genes we have developed a simple in silico method based on the data publicly available in the wheat databases Unigene and TIGR. Results The expression stability of 32 genes was assessed by qRT-PCR using a set of cDNAs from 24 different plant samples, which included different tissues, developmental stages and temperature stresses. The selected sequences included 12 well-known HKGs representing different functional classes and 20 genes novel with reference to the normalization issue. The expression stability of the 32 candidate genes was tested by the computer programs geNorm and NormFinder using five different data-sets. Some discrepancies were detected in the ranking of the candidate reference genes, but there was substantial agreement between the groups of genes with the most and least stable expression. Three new identified reference genes appear more effective than the well-known and frequently used HKGs to normalize gene expression in wheat. Finally, the expression study of a gene encoding a PDI-like protein showed that its correct evaluation relies on the adoption of suitable normalization genes and can be negatively affected by the use of traditional HKGs with unstable expression, such as actin and ?-tubulin. Conclusion The present research represents the first wide screening aimed to the identification of reference genes and of the corresponding primer pairs specifically designed for gene expression studies in wheat, in particular for qRT-PCR analyses. Several of the new identified reference genes outperformed the traditional HKGs in terms of expression stability under all the tested conditions. The new reference genes will enable more accurate normalization and quantification of gene expression in wheat and will be helpful for designing primer pairs targeting orthologous genes in other plant species. PMID:19232096

Paolacci, Anna R; Tanzarella, Oronzo A; Porceddu, Enrico; Ciaffi, Mario

2009-01-01

81

Identification of Phosphoglycerate Kinase 1 (PGK1) as a reference gene for quantitative gene expression measurements in human blood RNA  

PubMed Central

Background Blood is a convenient sample and increasingly used for quantitative gene expression measurements with a variety of diseases including chronic fatigue syndrome (CFS). Quantitative gene expression measurements require normalization of target genes to reference genes that are stable and independent from variables being tested in the experiment. Because there are no genes that are useful for all situations, reference gene selection is an essential step to any quantitative reverse transcription-PCR protocol. Many publications have described appropriate genes for a wide variety of tissues and experimental conditions, however, reference genes that may be suitable for the analysis of CFS, or human blood RNA derived from whole blood as well as isolated peripheral blood mononuclear cells (PBMCs), have not been described. Findings Literature review and analyses of our unpublished microarray data were used to narrow down the pool of candidate reference genes to six. We assayed whole blood RNA from Tempus tubes and cell preparation tube (CPT)-collected PBMC RNA from 46 subjects, and used the geNorm and NormFinder algorithms to select the most stable reference genes. Phosphoglycerate kinase 1 (PGK1) was one of the optimal normalization genes for both whole blood and PBMC RNA, however, additional genes differed for the two sample types; Ribosomal protein large, P0 (RPLP0) for PBMC RNA and Peptidylprolyl isomerase B (PPIB) for whole blood RNA. We also show that the use of a single reference gene is sufficient for normalization when the most stable candidates are used. Conclusions We have identified PGK1 as a stable reference gene for use with whole blood RNA and RNA derived from PBMC. When stable genes are selected it is possible to use a single gene for normalization rather than two or three. Optimal normalization will improve the ability of results from PBMC RNA to be compared with those from whole blood RNA and potentially allows comparison of gene expression results from blood RNA collected and processed by different methods with the intention of biomarker discovery. Results of this study should facilitate large-scale molecular epidemiologic studies using blood RNA as the target of quantitative gene expression measurements. PMID:21896205

2011-01-01

82

Validation of reference genes in Penicillium echinulatum to enable gene expression study using real-time quantitative RT-PCR.  

PubMed

Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) is a methodology that facilitates the quantification of mRNA expression in a given sample. Analysis of relative gene expression by qRT-PCR requires normalization of the data using a reference gene that is expressed at a similar level in all evaluated conditions. Determining an internal control gene is essential for gene expression studies. Gene expression studies in filamentous fungi frequently use the ?-actin gene (actb), ?-tubulin, and glyceraldehyde-3-phosphate dehydrogenase as reference genes because they are known to have consistent expression levels. Until now, no study has been performed to select an internal control gene for the filamentous fungal species Penicillium echinulatum. The aim of this study was to evaluate and validate internal control genes to enable the study of gene expression in P. echinulatum using qRT-PCR. P. echinulatum strain S1M29 was grown in conditions to either induce (cellulose and sugar cane bagasse) or repress (glucose) gene expression to analyze 23 candidate normalization genes for stable expression. Two software programs, BestKeeper and geNorm, were used to assess the expression of the candidate normalization genes. The results indicate that the actb reference gene is more stably expressed in P. echinulatum. This is the first report in the literature that determines a normalization gene for this fungus. From the results obtained, we recommend the use of the P. echinulatum actb gene as an endogenous control for gene expression studies of cellulases and hemicellulases by qRT-PCR. PMID:24509829

Zampieri, Denise; Nora, Luísa C; Basso, Vanessa; Camassola, Marli; Dillon, Aldo J P

2014-08-01

83

Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum.  

PubMed

Quantitative real-time reverse transcription PCR (qRT-PCR) is a rapid, sensitive, and reliable technique for gene expression studies. The accuracy and reliability of qRT-PCR results depend on the stability of the reference genes used for gene normalization. Therefore, a systematic process of reference gene evaluation is needed. Ganoderma lucidum is a famous medicinal mushroom in East Asia. In the current study, 10 potential reference genes were selected from the G. lucidum genomic data. The sequences of these genes were manually curated, and primers were designed following strict criteria. The experiment was conducted using qRT-PCR, and the stability of each candidate gene was assessed using four commonly used statistical programs-geNorm, NormFinder, BestKeeper, and RefFinder. According to our results, PP2A was expressed at the most stable levels under different fermentation conditions, and RPL4 was the most stably expressed gene in different tissues. RPL4, PP2A, and ?-tubulin are the most commonly recommended reference genes for normalizing gene expression in the entire sample set. The current study provides a foundation for the further use of qRT-PCR in G. lucidum gene analysis. PMID:24013612

Xu, Jiang; Xu, ZhiChao; Zhu, YingJie; Luo, HongMei; Qian, Jun; Ji, AiJia; Hu, YuanLei; Sun, Wei; Wang, Bo; Song, JingYuan; Sun, Chao; Chen, ShiLin

2014-01-01

84

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation  

NASA Astrophysics Data System (ADS)

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the ?-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was ?-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was ?-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 ? subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

2014-07-01

85

Selection of reference genes for qRT-PCR analysis of gene expression in sea cucumber Apostichopus japonicus during aestivation  

NASA Astrophysics Data System (ADS)

Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is a technique that is widely used for gene expression analysis, and its accuracy depends on the expression stability of the internal reference genes used as normalization factors. However, many applications of qRT-PCR used housekeeping genes as internal controls without validation. In this study, the expression stability of eight candidate reference genes in three tissues (intestine, respiratory tree, and muscle) of the sea cucumber Apostichopus japonicus was assessed during normal growth and aestivation using the geNorm, NormFinder, delta CT, and RefFinder algorithms. The results indicate that the reference genes exhibited significantly different expression patterns among the three tissues during aestivation. In general, the ?-tubulin (TUBB) gene was relatively stable in the intestine and respiratory tree tissues. The optimal reference gene combination for intestine was 40S ribosomal protein S18 (RPS18), TUBB, and NADH dehydrogenase (NADH); for respiratory tree, it was ?-actin (ACTB), TUBB, and succinate dehydrogenase cytochrome B small subunit (SDHC); and for muscle it was ?-tubulin (TUBA) and NADH dehydrogenase [ubiquinone] 1 ? subcomplex subunit 13 (NDUFA13). These combinations of internal control genes should be considered for use in further studies of gene expression in A. japonicus during aestivation.

Zhao, Ye; Chen, Muyan; Wang, Tianming; Sun, Lina; Xu, Dongxue; Yang, Hongsheng

2014-11-01

86

Identification of Reference Genes for qRT-PCR Analysis in Yesso Scallop Patinopecten yessoensis  

PubMed Central

Background Bivalves comprise around 30,000 extant species and have received much attention for their importance in ecosystems, aquaculture and evolutionary studies. Despite the increasing application of real-time quantitative reverse transcription PCR (qRT-PCR) in gene expression studies on bivalve species, little research has been conducted on reference gene selection which is critical for reliable and accurate qRT-PCR analysis. For scallops, systematic evaluation of reference genes that can be used among tissues or embryo/larva stages is lacking, and ?-actin (ACT) is most frequently used as qRT-PCR reference gene without validation. Results In this study, 12 commonly used candidate reference genes were selected from the transcriptome data of Yesso scallop (Patinopecten yessoensis) for suitable qRT-PCR reference genes identification. The expression of these genes in 36 tissue samples and 15 embryo/larva samples under normal physiological conditions was examined by qRT-PCR, and their expression stabilities were evaluated using three statistic algorithms, geNorm, NormFinder, and comparative ?Ct method. Similar results were obtained by the three approaches for the most and the least stably expressed genes. Final comprehensive ranking for the 12 genes combing the results from the three programs showed that, for different tissues, DEAD-box RNA helicase (HELI), ubiquitin (UBQ), and 60S ribosomal protein L16 (RPL16) were the optimal reference genes combination, while for different embryo/larva stages, gene set containing Cytochrome B (CB), Cytochrome C (CC), Histone H3.3 (His3.3), and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were recommended for qRT-PCR normalization. ACT was among the least stable genes for both adult tissues and embryos/larvae. Conclusions This work constitutes the first systematic analysis on reference genes selection for qRT-PCR normalization in scallop under normal conditions. The suitable reference genes we recommended will be useful for the identification of genes related to biological processes in Yesso scallop, and also in the reference gene selection for other scallop or bivalve species. PMID:24069432

Feng, Liying; Yu, Qian; Li, Xue; Ning, Xianhui; Wang, Jing; Zou, Jiajun; Zhang, Lingling; Wang, Shi; Hu, Jingjie; Hu, Xiaoli; Bao, Zhenmin

2013-01-01

87

SYBR®Green qPCR Salmonella detection system allowing discrimination at the genus, species and subspecies levels.  

PubMed

In this work, a three-level Salmonella detection system based on a combination of seven SYBR®Green qPCR was developed. This detection system discriminates Salmonella at the genus, species and subspecies levels using a single 96-well plate. The SYBR®Green qPCR assays target the invA, rpoD, iroB and safC genes, as well as the STM0296 locus, putatively coding for a cytoplasmic protein. This study includes the design of primer pairs, in silico and in situ selectivity, sensitivity, repeatability and reproducibility evaluations of the seven SYBR®Green qPCR assays. Each detection level displayed a selectivity of 100 %. This combinatory SYBR®Green qPCR system was also compared with three commercially available Salmonella qPCR detection kits. This comparison highlighted the importance of using a multi-gene detection system to be able to detect every target strain, even those with deletion or mutation of important genes. PMID:24113820

Barbau-Piednoir, Elodie; Bertrand, Sophie; Mahillon, Jacques; Roosens, Nancy H; Botteldoorn, Nadine

2013-11-01

88

Reference Genes for Expression Studies in Hypoxia and Hyperglycemia Models in Human Umbilical Vein Endothelial Cells  

PubMed Central

Human umbilical vein endothelial cell (HUVEC)-based gene expression studies performed under hypoxia and/or hyperglycemia show huge potential for modeling endothelial cell response in cardiovascular disease and diabetes. However, such studies require reference genes that are stable across the whole range of experimental conditions. These reference genes have not been comprehensively defined to date. We applied human genome-wide microarrays and quantitative real-time PCR (qRT-PCR) on RNA obtained from primary HUVEC cultures that were incubated for 24 hr either in euglycemic or in hyperglycemic conditions and then subjected to short-term CoCl2-induced hypoxia for 1, 3, or 12 hr. Using whole-transcript arrays, we selected 10 commonly used reference genes with no significant expression variation across eight different conditions. These genes were ranked using NormFinder software according to their stability values. Consequently, five genes were selected for validation by qRT-PCR. These were ribosomal protein large P0 (RPLP0), transferrin receptor (TFRC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ?-glucuronidase (GUSB), and ?-actin (ACTB). All five genes displayed stable expression under hyperglycemia. However, only RPLP0 and TFRC genes were stable under hypoxia up to 12 hr. Under hyperglycemia combined with hypoxia up to 12 hr, the expression of RPLP0, TFRC, GUSB, and ACTB genes remained unchanged. Our findings strongly confirm that RPLP0 and TFRC are the most suitable reference genes for HUVEC gene expression experiments subjected to hypoxia and/or hyperglycemia for the given experimental conditions. We provide further evidence that even commonly known references genes require experimental validation for all conditions involved. PMID:25193495

Bakhashab, Sherin; Lary, Sahira; Ahmed, Farid; Schulten, Hans-Juergen; Bashir, Ayat; Ahmed, Fahad W.; Al-Malki, Abdulrahman L.; Jamal, Hasan S.; Gari, Mamdooh A.; Weaver, Jolanta U.

2014-01-01

89

Identification and Validation of Reference Genes for Gene Expression Analysis Using Quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae)  

PubMed Central

Reverse transcription quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most sensitive and accurate method for the quantification of gene expression. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable housekeeping genes is required. These housekeeping genes need to show stable expression under the given experimental conditions for the qRT-PCR results to be accurate. Unfortunately, there are no studies on the stability of housekeeping genes used in Spodoptera litura. In this study, eight candidate reference genes, elongation factor 1 alpha (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L10 (RPL10), ribosomal protein S3 (RPS3), beta actin (ACTB), beta FTZ-F1 (FTZF1), ubiquinol-cytochrome c reductase (UCCR), and arginine kinase (AK), were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs, BestKeeper, geNorm and Normfinder, and the comparative ?Ct method. We determined the expression levels of the candidate reference genes for three biotic factors (developmental stage, tissue and population), and four abiotic treatments (temperature, insecticide, food and starvation). The results indicated that the best sets of candidates as reference genes were as follows: GAPDH and UCCR for developmental stages; RPL10, AK and EF1 for different tissues; RPL10 and EF1 for different populations in China; GAPDH and EF1 for temperature-stressed larvae; AK and ACTB for larvae treated with different insecticides; RPL10, GAPDH and UCCR for larvae fed different diets; RPS3 and ACTB for starved larvae. We believe that these results make an important contribution to gene analysis studies in S. litura and form the basis of further research on stable reference genes in S. litura and other organisms. PMID:23874494

Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong

2013-01-01

90

Validation of reference genes for ovarian tissue from capuchin monkeys (Cebus apella).  

PubMed

There is no tradition in studies reporting the effect of exposure to cryoprotectants or simply hypoxia and hypothermia on gene expression in the ovarian tissue and there has been only one study on reference or target genes quantification, and comparisons of normoxic with hypoxic, hypothermic and toxic conditions. Our aim in the present study was to investigate the stability of three reference genes in the ovarian tissue of capuchin monkeys (Cebus apella). To this end, fresh and cryoprotectant-exposed ovarian biopsies were used. Both fresh and exposed ovarian tissues were subjected to total RNA extraction and synthesis of cDNA. cDNA was amplified by real-time polymerase chain reaction (PCR), and GeNorm, BestKeeper and NormFinder software were used to evaluate the stability of glyceraldehyde-2-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and TATA-binding protein (TBP). Results demonstrated that, in the ovarian tissue from capuchin monkeys, HPRT1 and TBP were the most suitable reference genes and thus could be used as parameters to normalize data in future studies. In contrast, GAPDH appeared as the least stable gene among the tested reference genes. In conclusion, HPRT1 and TBP were the most stable reference genes in fresh and cryoprotectant-exposed ovarian tissue from capuchin monkeys. PMID:22475447

Brito, A B; Lima, J S; Brito, D C; Santana, L N; Costa, N N; Miranda, M S; Ohashi, O M; Santos, R R; Domingues, S F S

2013-05-01

91

Selection of plastid- and nuclear-encoded reference genes to study the effect of altered endogenous cytokinin content on photosynthesis genes in Nicotiana   tabacum  

Microsoft Academic Search

Selection and use of appropriate reference genes as internal controls in real-time reverse transcription PCR (RT-PCR) assays\\u000a is highly important for accurate quantification of gene expression levels. Since some photosynthetic genes are encoded in\\u000a the nuclear genome and others in the chloroplast genome, we evaluated both nuclear- and plastid-encoded candidate reference\\u000a genes. Six plastid-encoded candidate reference genes were derived from

Anne Cortleven; Tony Remans; Wolfram G. Brenner; Roland Valcke

2009-01-01

92

Reference Gene Validation for Quantitative RT-PCR during Biotic and Abiotic Stresses in Vitis vinifera  

PubMed Central

Grapevine is one of the most cultivated fruit crop worldwide with Vitis vinifera being the species with the highest economical importance. Being highly susceptible to fungal pathogens and increasingly affected by environmental factors, it has become an important agricultural research area, where gene expression analysis plays a fundamental role. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) is currently amongst the most powerful techniques to perform gene expression studies. Nevertheless, accurate gene expression quantification strongly relies on appropriate reference gene selection for sample normalization. Concerning V. vinifera, limited information still exists as for which genes are the most suitable to be used as reference under particular experimental conditions. In this work, seven candidate genes were investigated for their stability in grapevine samples referring to four distinct stresses (Erysiphe necator, wounding and UV-C irradiation in leaves and Phaeomoniella chlamydospora colonization in wood). The expression stability was evaluated using geNorm, NormFinder and BestKeeper. In all cases, full agreement was not observed for the three methods. To provide comprehensive rankings integrating the three different programs, for each treatment, a consensus ranking was created using a non-weighted unsupervised rank aggregation method. According to the last, the three most suitable reference genes to be used in grapevine leaves, regardless of the stress, are UBC, VAG and PEP. For the P. chlamydospora treatment, EF1, CYP and UBC were the best scoring genes. Acquaintance of the most suitable reference genes to be used in grapevine samples can contribute for accurate gene expression quantification in forthcoming studies. PMID:25340748

Borges, Alexandre Filipe; Fonseca, Catarina; Ferreira, Ricardo Boavida; Lourenco, Ana Maria; Monteiro, Sara

2014-01-01

93

Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.  

PubMed

Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. PMID:23954301

Kubi?, Piotr; Materniak, Magdalena; Ku?mak, Jacek

2013-12-01

94

Selection of reference genes for quantitative real-time PCR in bovine preimplantation embryos  

PubMed Central

Background Real-time quantitative PCR is a sensitive and very efficient technique to examine gene transcription patterns in preimplantation embryos, in order to gain information about embryo development and to optimize assisted reproductive technologies. Critical to the succesful application of real-time PCR is careful assay design, reaction optimization and validation to maximize sensitivity and accuracy. In most of the studies published GAPD, ACTB or 18S rRNA have been used as a single reference gene without prior verification of their expression stability. Normalization of the data using unstable controls can result in erroneous conclusions, especially when only one reference gene is used. Results In this study the transcription levels of 8 commonly used reference genes (ACTB, GAPD, Histone H2A, TBP, HPRT1, SDHA, YWHAZ and 18S rRNA) were determined at different preimplantation stages (2-cell, 8-cell, blastocyst and hatched blastocyst) in order to select the most stable genes to normalize quantitative data within different preimplantation embryo stages. Conclusion Using the geNorm application YWHAZ, GAPD and SDHA were found to be the most stable genes across the examined embryonic stages, while the commonly used ACTB was shown to be highly regulated. We recommend the use of the geometric mean of those 3 reference genes as an accurate normalization factor, which allows small expression differences to be reliably measured. PMID:16324220

Goossens, Karen; Van Poucke, Mario; Van Soom, Ann; Vandesompele, Jo; Van Zeveren, Alex; Peelman, Luc J

2005-01-01

95

Quantitative detection and identification of tyramine-producing enterococci and lactobacilli in cheese by multiplex qPCR  

Microsoft Academic Search

Tyramine is the most abundant biogenic amine in fermented dairy products, in which it is produced through the microbial enzymatic decarboxylation of tyrosine. This activity has been detected in a variety of lactic acid bacteria mainly belonging to the genera Enterococcus and Lactobacillus. This paper describes a culture-independent qPCR method, based on the specific amplification of the tdc gene, for

Victor Ladero; María Fernández; Isabel Cuesta; Miguel A. Alvarez

2010-01-01

96

A Brain Region-Specific Predictive Gene Map for Autism Derived by Profiling a Reference Gene Set  

PubMed Central

Molecular underpinnings of complex psychiatric disorders such as autism spectrum disorders (ASD) remain largely unresolved. Increasingly, structural variations in discrete chromosomal loci are implicated in ASD, expanding the search space for its disease etiology. We exploited the high genetic heterogeneity of ASD to derive a predictive map of candidate genes by an integrated bioinformatics approach. Using a reference set of 84 Rare and Syndromic candidate ASD genes (AutRef84), we built a composite reference profile based on both functional and expression analyses. First, we created a functional profile of AutRef84 by performing Gene Ontology (GO) enrichment analysis which encompassed three main areas: 1) neurogenesis/projection, 2) cell adhesion, and 3) ion channel activity. Second, we constructed an expression profile of AutRef84 by conducting DAVID analysis which found enrichment in brain regions critical for sensory information processing (olfactory bulb, occipital lobe), executive function (prefrontal cortex), and hormone secretion (pituitary). Disease specificity of this dual AutRef84 profile was demonstrated by comparative analysis with control, diabetes, and non-specific gene sets. We then screened the human genome with the dual AutRef84 profile to derive a set of 460 potential ASD candidate genes. Importantly, the power of our predictive gene map was demonstrated by capturing 18 existing ASD-associated genes which were not part of the AutRef84 input dataset. The remaining 442 genes are entirely novel putative ASD risk genes. Together, we used a composite ASD reference profile to generate a predictive map of novel ASD candidate genes which should be prioritized for future research. PMID:22174805

Kumar, Ajay; Swanwick, Catherine Croft; Johnson, Nicole; Menashe, Idan; Basu, Saumyendra N.; Bales, Michael E.; Banerjee-Basu, Sharmila

2011-01-01

97

GeneChip, geNorm, and gastrointestinal tumors: novel reference genes for real-time PCR  

Microsoft Academic Search

themselves ( R2 .0.86, P ,0.0001), with low variability (coefficient of variation (CV) ,12.7%) and high interassay reproducibility ( r 5 0.93, P 5 0.001). In comparison, the alternative control gene, GAPDH , exhibited the highest variability (CV 518.1%), was signif- icantly differently expressed between tissue types ( P 5 0.05), was poorly correlated with the three reference genes (

Mark Kidd; Boaz Nadler; Shrikant Mane; Geeta Eick; Maximillian Malfertheiner; Manish Champaneria; Roswitha Pfragner; Irvin Modlin

2007-01-01

98

Applicability of the chymopapain gene used as endogenous reference gene for transgenic huanong no. 1 papaya detection.  

PubMed

The virus-resistant papaya (Carica papaya L.), Huanong no. 1, was the genetically modified (GM) fruit approved for growing in China in 2006. To implement the labeling regulation of GM papaya and its derivates, the development of papaya endogenous reference gene is very necessary for GM papaya detection. Herein, we reported one papaya specific gene, Chymopapain (CHY), as one suitable endogenous reference gene, used for GM papaya identification. Thereafter, we established the conventional and real-time quantitative PCR assays of the CHY gene. In the CHY conventional PCR assay, the limit of detection (LOD) was 25 copies of haploid papaya genome. In the CHY real-time quantitative PCR assay, both the LOD and the limit of quantification (LOQ) were as low as 12.5 copies of haploid papaya genome. Furthermore, we revealed the construct-specific sequence of Chinese GM papaya Huanong no. 1 and developed its conventional and quantitative PCR systems employing the CHY gene as endogenous reference gene. This work is useful for papaya specific identification and GM papaya detection. PMID:19722561

Guo, Jinchao; Yang, Litao; Liu, Xin; Zhang, Haibo; Qian, Bingjun; Zhang, Dabing

2009-08-12

99

Improved group-specific primers based on the full SILVA 16S rRNA gene reference database.  

PubMed

Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics. PMID:25229098

Pfeiffer, Stefan; Pastar, Milica; Mitter, Birgit; Lippert, Kathrin; Hackl, Evelyn; Lojan, Paul; Oswald, Andreas; Sessitsch, Angela

2014-08-01

100

An Internal Reference Technique for Accurately Quantifying Specific mRNAs by Real-Time PCR with Application to the tceA Reductive Dehalogenase Gene  

Microsoft Academic Search

The accuracy of mRNA quantification by reverse transcription (RT) in conjunction with real-time PCR (qPCR) is limited by mRNA losses during sample preparation (cell lysis, RNA isolation, and DNA removal) and by inefficiencies in reverse transcription. To control for these losses and inefficiencies, a technique was developed that utilizes an exogenous internal reference mRNA (ref mRNA) along with mRNA absolute

David R. Johnson; Patrick K. H. Lee; Victor F. Holmes; Lisa Alvarez-Cohen

2005-01-01

101

Determination of viable Salmonellae from potable and source water through PMA assisted qPCR.  

PubMed

Resource constrained countries identified as endemic zones for pathogenicity of Salmonella bear an economic burden due to recurring expenditure on medical treatment. qPCR used for Salmonella detection could not discriminate between viable and nonviable cells. Propidium monoazide (PMA) that selectively penetrates nonviable cells to cross-link their DNA, was coupled with ttr gene specific qPCR for quantifying viable salmonellae in source/potable waters collected from a north Indian city. Source water (raw water for urban potable water supply) and urban potable water exhibited viable salmonellae in the range of 2.1×10(4)-2.6×10(6) and 2-7160CFU/100mL, respectively. Potable water at water works exhibited DNA from dead cells but no viable cells were detected. PMA assisted qPCR could specifically detect low numbers of live salmonellae in Source and potable waters. This strategy can be used in surveillance of urban potable water distribution networks to map contamination points for better microbial risk management. PMID:23623706

Singh, Gulshan; Vajpayee, Poornima; Bhatti, Saurabh; Ronnie, Nirmala; Shah, Nimish; McClure, Peter; Shanker, Rishi

2013-07-01

102

Quantification of Salmonella Typhi in water and sediments by molecular-beacon based qPCR.  

PubMed

A molecular-beacon based qPCR assay targeting staG gene was designed for specific detection and quantification of S. Typhi and validated against water and sediment samples collected from the river Ganga, Yamuna and their confluence on two days during Mahakumbha mela 2012-2013 (a) 18 December, 2012: before six major religious holy dips (Makar Sankranti, Paush Poornima, Mauni Amavasya, Basant Panchami, Maghi Poornima and Mahashivratri) (b) 10 February, 2013: after the holy dip was taken by over 3,00,00,000 devotees led by ascetics of Hindu sects at Sangam on 'Mauni Amavasya' (the most auspicious day of ritualistic mass bathing). The assay could detect linearly lowest 1 genomic equivalent per qPCR and is highly sensitive and selective for S. Typhi detection in presence of non specific DNA from other bacterial strains including S. Paratyphi A and S. Typhimurium. It has been observed that water and sediment samples exhibit S. Typhi. The mass holy dip by devotees significantly affected the water and sediment quality by enhancing the number of S. Typhi in the study area. The qPCR developed in the study might be helpful in planning the intervention and prevention strategies for control of enteric fever outbreaks in endemic regions. PMID:25042245

Rani, Neetika; Vajpayee, Poornima; Bhatti, Saurabh; Singh, Smriti; Shanker, Rishi; Gupta, Kailash Chand

2014-10-01

103

Finding relevant references to genes and proteins in Medline using a Bayesian approach  

Microsoft Academic Search

Motivation: Mining the biomedical literature for references to genes and proteins always involves a tradeoff between high precision with false negatives, and high recall with false positives. Having a reliable method for assessing the relevance of literature mining results is crucial to finding ways to balance precision and recall, and for subsequently building automated systems to analyze these results. We

Julie E. Leonard; Jeffrey B. Colombe; Joshua L. Levy

2002-01-01

104

Evaluation of four genes in rice for their suitability as endogenous reference standards in quantitative PCR.  

PubMed

The genetically modified (GM) food/feed quantification depends on the reliable detection systems of endogenous reference genes. Currently, four endogenous reference genes including sucrose phosphate synthase (SPS), GOS9, phospholipase D (PLD), and ppi phosphofructokinase (ppi-PPF) of rice have been used in GM rice detection. To compare the applicability of these four rice reference genes in quantitative PCR systems, we analyzed the target nucleotide sequence variation in 58 conventional rice varieties from various geographic and phylogenic origins, also their quantification performances were evaluated using quantitative real-time PCR and GeNorm analysis via a series of statistical calculation to get a "M value" which is negative correlation with the stability of genes. The sequencing analysis results showed that the reported GOS9 and PLD taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested rice cultivars, while no SNPs were observed for SPS and ppi-PPF amplicons. Also, poor quantitative performance was detectable in these cultivars with SNPs using GOS9 and PLD quantitative PCR systems. Even though the PCR efficiency of ppi-PPF system was slightly lower, the SPS and ppi-PPF quantitative PCR systems were shown to be applicable for rice endogenous reference assay with less variation among the C(t) values, good reproducibility in quantitative assays, and the low M values by the comprehensive quantitative PCR comparison and GeNorm analysis. PMID:20961039

Wang, Chong; Jiang, Lingxi; Rao, Jun; Liu, Yinan; Yang, Litao; Zhang, Dabing

2010-11-24

105

Identification of Reference Genes for Quantitative RT-PCR in Ascending Aortic Aneurysms  

PubMed Central

Hypertension and congenital aortic valve malformations are frequent causes of ascending aortic aneurysms. The molecular mechanisms of aneurysm formation under these circumstances are not well understood. Reference genes for gene activity studies in aortic tissue that are not influenced by aortic valve morphology and its hemodynamic consequences, aortic dilatation, hypertension, or antihypertensive medication are not available so far. This study determines genes in ascending aortic tissue that are independent of these parameters. Tissue specimens from dilated and undilated ascending aortas were obtained from 60 patients (age ?70 years) with different morphologies of the aortic valve (tricuspid undilated n?=?24, dilated n?=?11; bicuspid undilated n?=?6, dilated n?=?15; unicuspid dilated n?=?4). Of the studied individuals, 36 had hypertension, and 31 received ACE inhibitors or AT1 receptor antagonists. The specimens were obtained intraoperatively from the wall of the ascending aorta. We analyzed the expression levels of 32 candidate reference genes by quantitative RT-PCR (RT-qPCR). Differential expression levels were assessed by parametric statistics. The expression analysis of these 32 genes by RT-qPCR showed that EIF2B1, ELF1, and PPIA remained constant in their expression levels in the different specimen groups, thus being insensitive to aortic valve morphology, aortic dilatation, hypertension, and medication with ACE inhibitors or AT1 receptor antagonists. Unlike many other commonly used reference genes, the genes EIF2B1, ELF1, and PPIA are neither confounded by aortic comorbidities nor by antihypertensive medication and therefore are most suitable for gene expression analysis of ascending aortic tissue. PMID:23326585

Henn, Dominic; Bandner-Risch, Doris; Perttunen, Hilja; Schmied, Wolfram; Porras, Carlos; Ceballos, Francisco; Rodriguez-Losada, Noela; Schafers, Hans-Joachim

2013-01-01

106

Construction of a standard reference plasmid containing seven target genes for the detection of transgenic cotton.  

PubMed

Insect resistance and herbicide tolerance are the dominant traits of commercialized transgenic cotton. In this study, we constructed a general standard reference plasmid for transgenic cotton detection. Target genes, including the cowpea trypsin gene cptI, the insect resistance gene cry1Ab/1Ac, the herbicide tolerance gene cp4-epsps, the Agrobacterium tumefaciens nopaline synthase (Nos) terminator that exists in transgenic cotton and part of the endogenous cotton SadI gene were amplified from plasmids pCPT1, pBT, pCP4 and pBI121 and from DNA of the nontransgenic cotton line K312, respectively. The genes cry1Ab/1Ac and cptI, as well as cp4-epsps and the Nos terminator gene, were ligated together to form the fusion genes cptI-Bt and cp4-Nos, respectively, by overlapping PCR. We checked the validity of genes Sad1, cptI-Bt and cp4-Nos by DNA sequencing. Then, positive clones of cptI-Bt, cp4-Nos and Sad1 were digested with the corresponding restriction enzymes and ligated sequentially into vector pCamBIA2300, which contains the CAMV 35S promoter and nptII gene, to form the reference plasmid pMCS. Qualitative detection showed that pMCS is a good positive control for transgenic cotton detection. Real-time PCR detection efficiencies with pMCS as a calibrator ranged from 94.35% to 98.67% for the standard curves of the target genes (R(2)?0.998). The relative standard deviation of the mean value for the known sample was 11.95%. These results indicate that the strategy of using the pMCS plasmid as a reference material is feasible and reliable for the detection of transgenic cotton. Therefore, this plasmid can serve as a useful reference tool for qualitative and quantitative detection of single or stacked trait transgenic cotton, thus paving the way for the identification of various products containing components of transgenic cotton. PMID:24929128

Wang, Xujing; Tang, Qiaoling; Dong, Lei; Dong, Yufeng; Su, Yueyan; Jia, Shirong; Wang, Zhixing

2014-07-01

107

Optimisation of region-specific reference gene selection and relative gene expression analysis methods for pre-clinical trials of Huntington's disease  

PubMed Central

Background Transcriptional dysregulation is an early, key pathogenic mechanism in Huntington's disease (HD). Therefore, gene expression analyses have biomarker potential for measuring therapeutic efficacy in pre-clinical trials, particularly those aimed at correcting gene expression abnormalities. Housekeeping genes are commonly used as endogenous references in gene expression studies. However, a systematic study comparing the suitability of candidate reference genes for use in HD mouse models has not been performed. To remedy this situation, 12 housekeeping genes were examined to identify suitable reference genes for use in expression assays. Results We found that commonly used reference genes are dysregulated at later time points in the R6/2 mouse model of HD. Therefore, in order to reliably measure gene expression changes for use as pre-clinical trial biomarkers, we set out to identify suitable reference genes for use in R6/2 mice. The expression of potential reference genes was examined in striatum, cortex and cerebellum from 15 week old R6/2 and matched wild-type littermates. Expression levels of candidate reference genes varied according to genotype and brain region. GeNorm software was used to identify the three most stably expressed genes for each brain region. Relative quantification methods using the geometric mean of three reference genes for normalisation enables accurate determination of gene expression levels in wild-type and R6/2 mouse brain regions. Conclusion Our study has identified a reproducible, reliable method by which we able to accurately determine the relative expression level of target genes in specific brain regions, thus increasing the potential of gene expression analysis as a biomarker in HD pre-clinical trials. PMID:18954449

Benn, Caroline L; Fox, Helen; Bates, Gillian P

2008-01-01

108

qPCR analysis of carbon, nitrogen, and arsenic cycling in Zetaproteobacteria-dominated microbial mats  

NASA Astrophysics Data System (ADS)

The recently discovered Zetaproteobacteria represent a novel class of Proteobacteria which oxidize Fe(II) to Fe(III) to fix CO2 at hydrothermal vents. Zetaproteobacteria were first discovered at Lo'ihi Seamount, located 35 km southeast of the big island of Hawai'i and characterized by low-temperature diffuse hydrothermal vents. The hydrothermal vents at Lo'ihi are surrounded by luxuriant iron-rich microbial mats dominated by Zetaproteobacteria. We aim to use real-time quantitative PCR (qPCR) to quantify functional genes associated with the microbial carbon, nitrogen, and arsenic cycles in complex Zetaproteobacteria- dominated iron mat communities. Unique qPCR primer sets have been developed based on Illumina next-generation sequence data from an iron mat collected in 2009 at Lo'ihi. These primers target the sequences for arsenate reductase and nitrite reductase, genes associated with arsenic detoxification and denitrification, respectively. Additionally, we are utilizing published primer sets to quantify genes associated with autotrophic carbon and nitrogen fixation pathways. Genomic DNA was isolated from microbial mats at multiple vent sites with varying temperatures and fluid flow during our 2013 expedition to Lo'ihi. The qPCR data for these samples can be used to draw correlations among fine scale mat structures and nutrient cycling processes across diverse mat morphologies, as previous research has identified unique microbial communities and metabolic strategies associated with distinct mat morphologies. This work will enable us to better identify samples for further molecular analysis, and may provide insights into the evolutionary history and metabolic functionality of various mat morphotypes. We hypothesize that Zetaproteobacteria act as ecosystem engineers, driving the structure and function of iron mat ecosystems.

Jesser, K. J.; Fullerton, H.; Hilton, T. S.; Kimber, J.; Hager, K.; Moyer, C. L.

2013-12-01

109

Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.)  

Microsoft Academic Search

BACKGROUND: Quantitative real-time PCR (qRT-PCR) is currently the most accurate method for detecting differential gene expression. Such an approach depends on the identification of uniformly expressed 'housekeeping genes' (HKGs). Extensive transcriptomic data mining and experimental validation in different model plants have shown that the reliability of these endogenous controls can be influenced by the plant species, growth conditions and organs\\/tissues

Rudy Huis; Simon Hawkins; Godfrey Neutelings

2010-01-01

110

Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.  

PubMed

Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1? for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium. PMID:24810586

Zeng, Shaohua; Liu, Yongliang; Wu, Min; Liu, Xiaomin; Shen, Xiaofei; Liu, Chunzhao; Wang, Ying

2014-01-01

111

Identification and Validation of Reference Genes for Quantitative Real-Time PCR Normalization and Its Applications in Lycium  

PubMed Central

Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1? for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium. PMID:24810586

Zeng, Shaohua; Liu, Yongliang; Wu, Min; Liu, Xiaomin; Shen, Xiaofei; Liu, Chunzhao; Wang, Ying

2014-01-01

112

Novel and sensitive qPCR assays for the detection and identification of aspergillosis causing species.  

PubMed

Despite concerted efforts, diagnosis of aspergillosis is still a great challenge to clinical microbiology laboratories. Along with the requirement for high sensitivity and specificity, species-specific identification is important. We developed rapid, sensitive and species-specific qPCR assays using the TaqMan technology for the detection and identification of Aspergillus fumigatus and Aspergillus terreus. The assays were designed to target orthologs of the Streptomyces factor C gene that are only found in a few species of filamentous fungi. Fungi acquired this gene through horizontal gene transfer and divergence of the gene allows identification of species. The assays have potential as a molecular diagnosis tool for the early detection of fungal infection caused by Aspergillus fumigatus and Aspergillus terreus, which merits future diagnostic studies. The assays were sensitive enough to detect a few genomic equivalents in blood samples. PMID:25261942

Paholcsek, Melinda; Leiter, Eva; Markovics, Arnold; Biró, Sándor

2014-09-01

113

Identification and Validation of Reference Genes for Quantitative Real-Time PCR in Drosophila suzukii (Diptera: Drosophilidae).  

PubMed

To accurately evaluate gene expression levels and obtain more accurate quantitative real-time RT-PCR (qRT-PCR) data, normalization relative to reliable reference gene(s) is required. Drosophila suzukii, is an invasive fruit pest native to East Asia, and recently invaded Europe and North America, the stability of its reference genes have not been previously investigated. In this study, ten candidate reference genes (RPL18, RPS3, AK, EF-1?, TBP, NADH, HSP22, GAPDH, Actin, ?-Tubulin), were evaluated for their suitability as normalization genes under different biotic (developmental stage, tissue and population), and abiotic (photoperiod, temperature) conditions. The three statistical approaches (geNorm, NormFinder and BestKeeper) and one web-based comprehensive tool (RefFinder) were used to normalize analysis of the ten candidate reference genes identified ?-Tubulin, TBP and AK as the most stable candidates, while HSP22 and Actin showed the lowest expression stability. We used three most stable genes (?-Tubulin, TBP and AK) and one unstably expressed gene to analyze the expression of P-glycoprotein in abamectin-resistant and sensitive strains, and the results were similar to reference genes ?-Tubulin, TBP and AK, which show good stability, while the result of HSP22 has a certain bias. The three validated reference genes can be widely used for quantification of target gene expression with qRT-PCR technology in D.suzukii. PMID:25198611

Zhai, Yifan; Lin, Qingcai; Zhou, Xianhong; Zhang, Xiaoyan; Liu, Tingli; Yu, Yi

2014-01-01

114

Optimisation of reference genes for gene-expression analysis in a rabbit model of left ventricular diastolic dysfunction.  

PubMed

Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle's performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n?=?7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n?=?11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD. PMID:24558494

Nachar, Walid; Busseuil, David; Shi, Yanfen; Mihalache-Avram, Teodora; Mecteau, Mélanie; Rhéaume, Eric; Tardif, Jean-Claude

2014-01-01

115

Optimisation of Reference Genes for Gene-Expression Analysis in a Rabbit Model of Left Ventricular Diastolic Dysfunction  

PubMed Central

Left ventricular diastolic dysfunction (LVDD) is characterized by the disturbance of ventricle’s performance due to its abnormal relaxation or to its increased stiffness during the diastolic phase. The molecular mechanisms underlying LVDD remain unknown. We aimed to identify normalization genes for accurate gene-expression analysis of LVDD using quantitative real-time PCR (RT-PCR) in a new rabbit model of LVDD. Eighteen rabbits were fed with a normal diet (n?=?7) or a 0.5% cholesterol-enriched diet supplemented with vitamin D2 (n?=?11) for an average of 14.5 weeks. We validated the presence of LVDD in this model using echocardiography for diastolic function assessment. RT-PCR was performed using cDNA derived from left ventricle samples to measure the stability of 10 genes as candidate reference genes (Gapdh, Hprt1, Ppia, Sdha, Rpl5, Actb, Eef1e1, Ywhaz, Pgk1, and G6pd). Using geNorm analysis, we report that Sdha, Gapdh and Hprt1 genes had the highest stability (M <0.2). By contrast, Hprt1 and Rpl5 genes were found to represent the best combination for normalization when using the Normfinder algorithm (stability value of 0.042). Comparison of both normalization strategies highlighted an increase of natriuretic peptides (Bnp and Anp), monocytes chemotactic protein-1 (Mcp-1) and NADPH oxidase subunit (Nox-2) mRNA expressions in ventricle samples of the hypercholesterolemic rabbits compared to controls (P<0.05). This increase correlates with LVDD echocardiographic parameters and most importantly it molecularly validates the presence of the disease in our model. This is the first study emphasizing the selection of stable reference genes for RT-PCR normalization in a rabbit model of LVDD. PMID:24558494

Nachar, Walid; Busseuil, David; Shi, Yanfen; Mihalache-Avram, Teodora; Mecteau, Melanie; Rheaume, Eric; Tardif, Jean-Claude

2014-01-01

116

Quantification of Frankia in soils using SYBR Green based qPCR.  

PubMed

A SYBR Green based qPCR method was developed for the quantification of clusters 1 and 3 of the actinomycete Frankia in soils. Primer nifHr158 was designed to be used as reverse primer in combination with forward primer nifHf1 specifically amplifying a 191-bp fragment of the nifH gene of these Frankia. The primer combination was tested for specificity on selected pure cultures, and by comparative sequence analyses of randomly selected clones of a clone library generated with these primers from soil DNA extracts. After adjustments of DNA extraction conditions, and the determination of extraction efficiencies used for sample normalization, copy numbers of nifH genes representing Frankia of clusters 1 and 3 were quantified in different mineral soils, resulting in cell density estimates for these Frankia of up to 10(6) cells [g soil {dry weight}](-1) depending on the soil. Despite indications that the nifH gene is not a perfect target for the quantification of Frankia, the qPCR method described here provides a new tool for the quantification and thus a more complete examination of the ecology of Frankia in soils. PMID:22326815

Samant, Suvidha; Sha, Qiong; Iyer, Anita; Dhabekar, Priti; Hahn, Dittmar

2012-05-01

117

Robust RT-qPCR Data Normalization: Validation and Selection of Internal Reference Genes during Post-Experimental Data Analysis  

PubMed Central

Reverse transcription and real-time PCR (RT-qPCR) has been widely used for rapid quantification of relative gene expression. To offset technical confounding variations, stably-expressed internal reference genes are measured simultaneously along with target genes for data normalization. Statistic methods have been developed for reference validation; however normalization of RT-qPCR data still remains arbitrary due to pre-experimental determination of particular reference genes. To establish a method for determination of the most stable normalizing factor (NF) across samples for robust data normalization, we measured the expression of 20 candidate reference genes and 7 target genes in 15 Drosophila head cDNA samples using RT-qPCR. The 20 reference genes exhibit sample-specific variation in their expression stability. Unexpectedly the NF variation across samples does not exhibit a continuous decrease with pairwise inclusion of more reference genes, suggesting that either too few or too many reference genes may detriment the robustness of data normalization. The optimal number of reference genes predicted by the minimal and most stable NF variation differs greatly from 1 to more than 10 based on particular sample sets. We also found that GstD1, InR and Hsp70 expression exhibits an age-dependent increase in fly heads; however their relative expression levels are significantly affected by NF using different numbers of reference genes. Due to highly dependent on actual data, RT-qPCR reference genes thus have to be validated and selected at post-experimental data analysis stage rather than by pre-experimental determination. PMID:21423626

Ling, Daijun; Salvaterra, Paul M.

2011-01-01

118

Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae)  

PubMed Central

Background Quantitative real-time reverse transcriptase PCR (RT-qPCR) has been widely used for quantification of mRNA as a way to determine key genes involved in different biological processes. For accurate gene quantification analysis, normalization of RT-qPCR data is absolutely essential. To date, normalization is most frequently achieved by the use of internal controls, often referred to as reference genes. However, several studies have shown that the reference genes used for the quantification of mRNA expression can be affected by the experimental set-up or cell type resulting in variation of the expression level of these key genes. Therefore, the evaluation of reference genes is critical for gene expression profiling, which is often neglected in gene expression studies of insects. For this purpose, ten candidate reference genes were investigated in three different tissues (midgut, Malpighian tubules, and fat body) of the oriental fruit fly, Bactrocera dorsalis (Hendel). Results Two different programs, geNorm and Normfinder, were used to analyze the data. According to geNorm, ?-TUB + ACT5 are the most appropriate reference genes for gene expression profiling across the three different tissues in the female flies, while ACT3 + ?-TUB are considered as the best for males. Furthermore, we evaluated the stability of the candidate reference genes to determine the sexual differences in the same tissue. In the midgut and Malpighian tubules, ACT2 + ?-TUB are the best choice for both males and females. However, ?-TUB + ACT1 are the best pair for fat body. Meanwhile, the results calculated by Normfinder are quite the same as the results with geNorm; ?-TUB is always one of the most stable genes in each sample validated by the two programs. Conclusions In this study, we validated the suitable reference genes for gene expression profiling in different tissues of B. dorsalis. Moreover, appropriate reference genes were selected out for gene expression profiling of the same tissues taking the sexual differences into consideration. This work not only formed a solid basis for future gene expression study in B. dorsalis, but also will serve as a resource to screen reference genes for gene expression studies in any other insects. PMID:20923571

2010-01-01

119

SYBR ® Green qPCR methods for detection of endogenous reference genes in commodity crops: a step ahead in combinatory screening of genetically modified crops in food and feed products  

Microsoft Academic Search

Identification of crops present in food and\\/or feed matrices represents an important step in the screening strategies targeting\\u000a genetically modified organisms (GMO). Soybean, maize, oilseed rape, rice, cotton, sugar beet and potato are to date the most\\u000a important sources of genetically modified materials imported in the European Union (EU). In order to allow detection of their\\u000a presence in an integrated

E. Guillaume Mbongolo Mbella; Antoon Lievens; Elodie Barbau-Piednoir; Myriam Sneyers; Amaya Leunda-Casi; Nancy Roosens; Marc Van den Bulcke

2011-01-01

120

Selection and Validation of Reference Genes for Gene Expression Analysis in Switchgrass (Panicum virgatum) Using Quantitative Real-Time RT-PCR  

PubMed Central

Switchgrass (Panicum virgatum) has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1?, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1?, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass. PMID:24621568

Gimeno, Jacinta; Eattock, Nicholas; Van Deynze, Allen; Blumwald, Eduardo

2014-01-01

121

Selection and validation of reference genes for gene expression analysis in switchgrass (Panicum virgatum) using quantitative real-time RT-PCR.  

PubMed

Switchgrass (Panicum virgatum) has received a lot of attention as a forage and bioenergy crop during the past few years. Gene expression studies are in progress to improve new traits and develop new cultivars. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to study gene expression analysis. For accurate and reliable results, normalization of data with reference genes is essential. In this work, we evaluate the stability of expression of genes to use as reference for qRT-PCR in the grass P. virgatum. Eleven candidate reference genes, including eEF-1?, UBQ6, ACT12, TUB6, eIF-4a, GAPDH, SAMDC, TUA6, CYP5, U2AF, and FTSH4, were validated for qRT-PCR normalization in different plant tissues and under different stress conditions. The expression stability of these genes was verified by the use of two distinct algorithms, geNorm and NormFinder. Differences were observed after comparison of the ranking of the candidate reference genes identified by both programs but eEF-1?, eIF-4a, CYP5 and U2AF are ranked as the most stable genes in the samples sets under study. Both programs discard the use of SAMDC and TUA6 for normalization. Validation of the reference genes proposed by geNorm and NormFinder were performed by normalization of transcript abundance of a group of target genes in different samples. Results show similar expression patterns when the best reference genes selected by both programs were used but differences were detected in the transcript abundance of the target genes. Based on the above research, we recommend the use of different statistical algorithms to identify the best reference genes for expression data normalization. The best genes selected in this study will help to improve the quality of gene expression data in a wide variety of samples in switchgrass. PMID:24621568

Gimeno, Jacinta; Eattock, Nicholas; Van Deynze, Allen; Blumwald, Eduardo

2014-01-01

122

Reference gene selection for real-time RT-PCR normalization in rice field eel (Monopterus albus) during gonad development.  

PubMed

Real-time reverse transcriptase (RT) polymerase chain reaction (PCR) requires data normalization using an appropriate reference gene in order to obtain more reliable results with biological significance. We cloned a partial sequence of elongation factor-1-? (EF1?) and ribosomal protein L17 (RPL17) from Monopterus albus. We investigated the suitability of five commonly used reference genes [18S ribosomal RNA (18S), cytoskeletal protein (?-actin), glyceraldehyde phosphate dehydrogenase (GAPDH), EF1? and RPL17] as potential quantitative reference genes for normalizing real-time RT-PCR data generated in gonads of different developmental stages and in other tissues of M. albus. Analysis of the data indicated that 18S, ?-actin and GAPDH are not suitable as reference genes because of their levels of variations of expression. EF1? and RPL17 might be suitable as reference genes in the gonads of different developmental stages as well as in other tissues of M. albus. PMID:25079246

Hu, Qing; Guo, Wei; Gao, Yu; Tang, Rong; Li, Dapeng

2014-12-01

123

Evaluation of Candidate Reference Genes for Normalization of Quantitative RT-PCR in Soybean Tissues under Various Abiotic Stress Conditions  

PubMed Central

Quantitative RT-PCR can be a very sensitive and powerful technique for measuring differential gene expression. Changes in gene expression induced by abiotic stresses are complex and multifaceted, which make determining stably expressed genes for data normalization difficult. To identify the most suitable reference genes for abiotic stress studies in soybean, 13 candidate genes collected from literature were evaluated for stability of expression under dehydration, high salinity, cold and ABA (abscisic acid) treatments using delta CT and geNorm approaches. Validation of reference genes indicated that the best reference genes are tissue- and stress-dependent. With respect to dehydration treatment, the Fbox/ABC, Fbox/60s gene pairs were found to have the highest expression stability in the root and shoot tissues of soybean seedlings, respectively. Fbox and 60s genes are the most suitable reference genes across dehydrated root and shoot tissues. Under salt stress the ELF1b/IDE and Fbox/ELF1b are the most stably expressed gene pairs in roots and shoots, respectively, while 60s/Fbox is the best gene pair in both tissues. For studying cold stress in roots or shoots, IDE/60s and Fbox/Act27 are good reference gene pairs, respectively. With regard to gene expression analysis under ABA treatment in either roots, shoots or across these tissues, 60s/ELF1b, ELF1b/Fbox and 60s/ELF1b are the most suitable reference genes, respectively. The expression of ELF1b/60s, 60s/Fbox and 60s/Fbox genes was most stable in roots, shoots and both tissues, respectively, under various stresses studied. Among the genes tested, 60s was found to be the best reference gene in different tissues and under various stress conditions. The highly ranked reference genes identified from this study were proved to be capable of detecting subtle differences in expression rates that otherwise would be missed if a less stable reference gene was used. PMID:23029532

Le, Dung Tien; Watanabe, Yasuko; Ha, Chien Van; Nishiyama, Rie; Guttikonda, Satish K.; Quach, Truyen N.; Gutierrez-Gonzalez, Juan J.; Tran, Lam-Son Phan; Nguyen, Henry T.

2012-01-01

124

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp.  

PubMed

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species. PMID:25249772

Mo, Fei; Zhao, Jie; Liu, Na; Cao, Li-Hua; Jiang, Shan-Xiang

2014-09-01

125

Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp  

PubMed Central

Reference genes are commonly used for normalization of target gene expression during RT-qPCR analysis. However, no housekeeping genes or reference genes have been identified to be stable across different tissue types or under different experimental conditions. To identify the most suitable reference genes for RT-qPCR analysis of target gene expression in the hepatopancreas of crucian carp (Carassius auratus) under various conditions (sex, age, water temperature, and drug treatments), seven reference genes, including beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), alpha tubulin (TUBA), ribosomal protein l8 (RPL8) and glucose-6-phosphate dehydrogenase (G6PDH), were evaluated in this study. The stability and ranking of gene expression were analyzed using three different statistical programs: GeNorm, Normfinder and Bestkeeper. The expression errors associated with selection of the genes were assessed by the relative quantity of CYP4T. The results indicated that all the seven genes exhibited variability under the experimental conditions of this research, and the combination of ACTB/TUBA/EEF1A or of ACTB/EEF1A was the best candidate that raised the accuracy of quantitative analysis of gene expression. The findings highlighted the importance of validation of housekeeping genes for research on gene expression under different conditions of experiment and species. PMID:25249772

Mo, Fei; Zhao, Jie; Liu, Na; Cao, Li-hua; Jiang, Shan-xiang

2014-01-01

126

Physical lysis only (PLO) methods suitable as rapid sample pretreatment for qPCR assay.  

PubMed

Quantitative PCR (qPCR) enables rapid and sensitive gene quantification and is widely used in genomics, such as biological, medical, environmental, and food sciences. However, sample pretreatment requires the use of conventional DNA extraction kits which are time-consuming and labor intensive. In this study, we investigated four physical lysis only (PLO) methods which are rapid and could serve as alternatives to conventional DNA extraction kits. These PLO methods are bead mill, heating, sonication, and freeze-thaw. Using ethidium bromide-based assay, their performance was evaluated and compared. The effects of cell debris and its removal were also investigated. Bead mill method without cell debris removal appeared to yield the best qPCR results among the four PLO methods. In addition, bead mill method also performed better than conventional DNA extraction kits. It is probably due to the substantial loss of DNA material during the extensive purification of the conventional DNA extraction kits. The bead mill method has been demonstrated to successfully quantify 10(2) to 10(7) copies of the PAH-RHD? gene of Pseudomonas putida. PMID:25219535

Wang, Xiaofang; Lee, Byung-Tae; Son, Ahjeong

2014-10-01

127

Evaluation and Validation of Reference Genes for qRT-PCR Normalization in Frankliniella occidentalis (Thysanoptera:Thripidae)  

PubMed Central

Quantitative real time PCR (qRT-PCR) has emerged as a reliable and reproducible technique for studying gene expression analysis. For accurate results, the normalization of data with reference genes is particularly essential. Once the transcriptome sequencing of Frankliniella occidentalis was completed, numerous unigenes were identified and annotated. Unfortunately, there are no studies on the stability of reference genes used in F. occidentalis. In this work, seven candidate reference genes, including actin, 18S rRNA, H3, tubulin, GAPDH, EF-1 and RPL32, were evaluated for their suitability as normalization genes under different experimental conditions using the statistical software programs BestKeeper, geNorm, Normfinder and the comparative ?Ct method. Because the rankings of the reference genes provided by each of the four programs were different, we chose a user-friendly web-based comprehensive tool RefFinder to get the final ranking. The result demonstrated that EF-1 and RPL32 displayed the most stable expression in different developmental stages; RPL32 and GAPDH showed the most stable expression at high temperatures, while 18S and EF-1 exhibited the most stable expression at low temperatures. In this study, we validated the suitable reference genes in F. occidentalis for gene expression profiling under different experimental conditions. The choice of internal standard is very important in the normalization of the target gene expression levels, thus validating and selecting the best genes will help improve the quality of gene expression data of F. occidentalis. What is more, these validated reference genes could serve as the basis for the selection of candidate reference genes in other insects. PMID:25356721

Zheng, Yu-Tao; Li, Hong-Bo; Lu, Ming-Xing; Du, Yu-Zhou

2014-01-01

128

Reference Gene Selection for qRT-PCR Analysis in the Sweetpotato Whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae)  

PubMed Central

Background Accurate evaluation of gene expression requires normalization relative to the expression of reliable reference genes. Expression levels of “classical” reference genes can differ, however, across experimental conditions. Although quantitative real-time PCR (qRT-PCR) has been used extensively to decipher gene function in the sweetpotato whitefly Bemisia tabaci, a world-wide pest in many agricultural systems, the stability of its reference genes has rarely been validated. Results In this study, 15 candidate reference genes from B. tabaci were evaluated using two Excel-based algorithms geNorm and Normfinder under a diverse set of biotic and abiotic conditions. At least two reference genes were selected to normalize gene expressions in B. tabaci under experimental conditions. Specifically, for biotic conditions including host plant, acquisition of a plant virus, developmental stage, tissue (body region of the adult), and whitefly biotype, ribosomal protein L29 was the most stable reference gene. In contrast, the expression of elongation factor 1 alpha, peptidylprolyl isomerase A, NADH dehydrogenase, succinate dehydrogenase complex subunit A and heat shock protein 40 were consistently stable across various abiotic conditions including photoperiod, temperature, and insecticide susceptibility. Conclusion Our finding is the first step toward establishing a standardized quantitative real-time PCR procedure following the MIQE (Minimum Information for publication of Quantitative real time PCR Experiments) guideline in an agriculturally important insect pest, and provides a solid foundation for future RNA interference based functional study in B. tabaci. PMID:23308130

Li, Rumei; Xie, Wen; Wang, Shaoli; Wu, Qingjun; Yang, Nina; Yang, Xin; Pan, Huipeng; Zhou, Xiaomao; Bai, Lianyang; Xu, Baoyun; Zhou, Xuguo; Zhang, Youjun

2013-01-01

129

Selection of New Appropriate Reference Genes for RT-qPCR Analysis via Transcriptome Sequencing of Cynomolgus Monkeys (Macaca fascicularis)  

PubMed Central

In the investigation of the expression levels of target genes, reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is the most accurate and widely used method. However, a normalization step is a prerequisite to obtain accurate quantification results from RT-qPCR data. Therefore, many studies regarding the selection of reference genes have been carried out. Recently, these studies have involved large-scale gene analysis methods such as microarray and next generation sequencing. In our previous studies, we analyzed large amounts of transcriptome data from the cynomolgus monkey. Using a modification of this large-scale transcriptome sequencing dataset, we selected and compared 12 novel candidate reference genes (ARFGAP2, ARL1, BMI1, CASC3, DDX3X, MRFAP1, ORMDL1, RSL24D1, SAR1A, USP22, ZC3H11A, and ZRANB2) and 4 traditionally used reference genes (ACTB, GAPDH, RPS19, and YWHAZ) in 13 different whole-body tissues by the 3 well-known programs geNorm, NormFinder, and BestKeeper. Combined analysis by these 3 programs showed that ADP-ribosylation factor GTPase activating protein 2 (ARFGAP2), morf4 family associated protein 1 (MRFAP1), and ADP-ribosylation factor-like 1 (ARL1) are the most appropriate reference genes for accurate normalization. Interestingly, 4 traditionally used reference genes were the least stably expressed in this study. For this reason, selection of appropriate reference genes is vitally important, and large-scale analysis is a good method for finding new candidate reference genes. Our results could provide reliable reference gene lists for future studies on the expression of various target genes in the cynomolgus monkey. PMID:23613744

Lee, Sang-Rae; Kim, Sang-Hyun; Kim, Sun-Uk; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Kyoung-Min; Kim, Heui-Soo; Chang, Kyu-Tae

2013-01-01

130

Evaluation of reference genes for real-time quantitative PCR studies in Candida glabrata following azole treatment  

PubMed Central

Background The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata. Results The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1?, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2-??CT method and three other software packages. The stability rankings of the reference genes by geNorm and the 2-??CT method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments. Conclusions We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model. PMID:22747760

2012-01-01

131

Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions  

PubMed Central

Background Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied. Methodology/Principal Findings In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments. Conclusions/Significance These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies. PMID:24699822

Jiang, Xiaomei; Yin, Guohua; Zhang, Xinquan; Qi, Xiao; Zhang, Yu; Yan, Yanhong; Ma, Xiao; Peng, Yan

2014-01-01

132

Selection and Validation of Endogenous Reference Genes for qRT-PCR Analysis in Leafy Spurge (Euphorbia esula)  

PubMed Central

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis “general purpose” traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the CT values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ?CT method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species. PMID:22916167

Chao, Wun S.; Dogramaci, Munevver; Foley, Michael E.; Horvath, David P.; Anderson, James V.

2012-01-01

133

Selection and validation of endogenous reference genes for qRT-PCR analysis in leafy spurge (Euphorbia esula).  

PubMed

Quantitative real-time polymerase chain reaction (qRT-PCR) is the most important tool in measuring levels of gene expression due to its accuracy, specificity, and sensitivity. However, the accuracy of qRT-PCR analysis strongly depends on transcript normalization using stably expressed reference genes. The aim of this study was to find internal reference genes for qRT-PCR analysis in various experimental conditions for seed, adventitious underground bud, and other organs of leafy spurge. Eleven candidate reference genes (BAM4, PU1, TRP-like, FRO1, ORE9, BAM1, SEU, ARF2, KAPP, ZTL, and MPK4) were selected from among 171 genes based on expression stabilities during seed germination and bud growth. The other ten candidate reference genes were selected from three different sources: (1) 3 stably expressed leafy spurge genes (60S, bZIP21, and MD-100) identified from the analyses of leafy spurge microarray data; (2) 3 orthologs of Arabidopsis "general purpose" traditional reference genes (GAPDH_1, GAPDH_2, and UBC); and (3) 4 orthologs of Arabidopsis stably expressed genes (UBC9, SAND, PTB, and F-box) identified from Affymetrix ATH1 whole-genome GeneChip studies. The expression stabilities of these 21 genes were ranked based on the C(T) values of 72 samples using four different computation programs including geNorm, Normfinder, BestKeeper, and the comparative ?C(T) method. Our analyses revealed SAND, PTB, ORE9, and ARF2 to be the most appropriate reference genes for accurate normalization of gene expression data. Since SAND and PTB were obtained from 4 orthologs of Arabidopsis, while ORE9 and ARF2 were selected from 171 leafy spurge genes, it was more efficient to identify good reference genes from the orthologs of other plant species that were known to be stably expressed than that of randomly testing endogenous genes. Nevertheless, the two newly identified leafy spurge genes, ORE9 and ARF2, can serve as orthologous candidates in the search for reference genes from other plant species. PMID:22916167

Chao, Wun S; Do?ramaci, Münevver; Foley, Michael E; Horvath, David P; Anderson, James V

2012-01-01

134

Selection of reference genes for quantitative reverse-transcription polymerase chain reaction normalization in Brassica napus under various stress conditions.  

PubMed

Data normalization is essential for reliable output of quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assays, as the unsuitable choice of reference gene(s), whose expression might be influenced by exogenous treatments in plant tissues, could cause misinterpretation of results. To date, no systematic studies on reference genes have been performed in stressed Brassica napus. In this study, we investigated the expression variations of nine candidate reference genes in 40 samples of B. napus leaves subjected to various exogenous treatments. Parallel analyses by geNorm and NormFinder revealed that optimal reference genes differed across the different sets of samples. The best-ranked reference genes were PP2A and TIP41 for salt stress, TIP41 and ACT7 for heavy metal (Cr(6+)) stress, PP2A and UBC21 for drought stress, F-box and SAND for cold stress, F-box and ZNF for salicylic acid stress, TIP41, ACT7, and PP2A for methyl jasmonate stress, TIP41 and ACT7 for abscisic acid stress, and TIP41, UBC21, and PP2A for Sclerotinia sclerotiorum stress. Two newly employed reference genes, TIP41 and PP2A, showed better performances, suggesting their suitability in multiple conditions. To further validate the suitability of the reference genes, the expression patterns of BnWRKY40 and BnMKS1 were studied in parallel. This study is the first systematic analysis of reference gene selection for qRT-PCR normalization in B. napus, an agriculturally important crop, under different stress conditions. The results will contribute toward more accurate and widespread use of qRT-PCR in gene analysis of the genus Brassica. PMID:24770781

Wang, Zheng; Chen, Yu; Fang, Hedi; Shi, Haifeng; Chen, Keping; Zhang, Zhiyan; Tan, Xiaoli

2014-10-01

135

Methamphetamine alters reference gene expression in nigra and striatum of adult rat brain.  

PubMed

The nigrostriatal dopaminergic system is a major lesion target for methamphetamine (MA), one of the most addictive and neurotoxic drugs of abuse. High doses of MA alter the expression of a large number of genes. Reference genes (RGs) are considered relatively stable and are often used as standards for quantitative real-time PCR (qRT-PCR) reactions. The purpose of this study was to determine whether MA altered the expression of RGs and to identify the appropriate RGs for gene expression studies in animals receiving MA. Adult male Sprague-Dawley rats were treated with high doses of MA or saline. Striatum and substantia nigra were harvested at 2h or 24h after MA administration. The expression and stability of 10 commonly used RGs were examined using qRT-PCR and then evaluated by geNorm and Normfinder. We found that MA altered the expression of selected RGs. These candidate RGs presented differential stability in the striatum and in substantia nigra at both 2h and 24h after MA injection. Selection of an unstable RG as a standard altered the significance of tyrosine hydroxylase (TH) mRNA expression after MA administration. In conclusion, our data show that MA site- and time-dependently altered the expression of RGs in nigrostriatal dopaminergic system. These temporal and spatial factors should be considered when selecting appropriate RGs for interpreting the expression of target genes in animals receiving MA. PMID:24042092

He, Yi; Yu, Seongjin; Bae, Eunkyung; Shen, Hui; Wang, Yun

2013-12-01

136

Validation of Novel Reference Genes for Reverse Transcription Quantitative Real-Time PCR in Drought-Stressed Sugarcane  

PubMed Central

One of the most challenging aspects of RT-qPCR data analysis is the identification of reliable reference genes. Ideally, they should be neither induced nor repressed under different experimental conditions. To date, few reference genes have been adequately studied for sugarcane (Saccharum spp.) using statistical approaches. In this work, six candidate genes (?TUB, GAPDH, H1, SAMDC, UBQ, and 25S rRNA) were tested for gene expression normalization of sugarcane root tissues from drought-tolerant and -sensitive accessions after continuous dehydration (24?h). By undergoing different approaches (GeNorm, NormFinder, and BestKeeper), it was shown that most of them could be used in combinations for normalization purposes, with the exception of SAMDC. Nevertheless three of them (H1, ?TUB, and GAPDH) were considered the most reliable reference genes. Their suitability as reference genes validated the expression profiles of two targets (AS and PFP?1), related to SuperSAGE unitags, in agreement with results revealed by previous in silico analysis. The other two sugarcane unitags (ACC oxidase and PIP1-1), after salt stress (100?mM NaCl), presented their expressions validated in the same way. In conclusion, these reference genes will be useful for dissecting gene expression in sugarcane roots under abiotic stress, especially in transcriptomic studies using SuperSAGE or RNAseq approaches. PMID:24987730

Silva, Roberta Lane de Oliveira; Silva, Manasses Daniel; Ferreira Neto, Jose Ribamar Costa; de Nardi, Claudia Huerta; Chabregas, Sabrina Moutinho; Burnquist, William Lee; Kahl, Gunter; Benko-Iseppon, Ana Maria; Kido, Ederson Akio

2014-01-01

137

Evaluation of Four Endogenous Reference Genes and Their Real-Time PCR Assays for Common Wheat Quantification in GMOs Detection  

PubMed Central

Proper selection of endogenous reference genes and their real-time PCR assays is quite important in genetically modified organisms (GMOs) detection. To find a suitable endogenous reference gene and its real-time PCR assay for common wheat (Triticum aestivum L.) DNA content or copy number quantification, four previously reported wheat endogenous reference genes and their real-time PCR assays were comprehensively evaluated for the target gene sequence variation and their real-time PCR performance among 37 common wheat lines. Three SNPs were observed in the PKABA1 and ALMT1 genes, and these SNPs significantly decreased the efficiency of real-time PCR amplification. GeNorm analysis of the real-time PCR performance of each gene among common wheat lines showed that the Waxy-D1 assay had the lowest M values with the best stability among all tested lines. All results indicated that the Waxy-D1 gene and its real-time PCR assay were most suitable to be used as an endogenous reference gene for common wheat DNA content quantification. The validated Waxy-D1 gene assay will be useful in establishing accurate and creditable qualitative and quantitative PCR analysis of GM wheat. PMID:24098735

Huang, Huali; Cheng, Fang; Wang, Ruoan; Zhang, Dabing; Yang, Litao

2013-01-01

138

Reference Genes Selection for Quantitative Real-Time PCR Using RankAggreg Method in Different Tissues of Capra hircus  

PubMed Central

Identification of reference genes with stable levels of gene expression is an important prerequisite for obtaining reliable results in analysis of gene expression data using quantitative real time PCR (RT-qPCR). Since the underlying assumption of reference genes is that expressed at the exact same level in all sample types, in this study, we evaluated the expression stability of nine most commonly used endogenous controls (GAPDH, ACTB, 18S rRNA, RPS18, HSP-90, ALAS, HMBS, ACAC, and B2M) in four different tissues of the domestic goat, Capra hircus, including liver, visceral, subcutaneous fat and longissimus muscles, across different experimental treatments (a standard diet prepared using the NRC computer software as control and the same diet plus one mg chromium/day). We used six different software programs for ranking of reference genes and found that individual rankings of the genes differed among them. Additionally, there was a significant difference in ranking patterns of the studied genes among different tissues. A rank aggregation method was applied to combine the ranking lists of the six programs to a consensus ranking. Our results revealed that HSP-90 was nearly always among the two most stable genes in all studied tissues. Therefore, it is recommended for accurate normalization of RT-qPCR data in goats, while GAPDH, ACTB, and RPS18 showed the most varied expressions and should be avoided as reference genes. PMID:24358246

Najafpanah, Mohammad Javad; Sadeghi, Mostafa; Bakhtiarizadeh, Mohammad Reza

2013-01-01

139

Selection of reference genes for qRT-PCR in high fat diet-induced hepatic steatosis mice model.  

PubMed

With the epidemic proportions of obesity worldwide and the concurrent prevalence of hepatic steatosis, there is an urgent need for better understanding the intrinsic mechanism of hepatic steatosis, especially the changes of gene expression underlying the development of hepatic steatosis and its associated abnormal liver function. Quantitative real-time PCR (qRT-PCR) is a sensitive and highly reproducible technique of gene expression analysis. However, for accurate and reliable gene expression results, it is vital to have an internal control gene expressed at constant levels under all the experimental conditions being analyzed for. In this study, the authors validated candidate reference genes suitable for qRT-PCR profiling experiments using livers from control mice and high fat diet-induced obese mice. Cross-validation of expression stability of ten selected reference genes using three popular algorithms, GeNorm, NormFinder, and BestKeeper found HPRT1 and GAPDH as most stable reference genes. Thus, HPRT1 and GAPDH are recommended as stable reference genes most suitable for gene expression studies in the development of hepatic steatosis. PMID:21184202

Xu, Lingyan; Ma, Xinran; Cui, Bin; Li, Xiaoying; Ning, Guang; Wang, Shu

2011-07-01

140

Validation of Reference Genes Aiming Accurate Normalization of qRT-PCR Data in Dendrocalamus latiflorus Munro  

PubMed Central

Background Dendrocalamus latiflorus Munro distributes widely in subtropical areas and plays vital roles as valuable natural resources. The transcriptome sequencing for D. latiflorus Munro has been performed and numerous genes especially those predicted to be unique to D. latiflorus Munro were revealed. qRT-PCR has become a feasible approach to uncover gene expression profiling, and the accuracy and reliability of the results obtained depends upon the proper selection of stable reference genes for accurate normalization. Therefore, a set of suitable internal controls should be validated for D. latiflorus Munro. Results In this report, twelve candidate reference genes were selected and the assessment of gene expression stability was performed in ten tissue samples and four leaf samples from seedlings and anther-regenerated plants of different ploidy. The PCR amplification efficiency was estimated, and the candidate genes were ranked according to their expression stability using three software packages: geNorm, NormFinder and Bestkeeper. GAPDH and EF1? were characterized to be the most stable genes among different tissues or in all the sample pools, while CYP showed low expression stability. RPL3 had the optimal performance among four leaf samples. The application of verified reference genes was illustrated by analyzing ferritin and laccase expression profiles among different experimental sets. The analysis revealed the biological variation in ferritin and laccase transcript expression among the tissues studied and the individual plants. Conclusions geNorm, NormFinder, and BestKeeper analyses recommended different suitable reference gene(s) for normalization according to the experimental sets. GAPDH and EF1? had the highest expression stability across different tissues and RPL3 for the other sample set. This study emphasizes the importance of validating superior reference genes for qRT-PCR analysis to accurately normalize gene expression of D. latiflorus Munro. PMID:24498321

Liu, Mingying; Jiang, Jing; Han, Xiaojiao; Qiao, Guirong; Zhuo, Renying

2014-01-01

141

Selection and validation of reference genes for quantitative gene expression studies by real-time PCR in eggplant (Solanum melongena L)  

PubMed Central

Background Analysis of gene expression patterns leads to functional understanding of biological processes. Quantitative real-time PCR has become the most commonly used technique for in-depth studies of gene expression. To quantify variation in specific gene expression, accurate and reliable normalization across different samples and tissues is necessary. This can be achieved by selecting one or more suitable reference genes to compare the target mRNA transcript levels. In the present work, we illustrate the first evaluation of potential internal control or reference genes across different developmental stages of eggplant for reliable quantification of transcripts by real-time PCR. Results We have evaluated the stability in expression of six candidate reference genes (18s rRNA, APRT, GAPDH, Cyclophilin, Actin, and RuBP) in a set of tissues representing six developmental stages of eggplant. The candidate genes were cloned from cDNA and analysed by real-time PCR. The expression data analyzed by three statistical methods (geNorm, NormFinder and BestKeeper) identified 18s rRNA, Cyclophilin and APRT as the most stable and suitable reference genes in eggplant. This was further confirmed in four different varieties, two representative lines of transgenic eggplant as well as in nematode infected eggplant. Conclusion 18s rRNA, Cyclophilin and APRT have been found to be appropriate for the normalization of real-time PCR data for gene expression studies in eggplant. PMID:23919495

2013-01-01

142

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)  

PubMed Central

Background Teak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak. Results Identification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), ?-Tubulin (?-Tub), ubiquitin (Ubq), elongation factor 1-? (Ef-1?), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1? are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1? as internal controls. These analyses exposed a consistent expression pattern with both reference genes. Conclusion This study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1? have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided. PMID:25048176

2014-01-01

143

Reference genes for RT-qPCR studies in Corynebacterium pseudotuberculosis identified through analysis of RNA-seq data.  

PubMed

Reference genes presenting stable expression profiles over a wide variety of conditions are required in relative expression studies of specific bacterial genes by quantitative reverse transcription PCR (RT-qPCR). High-throughput sequencing of bacterial transcriptomes using the RNA-seq methodology now provides a wealth of data that may be searched for identification of the most stably expressed genes of a given bacterium. Herein, we searched a RNA-seq dataset from various experiments with the pathogenic bacterium Corynebacterium pseudotuberculosis, grown under different stress conditions, in order to select appropriate candidate reference genes for this species. Nineteen genes involved in maintenance of basic cellular functions, so-called housekeeping genes, were chosen for study and their expression profiles in C. pseudotuberculosis were evaluated throughout all growth conditions. Eight of these genes (atpA, dnaG, efp, fusA, gyrA, gyrB, rpoB, and rpoC), mostly participating in DNA replication and transcription, matched the defined criteria to be included as candidate reference genes. Transcriptional levels of these genes were quantified by RT-qPCR assays after growth of C. pseudotuberculosis under two additional conditions. Expression stability analysis by NormFinder indicated the combination of genes encoding DNA gyrase subunit A (gyrA) and elongation factor P (fusA) as the most suitable for normalization of RT-qPCR studies in C. pseudotuberculosis. PMID:25017489

Carvalho, Daiane M; de Sá, Pablo H; Castro, Thiago L P; Carvalho, Rodrigo D; Pinto, Anne; Gil, Danilo J P; Bagano, Priscilla; Bastos, Bruno; Costa, Lilia F M; Meyer, Roberto; Silva, Artur; Azevedo, Vasco; Ramos, Rommel T J; Pacheco, Luis G C

2014-10-01

144

Reference Genes to Study Herbicide Stress Response in Lolium sp.: Up-Regulation of P450 Genes in Plants Resistant to Acetolactate-Synthase Inhibitors  

PubMed Central

Variation in the expression of numerous genes is at the basis of plant response to environmental stresses. Non-target-site-based resistance to herbicides (NTSR), the major threat to grass weed chemical control, is governed by a subset of the genes involved in herbicide stress response. Quantitative PCR assays allowing reliable comparison of gene expression are thus key to identify genes governing NTSR. This work aimed at identifying a set of reference genes with a stable expression to be used as an internal standard for the normalisation of quantitative PCR data in studies investigating NTSR to herbicides inhibiting acetolactate synthase (ALS) in the major grass weed Lolium sp. Gene expression stability was assessed in plants resistant or sensitive to two ALS inhibitors, subjected or not to herbicide stress. Using three complementary approaches implemented in the programs BestKeeper, NormFinder and geNorm, cap-binding protein, glyceraldehyde-3-phosphate-dehydrogenase and ubiquitin were identified as the most suitable reference genes. This reference gene set can probably be used to study herbicide response in other weed species. It was used to compare the expression of the genes encoding two herbicide target enzymes (ALS and acetyl-coenzyme A carboxylase) and five cytochromes P450 (CYP) with potential herbicide-degrading activity between plants resistant or sensitive to ALS inhibitors. Overall, herbicide application enhanced CYP gene expression. Constitutive up-regulation of all CYP genes observed in resistant plants compared to sensitive plants suggested enhanced secondary metabolism in the resistant plants. Comprehensive transcriptome studies associated to gene expression analyses using the reference gene set validated here are required to unravel NTSR genetic determinants. PMID:23696834

Duhoux, Arnaud; Delye, Christophe

2013-01-01

145

Identification and evaluation of suitable reference genes for gene expression studies in the whitefly Bemisia tabaci (Asia I) by reverse transcription quantitative realtime PCR.  

PubMed

This study presents a reliable method for performing reverse transcription quantitative realtime PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1? (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and ?-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data pre- sented here will assist future transcript profiling studies in whiteflies. PMID:25373210

Collins, Carl; Patel, Mitulkumar V; Colvin, John; Bailey, David; Seal, Susan

2014-01-01

146

Identification and Evaluation of Suitable Reference Genes for Gene Expression Studies in the Whitefly Bemisia tabaci (Asia I) by Reverse Transcription Quantitative Real-Time PCR  

PubMed Central

This study presents a reliable method for performing reverse transcription quantitative real-time PCR (RT-qPCR) to measure gene expression in the whitefly Bemisia tabaci (Asia I) (Gennadius) (Hemiptera: Aleyrodidae), utilising suitable reference genes for data normalisation. We identified orthologs of commonly used reference genes (actin (ACT), cyclophilin 1 (CYP1), elongation factor 1? (EF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ribosomal protein L13a (RPL13A), and ?-tubulin (TUB1A)), measured the levels of their transcripts by RT-qPCR during development and in response to thermal stress, and evaluated their suitability as endogenous controls using geNorm, BestKeeper, and NormFinder programs. Overall, TUB1A, RPL13A, and CYP1 were the most stable reference genes during B. tabaci development, and TUB1A, GAPDH, and RPL13A were the most stable reference genes in the context of thermal stress. An analysis of the effects of reference gene choice on the transcript profile of a developmentally-regulated gene encoding vitellogenin demonstrated the importance of selecting the correct endogenous controls for RT-qPCR studies. We propose the use of TUB1A, RPL13A, and CYP1 as endogenous controls for transcript profiling studies of B. tabaci development, whereas the combination of TUB1A, GAPDH, and RPL13A should be employed for studies into thermal stress. The data presented here will assist future transcript profiling studies in whiteflies. PMID:25373210

Collins, Carl; Patel, Mitulkumar V.; Colvin, John; Bailey, David; Seal, Susan

2014-01-01

147

Validation of a Tomato-Specific Gene, LAT52, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Transgenic Tomatoes  

Microsoft Academic Search

Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products

Litao Yang; AIHU PAN; JUNWEI JIA; JIAYU DING; JIANXIU CHEN; Cheng Huang; CHENGMEI ZHANG; Da-Bing Zhang

2005-01-01

148

Autonomous in-situ qPCR in the Deep Sea  

NASA Astrophysics Data System (ADS)

We are developing an instrument to autonomously detect microbial genes that mediate biogeochemical transformations in the deep sea as a step towards developing the scientific and technical capability for searching for life on other planets. This device, known as the Deep-sea Environmental Sample Processor (D-ESP), allows for autonomous collection of discrete 10L water samples at depths up to 4,000 m followed by application of DNA probe arrays as well as qPCR in support of microbial community ribotype analyses and detection of specific genes, respectively. The D-ESP can also be used to preserve particulate material for return to laboratory to validate information gleaned from in-situ sample processing as well as to support expanded studies of genomic diversity and gene expression. To provide a contextual framework for evaluating factors controlling microbial populations, the D-ESP is deployed with a custom suite of chemical and physical sensors including an in-situ mass spectrometer (ISMS), In Situ Underwater Spectrophotometer (ISUS) and CTD/optical sensor package. The D-ESP was deployed for 5 days during July 2010 on the crest of a methane-rich authigenic carbonate mound in Santa Monica Basin (~800 m depth) and two off-mound sites (70 m and 263 m due east of the mound). Bacterial mats mantle the mound, and streams of methane bubbles rise out of long linear cracks in the authigenic carbonate mound. Previous molecular investigations of the benthic water column over and surrounding the mound have demonstrated presence of a diverse assemblage of 16S rRNA and particulate methane monoxygenase subunit A (pMMO-A) genes belonging to putative aerobic methanotrophs making it a useful site for testing the D-ESP. For qPCR analysis, the D-ESP was thus configured to target the pMMO-A and 16S rRNA genes of two putative methanotrophic groups OPU1 and OPU3, the most widespread and abundant groups found in Santa Monica Basin. The pMMO-A and 16S rRNA genes of OPU1 and OPU3 were detected by D-ESP at the on- and off-mound sites as was expected based on previous work. The pMMO-A gene of OPU1 was more abundant than OPU3. In contrast, the 16S rRNA gene of OPU3 group was more abundant than OPU1. Shipboard measurements of on-mound methane show a high degree of meter-scale spatial and temporal heterogeneity (2.9 to 55,000 nM). At the two off-mound sites, methane was more uniform and substantially lower (70-m site = 8.4±0.3 nM; 263-m site = 2.2±0.1 nM). Water for background comparison collected by CTD rosette from 620 m depth at a site near Point Conception was processed by the D-ESP on the deck of the R/V Western Flyer. OPU1 and OPU3 were less abundant than at the off-mound sites, and although detected they were unquantifiable (<10 copies/mL seawater). Methane concentration was an order of magnitude lower than the off-mound sites (0.4±0.07 nM). Although the qPCR data indicate the increased presence of these microbes in a region of methane-enriched water relative to background, methane itself is not a good predictor of the abundance of genes involved with metabolizing that substrate highlighting the need to place biogeochemical analyses in a regional rather than site-specific context.

Ussler, W.; Tavormina, P.; Preston, C.; Shah, S.; Girguis, P. R.; Birch, J. M.; Orphan, V.; Scholin, C.

2010-12-01

149

Evaluation of reference genes for real-time PCR studies of Brazilian Somalis sheep infected by gastrointestinal nematodes  

PubMed Central

Precise normalization with reference genes is necessary, in order to obtain reliable relative expression data in response to gastrointestinal nematode infection. By using sheep from temperate regions as models, three reference genes, viz., ribosomal protein LO (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and succinate dehydrogenase complex subunit A (SDHA), were investigated in the abomasum, abomasal lymph nodes and small intestine of Brazilian Somalis sheep, either resistant or susceptible to gastrointestinal nematodes infections. Real time PCR was carried out by using SYBR Green I dye, and gene stability was tested by geNorm. RPLO was an ideal reference gene, since its expression was constant across treatments, presented lower variation, and was ranked as the most stable in abomasum and lymph node tissues. On the other hand, SDHA was the most stable in the small intestine followed by RPLO and GAPDH. These findings demonstrate the importance of correctly choosing reference genes prior to relative quantification. In addition, we determined that reference genes used in sheep from temperate regions, when properly tested, can be applied in animals from tropical regions such as the Brazilian Somalis sheep. PMID:21637421

2010-01-01

150

Selection of reference genes for normalization of quantitative polymerase chain reaction data in mouse models of heart failure.  

PubMed

The accurate measurement of mRNA expression levels is crucially dependent on the use of relevant reference genes for the normalization of data. Currently, heart failure is a serious and widespread disease, and multiple mouse models are utilized for the study of this complex disease. Although mouse models are commonly used to study cardiovascular disease, various studies have not employed the appropriate selection strategies. The present study investigated the expression stability of eight candidate reference genes (GAPDH, ACTB, B2M, CycA, TBP, PBGD, HTRP 1 and 18S) in two mouse models of heart failure, including the transverse aortic arch constriction (TAC) model and the myocardial infarction (MI) model, using GeNorm software. The expression of BNP was normalized using different reference gene strategies, and it was demonstrated that its induction following heart failure was most profound with the optimal reference gene combination. The most stable genes were identified as follows: TBP and CycA in the MI model, and PBGD and GAPDH in the TAC model. The present study provides important information for reference gene selection in mouse models of heart failure, and will aid further investigations of the transcriptome in cardiovascular research. PMID:25338732

Li, Qiaoling; Hu, Tingting; Chen, Liang; Sun, Jiayin; Xie, Jun; Li, Rang; Xu, Biao

2015-01-01

151

Selection of Reliable Reference Genes for Real-time qRT-PCR Analysis of Zi Geese (Anser anser domestica) Gene Expression  

PubMed Central

Zi geese (Anser anser domestica) belong to the white geese and are excellent layers with a superior feed-to-egg conversion ratio. Quantitative gene expression analysis, such as Real-time qRT-PCR, will provide a good understanding of ovarian function during egg-laying and consequently improve egg production. However, we still don’t know what reference genes in geese, which show stable expression, should be used for such quantitative analysis. In order to reveal such reference genes, the stability of seven genes were tested in five tissues of Zi geese. Methodology/Principal Findings: The relative transcription levels of genes encoding hypoxanthine guanine phosphoribosyl transferase 1 (HPRT1), ?-actin (ACTB), ?-tubulin (TUB), glyceraldehyde-3-phosphate-dehydrogenase (GADPH), succinate dehydrogenase flavoprotein (SDH), 28S rRNA (28S) and 18S rRNA (18S) have been quantified in heart, liver, kidney, muscle and ovary in Zi geese respectively at different developmental stages (1 d, 2, 4, 6 and 8 months). The expression stability of these genes was analyzed using geNorm, NormFinder and BestKeeper software. Conclusions: The expression of 28S in heart, GAPDH in liver and ovary, ACTB in kidney and HPRT1 in muscle are the most stable genes as identified by the three different analysis methods. Thus, these genes are recommended for use as candidate reference genes to compare mRNA transcription in various developmental stages of geese. PMID:25049806

Ji, Hong; Wang, Jianfa; Liu, Juxiong; Guo, Jingru; Wang, Zhongwei; Zhang, Xu; Guo, Li; Yang, Huanmin

2013-01-01

152

Selection and Assessment of Reference Genes for Quantitative PCR Normalization in Migratory Locust Locusta migratoria (Orthoptera: Acrididae)  

PubMed Central

Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1?, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1?, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1? and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts. PMID:24887329

Yang, Qingpo; Li, Zhen; Cao, Jinjun; Zhang, Songdou; Zhang, Huaijiang; Wu, Xiaoyun; Zhang, Qingwen; Liu, Xiaoxia

2014-01-01

153

Screening Suitable Reference Genes for Normalization in Reverse Transcription Quantitative Real-Time PCR Analysis in Melon  

PubMed Central

Melon (Cucumis melo. L) is not only an economically important cucurbitaceous crop but also an attractive model for studying many biological characteristics. Screening appropriate reference genes is essential to reverse transcription quantitative real-time PCR (RT-qPCR), which is key to many studies involving gene expression analysis. In this study, 14 candidate reference genes were selected, and the variations in their expression in roots and leaves of plants subjected to biotic stress, abiotic stress, and plant growth regulator treatment were assessed by RT-qPCR. The stability of the expression of the selected genes was determined and ranked using geNorm and NormFinder. geNorm identified the two most stable genes for each set of conditions: CmADP and CmUBIep across all samples, CmUBIep and CmRPL in roots, CmRAN and CmACT in leaves, CmADP and CmRPL under abiotic stress conditions, CmTUA and CmACT under biotic stress conditions, and CmRAN and CmACT under plant growth regulator treatments. NormFinder determined CmRPL to be the best reference gene in roots and under biotic stress conditions and CmADP under the other experimental conditions. CmUBC2 and CmPP2A were not found to be suitable under many experimental conditions. The catalase family genes CmCAT1, CmCAT2, and CmCAT3 were identified in melon genome and used as target genes to validate the reliability of identified reference genes. The catalase family genes showed the most upregulation 3 days after inoculation with Fusarium wilt in roots, after which they were downregulated. Their levels of expression were significantly overestimated when the unsuitable reference gene was used for normalization. These results not only provide guidelines for the selection of reference genes for gene expression analyses in melons but may also provide valuable information for studying the functions of catalase family genes in stress responses. PMID:24475250

Kong, Qiusheng; Yuan, Jingxian; Niu, Penghui; Xie, Junjun; Jiang, Wei; Huang, Yuan; Bie, Zhilong

2014-01-01

154

A Novel qPCR Assay for the Detection of African Animal Trypanosomosis in Trypanotolerant and Trypanosusceptible Cattle Breeds  

PubMed Central

This study was conducted to (i) determine the prevalence of African Animal Trypanosomosis (AAT) in tsetse challenged areas, (ii) compare conventional with qPCR detection systems and (iii) evaluate the host genetic background and biology as risk factors. AAT prevalence studies are often confronted with low levels of parasitaemia. Hence, we designed a novel qPCR assay using primers and species specific probes amplifying the Internal Transcribed Spacer 1 (ITS1) gene. Thereby all three AAT species could be detected simultaneously. 368 individuals from three cattle types (Baoulé, Zebu and hybrids) originating from 72 farms in Burkina Faso were analysed. Farmers were interviewed and morphometric measurements of the cattle taken. A chi-squared test and a logistic regression model were calculated to detect associations with infection. In our study, the overall rate of prevalence detected with the novel qPCR assay was 11.14%. Compared to conventional PCR we identified a concordance of 91.30%. We tested 41 animals positive for trypanosome DNA, five animals showed multiple infections. Zebus were twice as often infected (21.74%) compared to Baoulé (9.70%) and hybrids (9.57%). Trypanosoma vivax is the dominant species (9.24%), as compared to T. congolense (2.44%) and T. brucei (0.82%). The chi-squared tests linking the infection events to the breeds (Zebu vs. Baoulé and Zebu vs. hybrids) were on the border of significance. No significant association with other tested parameters could be detected. We introduce a novel qPCR technique for the fast, sensitive and simultaneous detection of the three AAT species. Our results suggest that associations with breed and infection exist since Zebu cattle are more likely to be infected compared to Baoulé and hybrids. Indigenous taurine cattle breeds, like the Baoulé, therefore provide a unique and valuable genetic resource. PMID:23967357

Silbermayr, Katja; Li, Fuyong; Soudré, Albert; Müller, Simone; Sölkner, Johann

2013-01-01

155

Sequencing and Validation of Reference Genes to Analyze Endogenous Gene Expression and Quantify Yellow Dwarf Viruses Using RT-qPCR in Viruliferous Rhopalosiphum padi  

PubMed Central

The bird cherry-oat aphid (Rhopalosiphum padi), an important pest of cereal crops, not only directly sucks sap from plants, but also transmits a number of plant viruses, collectively the yellow dwarf viruses (YDVs). For quantifying changes in gene expression in vector aphids, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is a touchstone method, but the selection and validation of housekeeping genes (HKGs) as reference genes to normalize the expression level of endogenous genes of the vector and for exogenous genes of the virus in the aphids is critical to obtaining valid results. Such an assessment has not been done, however, for R. padi and YDVs. Here, we tested three algorithms (GeNorm, NormFinder and BestKeeper) to assess the suitability of candidate reference genes (EF-1?, ACT1, GAPDH, 18S rRNA) in 6 combinations of YDV and vector aphid morph. EF-1? and ACT1 together or in combination with GAPDH or with GAPDH and 18S rRNA could confidently be used to normalize virus titre and expression levels of endogenous genes in winged or wingless R. padi infected with Barley yellow dwarf virus isolates (BYDV)-PAV and BYDV-GAV. The use of only one reference gene, whether the most stably expressed (EF-1?) or the least stably expressed (18S rRNA), was not adequate for obtaining valid relative expression data from the RT-qPCR. Because of discrepancies among values for changes in relative expression obtained using 3 regions of the same gene, different regions of an endogenous aphid gene, including each terminus and the middle, should be analyzed at the same time with RT-qPCR. Our results highlight the necessity of choosing the best reference genes to obtain valid experimental data and provide several HKGs for relative quantification of virus titre in YDV-viruliferous aphids. PMID:24810421

Wu, Keke; Liu, Wenwen; Mar, Thithi; Liu, Yan; Wu, Yunfeng; Wang, Xifeng

2014-01-01

156

RefPrimeCouch--a reference gene primer CouchApp  

PubMed Central

To support a quantitative real-time polymerase chain reaction standardization project, a new reference gene database application was required. The new database application was built with the explicit goal of simplifying not only the development process but also making the user interface more responsive and intuitive. To this end, CouchDB was used as the backend with a lightweight dynamic user interface implemented client-side as a one-page web application. Data entry and curation processes were streamlined using an OpenRefine-based workflow. The new RefPrimeCouch database application provides its data online under an Open Database License. Database URL: http://hpclife.th-wildau.de:5984/rpc/_design/rpc/view.html PMID:24368831

Silbermann, Jascha; Wernicke, Catrin; Pospisil, Heike; Frohme, Marcus

2013-01-01

157

Evaluation of Internal Reference Genes for Quantitative Expression Analysis by Real-Time PCR in Ovine Whole Blood  

PubMed Central

The use of reference genes is commonly accepted as the most reliable approach to normalize qRT-PCR and to reduce possible errors in the quantification of gene expression. The most suitable reference genes in sheep have been identified for a restricted range of tissues, but no specific data on whole blood are available. The aim of this study was to identify a set of reference genes for normalizing qRT-PCR from ovine whole blood. We designed 11 PCR assays for commonly employed reference genes belonging to various functional classes and then determined their expression stability in whole blood samples from control and disease-stressed sheep. SDHA and YWHAZ were considered the most suitable internal controls as they were stably expressed regardless of disease status according to both geNorm and NormFinder software; furthermore, geNorm indicated SDHA/HPRT, YWHAZ/GAPDH and SDHA/YWHAZ as the best reference gene combinations in control, disease-stressed and combined sheep groups, respectively. Our study provides a validated panel of optimal control genes which may be useful for the identification of genes differentially expressed by qRT-PCR in a readily accessible tissue, with potential for discovering new physiological and disease markers and as a tool to improve production traits (e.g., by identifying expression Quantitative Trait Loci). An additional outcome of the study is a set of intron-spanning primer sequences suitable for gene expression experiments employing SYBR Green chemistry on other ovine tissues and cells. PMID:22174628

Peletto, Simone; Bertuzzi, Simone; Campanella, Chiara; Modesto, Paola; Maniaci, Maria Grazia; Bellino, Claudio; Ariello, Dario; Quasso, Antonio; Caramelli, Maria; Acutis, Pier Luigi

2011-01-01

158

Evaluation of Reference Genes for Quantitative Real-Time PCR in Oil Palm Elite Planting Materials Propagated by Tissue Culture  

PubMed Central

Background The somatic embryogenesis tissue culture process has been utilized to propagate high yielding oil palm. Due to the low callogenesis and embryogenesis rates, molecular studies were initiated to identify genes regulating the process, and their expression levels are usually quantified using reverse transcription quantitative real-time PCR (RT-qPCR). With the recent release of oil palm genome sequences, it is crucial to establish a proper strategy for gene analysis using RT-qPCR. Selection of the most suitable reference genes should be performed for accurate quantification of gene expression levels. Results In this study, eight candidate reference genes selected from cDNA microarray study and literature review were evaluated comprehensively across 26 tissue culture samples using RT-qPCR. These samples were collected from two tissue culture lines and media treatments, which consisted of leaf explants cultures, callus and embryoids from consecutive developmental stages. Three statistical algorithms (geNorm, NormFinder and BestKeeper) confirmed that the expression stability of novel reference genes (pOP-EA01332, PD00380 and PD00569) outperformed classical housekeeping genes (GAPDH, NAD5, TUBULIN, UBIQUITIN and ACTIN). PD00380 and PD00569 were identified as the most stably expressed genes in total samples, MA2 and MA8 tissue culture lines. Their applicability to validate the expression profiles of a putative ethylene-responsive transcription factor 3-like gene demonstrated the importance of using the geometric mean of two genes for normalization. Conclusions Systematic selection of the most stably expressed reference genes for RT-qPCR was established in oil palm tissue culture samples. PD00380 and PD00569 were selected for accurate and reliable normalization of gene expression data from RT-qPCR. These data will be valuable to the research associated with the tissue culture process. Also, the method described here will facilitate the selection of appropriate reference genes in other oil palm tissues and in the expression profiling of genes relating to yield, biotic and abiotic stresses. PMID:24927412

Chan, Pek-Lan; Rose, Ray J.; Abdul Murad, Abdul Munir; Zainal, Zamri; Leslie Low, Eng-Ti; Ooi, Leslie Cheng-Li; Ooi, Siew-Eng; Yahya, Suzaini; Singh, Rajinder

2014-01-01

159

Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua l.) during temperature stress  

PubMed Central

Background One important physiological response to environmental stress in animals is change in gene expression. To obtain reliable data from gene expression studies using RT-qPCR it is important to evaluate a set of possible reference genes as normalizers for expression. The expression of these candidate genes should be analyzed in the relevant tissues during normal and stressed situations. To find suitable reference genes it was crucial that the genes were stably expressed also during a situation of physiological stress. For poikilotermic animals like cod, changes in temperature are normal, but if the changes are faster than physiological compensation, the animals respond with typical stress responses. It has previously been shown that Atlantic cod show stress responses when elevation of water temperature is faster than 1 degree/day, for this reason we chose hyperthermia as stress agent for this experiment. Findings We here describe the expression of eight candidate reference genes from Atlantic cod (Gadus morhua l.) and their stability during thermal stress (temperature elevation of one degree C/day for 5 days). The genes investigated were: Eukaryotic elongation factor 1 alpha, ef1a; 18s ribosomal RNA; 18s, Ubiquitin conjugate protein; ubiq, cytoskeletal beta-actin; actb, major histcompatibility complex I; MHC-I light chain, beta-2 -microglobulin; b2m, cytoskeletal alpha-tubulin; tba1c, acidic ribosomal phosphoprotein; rplp1, glucose-6-phosphate dehydrogenase; g6pd. Their expression were analyzed in 6 tissues (liver, head kidney, intestine, spleen, heart and gills) from cods exposed to elevated temperature and compared to a control group. Although there were variations between tissues with respect to reference gene stability, four transcripts were more consistent than the others: ubiq, ef1a, 18s and rplp1. We therefore used these to analyze the expression of stress related genes (heat shock proteins) induced during hyperthermia. We found that both transcripts were significantly upregulated in several tissues in fish exposed to increased temperature. Conclusion This is the first study comparing reference genes for RT-qPCR analyses of expression during hyperthermia in Atlantic cod. ef1a, 18s, rplp1 and ubiq transcripts were found to be well suited as reference genes during these experimental conditions. PMID:21466674

2011-01-01

160

International collaborative study of the endogenous reference gene LAT52 used for qualitative and quantitative analyses of genetically modified tomato.  

PubMed

One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates. PMID:18442244

Yang, Litao; Zhang, Haibo; Guo, Jinchao; Pan, Liangwen; Zhang, Dabing

2008-05-28

161

Distribution of aquaporin genes and selection of individual reference genes for quantitative real-time RT-PCR analysis in multiple tissues of the mouse.  

PubMed

Aquaporins (AQPs) are a family of water-transporting proteins that are selectively expressed in epithelial, endothelial, and many other cell types of various tissues, where they play important physiological functions. However, the accurate distribution of AQP gene expression has not yet been examined in various tissues of the mouse. We first evaluated the tissue distribution of AQP gene expression using tongue, nasal epithelium, bronchus, trachea, lung, esophagus, stomach, ileum, transverse colon, liver, pancreas, whole blood, thigh muscle, spinal cord, brain, thoracic aorta, heart, kidney, thymus, spleen, skin, eye, and testis of the mouse. Furthermore, for a quantitative analysis, we selected appropriate reference genes for normalized qRT-PCR data in various tissues. The stability of the reference genes was assessed using NormFinder. The stably expressed genes identified in the present study were 18s rRNA. When 18s rRNA was used, as the best reference gene in the present study, the genes for AQPs 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, and 12 were notably expressed in the eye, lung, testis, eye, spinal cord, trachea, kidney, testis, testis, testis, testis, and pancreas. These results, regarding the distribution of AQPs, suggest that AQPs may be involved in various physiological and pathophysiological processes. PMID:25188728

Sakai, Hiroyasu; Sato, Ken; Kai, Yuki; Shoji, Tetsuro; Hasegawa, Satoshi; Nishizaki, Maiko; Sagara, Atsunobu; Yamashita, Akira; Narita, Minoru

2014-09-01

162

Expression stabilities of candidate reference genes for RT-qPCR under different stress conditions in soybean.  

PubMed

Due to its accuracy, sensitivity and high throughput, real time quantitative PCR (RT-qPCR) has been widely used in analysing gene expression. The quality of data from such analyses is affected by the quality of reference genes used. Expression stabilities for nine candidate reference genes widely used in soybean were evaluated under different stresses in this study. Our results showed that EF1A and ACT11 were the best under salinity stress, TUB4, TUA5 and EF1A were the best under drought stress, ACT11 and UKN2 were the best under dark treatment, and EF1B and UKN2 were the best under virus infection. EF1B and UKN2 were the top two genes which can be reliably used in all of the stress conditions assessed. PMID:24124481

Ma, Shuhua; Niu, Hongwei; Liu, Chunji; Zhang, Jie; Hou, Chunyan; Wang, Dongmei

2013-01-01

163

Construction and Application of a Korean Reference Panel for Imputing Classical Alleles and Amino Acids of Human Leukocyte Antigen Genes  

PubMed Central

Genetic variations of human leukocyte antigen (HLA) genes within the major histocompatibility complex (MHC) locus are strongly associated with disease susceptibility and prognosis for many diseases, including many autoimmune diseases. In this study, we developed a Korean HLA reference panel for imputing classical alleles and amino acid residues of several HLA genes. An HLA reference panel has potential for use in identifying and fine-mapping disease associations with the MHC locus in East Asian populations, including Koreans. A total of 413 unrelated Korean subjects were analyzed for single nucleotide polymorphisms (SNPs) at the MHC locus and six HLA genes, including HLA-A, -B, -C, -DRB1, -DPB1, and -DQB1. The HLA reference panel was constructed by phasing the 5,858 MHC SNPs, 233 classical HLA alleles, and 1,387 amino acid residue markers from 1,025 amino acid positions as binary variables. The imputation accuracy of the HLA reference panel was assessed by measuring concordance rates between imputed and genotyped alleles of the HLA genes from a subset of the study subjects and East Asian HapMap individuals. Average concordance rates were 95.6% and 91.1% at 2-digit and 4-digit allele resolutions, respectively. The imputation accuracy was minimally affected by SNP density of a test dataset for imputation. In conclusion, the Korean HLA reference panel we developed was highly suitable for imputing HLA alleles and amino acids from MHC SNPs in East Asians, including Koreans. PMID:25398076

Kim, Kwangwoo; Bang, So-Young; Lee, Hye-Soon; Bae, Sang-Cheol

2014-01-01

164

Identification of reference genes suitable for qRT-PCR in grapevine and application for the study of the expression of genes involved in pterostilbene synthesis  

Microsoft Academic Search

The recent publication of the grapevine genome sequence facilitates the use of qRT-PCR to study gene expression changes. For\\u000a this approach, reference genes are commonly used to normalize data and their stability of expression should be systematically\\u000a validated. Among grapevine defenses is the production of the antimicrobial stilbenic phytoalexins, notably the highly fungitoxic\\u000a pterostilbene, which plays a crucial role in

Magdalena Gamm; Marie-Claire Héloir; Jani Kelloniemi; Benoît Poinssot; David Wendehenne; Marielle Adrian

2011-01-01

165

The Future of qPCR: Best practices, Standardization, and the MIQE Guidelines  

NSDL National Science Digital Library

Quantitative polymerase chain reaction (qPCR) has emerged as a powerful tool in molecular biology laboratories, both in research and in diagnostic settings. This webinar will address the best practices of qPCR, with the goal of providing researchers with more consistent and reliable data.

n/a n/a (AAAS;)

2010-09-30

166

Functional classification of genes using semantic distance and fuzzy clustering approach: evaluation with reference sets and overlap analysis.  

PubMed

Functional classification aims at grouping genes according to their molecular function or the biological process they participate in. Evaluating the validity of such unsupervised gene classification remains a challenge given the variety of distance measures and classification algorithms that can be used. We evaluate here functional classification of genes with the help of reference sets: KEGG (Kyoto Encyclopaedia of Genes and Genomes) pathways and Pfam clans. These sets represent ground truth for any distance based on GO (Gene Ontology) biological process and molecular function annotations respectively. Overlaps between clusters and reference sets are estimated by the F-score method. We test our previously described IntelliGO semantic distance with hierarchical and fuzzy C-means clustering and we compare results with the state-of-the-art DAVID (Database for Annotation Visualisation and Integrated Discovery) functional classification method. Finally, study of best matching clusters to reference sets leads us to propose a set-difference method for discovering missing information. PMID:23013652

Devignes, Marie-Dominique; Benabderrahmane, Sidahmed; Smaïl-Tabbone, Malika; Napoli, Amedeo; Poch, Olivier

2012-01-01

167

Identification and validation of reference genes to study the gene expression in Gluconacetobacter diazotrophicus grown in different carbon sources using RT-qPCR.  

PubMed

Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus. PMID:22814372

Galisa, Péricles S; da Silva, Helder A P; Macedo, Aline V M; Reis, Verônica M; Vidal, Márcia S; Baldani, José I; Simões-Araújo, Jean L

2012-10-01

168

Genome-wide identification of housekeeping genes in maize.  

PubMed

In the wake of recent progress of high throughput transcriptome profiling technologies, extensive housekeeping gene mining has been conducted in humans. However, very few studies have been reported in maize (Zea mays L.), an important crop plant, and none were conducted on a genome -wide level. In this study, we surveyed housekeeping genes throughout the maize transcriptome using RNA-seq and microarray techniques, and validated the housekeeping profile with quantitative polymerase chain reaction (qPCR) under a series of conditions including different genotypes and nitrogen supplies. Seven microarray datasets and two RNA-seq libraries representing 40 genotypes at more than 20 developmental stages were selected to screen for commonly expressed genes. A total of 1,661 genes showed constitutive expression in both microarray and RNA-seq datasets, serving as our starting housekeeping gene candidates. To determine for stably expressed housekeeping genes, NormFinder was used to select the top 20 % invariable genes to be the more likely candidates, which resulted in 48 and 489 entries from microarray and RNA-seq data, respectively. Among them, nine genes (2OG-Fe, CDK, DPP9, DUF, NAC, RPN, SGT1, UPF1 and a hypothetical protein coding gene) were expressed in all 40 maize diverse genotypes tested covering 16 tissues at more than 20 developmental stages under normal and stress conditions, implying these as being the most reliable reference genes. qPCR analysis confirmed the stable expression of selected reference gene candidates compared to two widely used housekeeping genes. All the reference gene candidates showed higher invariability than ACT and GAPDH. The hypothetical protein coding gene exhibited the most stable expression across 26 maize lines with different nitrogen treatments with qPCR, followed by CDK encoding the cyclin-dependent kinase. As the first study to systematically screen for housekeeping genes in maize, we identified candidates by examining the transcriptome atlas generated from RNA-seq and microarray technologies. The nine top-ranked qPCR-validated novel housekeeping genes provide a valuable resource of reference genes for maize gene expression analysis. PMID:25209110

Lin, Feng; Jiang, Lu; Liu, Yuhe; Lv, Yuanda; Dai, Huixue; Zhao, Han

2014-11-01

169

Application of absolute qPCR as a screening method to detect illicit 17?-oestradiol administration in male cattle.  

PubMed

It has been previously demonstrated that the progesterone receptor gene is up-regulated in the sex accessory glands of pre-pubertal and adult male bovines after 17?-oestradiol treatment. In the present study, a qualitative screening method was optimised to detect 17?-oestradiol treatment using absolute quantification by qPCR of the progesterone receptor gene to determine the amount of gene expression in bulbo-urethral glands. An external standard curve was generated and developed with TaqMan® technology. Based on two in vivo experiments, the decision limit CC?, sensitivity and specificity of this screening method were established. Trial 1 consisted of 32 Friesian veal calves divided into two groups: group A (n = 12), consisting of animals treated with four doses of 17?-oestradiol (5 mg week(-1) per animal); and group B (n = 20), consisting of control animals. Trial 2 was performed on 26 Charolaise beef cattle that either received five doses of 17?-oestradiol (group C; 20 mg week(-1) per animal; n = 6) or remained untreated (group D; n = 20). Further, progesterone receptor gene expression was evaluated in beef and veal calves for human consumption. A specific CC? on 20 Piedmontese control beef cattle was calculated to include these animals in a field investigation. Five out of 190 beef cattle and 26 out of 177 calves tested expressed the progesterone receptor gene above their respective CC? and they were classified as being suspected of 17?-oestradiol treatment. Additionally, 58% of veal calves that tested suspect via qPCR exhibited histological lesions of the bulbo-urethral gland tissue, which are typical of oestrogen administration and are consistent with hyperplasia and metaplasia of the glandular epithelium. PMID:23131142

Uslenghi, F; Divari, S; Cannizzo, F T; De Maria, R; Spada, F; Mulasso, C; Pezzolato, M; Bozzetta, E; Attucci, A; Giorgi, P; Biolatti, B

2013-01-01

170

Reference gene selection for real-time RT-PCR in regenerating mouse livers  

SciTech Connect

The liver has an intrinsic ability to undergo active proliferation and recover functional liver mass in response to an injury response. This regenerative process involves a complex yet well orchestrated change in the gene expression profile. To produce accurate and reliable gene expression of target genes during various stages of liver regeneration, the determination of internal control housekeeping genes (HKGs) those are uniformly expressed is required. In the present study, the gene expression of 8 commonly used HKGs, including GAPDH, ACTB, HPRT1, GUSB, PPIA, TBP, TFRC, and RPL4, were studied using mouse livers that were quiescent and actively regenerating induced by partial hepatectomy. The amplification of the HKGs was statistically analyzed by two different mathematical algorithms, geNorm and NormFinder. Using this method, PPIA and TBP gene expression found to be relatively stable regardless of the stages of liver regeneration and would be ideal for normalization to target gene expression.

Tatsumi, Kohei [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan); Ohashi, Kazuo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan)], E-mail: ohashi@abmes.twmu.ac.jp; Taminishi, Sanae [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan); Okano, Teruo [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Yoshioka, Akira; Shima, Midori [Department of Pediatrics, Nara Medical University, 840 Shijo-cho, Kashihara, Nara 634-8522 (Japan)

2008-09-12

171

Selection of Appropriate Reference Genes for RT-qPCR Analysis in a Streptozotocin-Induced Alzheimer’s Disease Model of Cynomolgus Monkeys (Macaca fascicularis)  

PubMed Central

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) has been widely used to quantify relative gene expression because of the specificity, sensitivity, and accuracy of this technique. In order to obtain reliable gene expression data from RT-qPCR experiments, it is important to utilize optimal reference genes for the normalization of target gene expression under varied experimental conditions. Previously, we developed and validated a novel icv-STZ cynomolgus monkey model for Alzheimer’s disease (AD) research. However, in order to enhance the reliability of this disease model, appropriate reference genes must be selected to allow meaningful analysis of the gene expression levels in the icv-STZ cynomolgus monkey brain. In this study, we assessed the expression stability of 9 candidate reference genes in 2 matched-pair brain samples (5 regions) of control cynomolgus monkeys and those who had received intracerebroventricular injection of streptozotocin (icv-STZ). Three well-known analytical programs geNorm, NormFinder, and BestKeeper were used to choose the suitable reference genes from the total sample group, control group, and icv-STZ group. Combination analysis of the 3 different programs clearly indicated that the ideal reference genes are RPS19 and YWHAZ in the total sample group, GAPDH and RPS19 in the control group, and ACTB and GAPDH in the icv-STZ group. Additionally, we validated the normalization accuracy of the most appropriate reference genes (RPS19 and YWHAZ) by comparison with the least stable gene (TBP) using quantification of the APP and MAPT genes in the total sample group. To the best of our knowledge, this research is the first study to identify and validate the appropriate reference genes in cynomolgus monkey brains. These findings provide useful information for future studies involving the expression of target genes in the cynomolgus monkey. PMID:23457495

Kim, Kyoung-Min; Kim, Heui-Soo; Lee, Sang-Rae; Kim, Sun-Uk; Kim, Sang-Hyun; Kim, Ji-Su; Jeong, Kang-Jin; Lee, Kyoung-Min; Huh, Jae-Won; Chang, Kyu-Tae

2013-01-01

172

MPN estimation of qPCR target sequence recoveries from whole cell calibrator samples  

EPA Science Inventory

DNA extracts from enumerated target organism cells (calibrator samples) have been used for estimating Enterococcus cell equivalent densities in surface waters by a comparative cycle threshold (Ct) qPCR analysis method. To compare surface water Enterococcus density estimates from ...

173

Quantitative detection and identification of tyramine-producing enterococci and lactobacilli in cheese by multiplex qPCR.  

PubMed

Tyramine is the most abundant biogenic amine in fermented dairy products, in which it is produced through the microbial enzymatic decarboxylation of tyrosine. This activity has been detected in a variety of lactic acid bacteria mainly belonging to the genera Enterococcus and Lactobacillus. This paper describes a culture-independent qPCR method, based on the specific amplification of the tdc gene, for the detection, quantification and identification of bacteria with the ability to produce tyramine. This method was found to be specific and to show a wide dynamic range, thus allowing the quantification of these tdc+ bacterial groups among the complex microbiota of cheese. tdc qPCR was used to follow the development of tdc+ microbiota during the manufacture of a blue-veined cheese (Cabrales) made from raw milk. In this type of cheese, tdc+ enterococci seem to be responsible for the high concentrations of tyramine detected. The method was also used to identify and quantify tdc+ enterococci and lactobacilli in 18 commercially available cheeses. Different types and numbers of these microorganisms were found. Their relationships with the concentration of tyramine and technological factors are discussed. PMID:20688235

Ladero, Victor; Fernández, María; Cuesta, Isabel; Alvarez, Miguel A

2010-10-01

174

Wheat-specific gene, ribosomal protein l21, used as the endogenous reference gene for qualitative and real-time quantitative polymerase chain reaction detection of transgenes.  

PubMed

Wheat-specific ribosomal protein L21 (RPL21) is an endogenous reference gene suitable for genetically modified (GM) wheat identification. This taxon-specific RPL21 sequence displayed high homogeneity in different wheat varieties. Southern blots revealed 1 or 3 copies, and sequence analyses showed one amplicon in common wheat. Combined analyses with sequences from common wheat (AABBDD) and three diploid ancestral species, Triticum urartu (AA), Aegilops speltoides (BB), and Aegilops tauschii (DD), demonstrated the presence of this amplicon in the AA genome. Using conventional qualitative polymerase chain reaction (PCR), the limit of detection was 2 copies of wheat haploid genome per reaction. In the quantitative real-time PCR assay, limits of detection and quantification were about 2 and 8 haploid genome copies, respectively, the latter of which is 2.5-4-fold lower than other reported wheat endogenous reference genes. Construct-specific PCR assays were developed using RPL21 as an endogenous reference gene, and as little as 0.5% of GM wheat contents containing Arabidopsis NPR1 were properly quantified. PMID:25325387

Liu, Yi-Ke; Li, He-Ping; Huang, Tao; Cheng, Wei; Gao, Chun-Sheng; Zuo, Dong-Yun; Zhao, Zheng-Xi; Liao, Yu-Cai

2014-10-29

175

Genetics of coronary heart disease with reference to ApoAI-CIII-AIV gene region.  

PubMed

Cardiovascular diseases are affected by multiple factors like genetic as well as environmental hence they reveal factorial nature. The evidences that genetic factors are susceptible for developing cardiovascular diseases come from twin studies and familial aggregation. Different ethnic populations reveal differences in the prevalence coronary artery disease (CAD) pointing towards the genetic susceptibility. With progression in molecular techniques different developments have been made to comprehend the disease physiology. Molecular markers have also assisted to recognize genes that may provide evidences to evaluate the role of genetic factors in causation of susceptibility towards CAD. Numerous studies suggest the contribution of specific "candidate genes", which correlate with various roles/pathways that are involved in the coronary heart disease. Different studies have revealed that there are large numbers of genes which are involved towards the predisposition of CAD. However, these reports are not consistent. One of the reasons could be weak contribution of genetic susceptibility of these genes. Genome wide associations show different chromosomal locations which dock, earlier unknown, genes which may attribute to CAD. In the present review different ApoAI-CIII-AIV gene clusters have been discussed. PMID:25228954

Agrawal, Suraksha; Mastana, Sarabjit

2014-08-26

176

A Java Program for LRE-Based Real-Time qPCR that Enables Large-Scale Absolute Quantification  

PubMed Central

Background Linear regression of efficiency (LRE) introduced a new paradigm for real-time qPCR that enables large-scale absolute quantification by eliminating the need for standard curves. Developed through the application of sigmoidal mathematics to SYBR Green I-based assays, target quantity is derived directly from fluorescence readings within the central region of an amplification profile. However, a major challenge of implementing LRE quantification is the labor intensive nature of the analysis. Findings Utilizing the extensive resources that are available for developing Java-based software, the LRE Analyzer was written using the NetBeans IDE, and is built on top of the modular architecture and windowing system provided by the NetBeans Platform. This fully featured desktop application determines the number of target molecules within a sample with little or no intervention by the user, in addition to providing extensive database capabilities. MS Excel is used to import data, allowing LRE quantification to be conducted with any real-time PCR instrument that provides access to the raw fluorescence readings. An extensive help set also provides an in-depth introduction to LRE, in addition to guidelines on how to implement LRE quantification. Conclusions The LRE Analyzer provides the automated analysis and data storage capabilities required by large-scale qPCR projects wanting to exploit the many advantages of absolute quantification. Foremost is the universal perspective afforded by absolute quantification, which among other attributes, provides the ability to directly compare quantitative data produced by different assays and/or instruments. Furthermore, absolute quantification has important implications for gene expression profiling in that it provides the foundation for comparing transcript quantities produced by any gene with any other gene, within and between samples. PMID:21407812

Rutledge, Robert G.

2011-01-01

177

Injury Modality, Survival Interval, and Sample Region Are Critical Determinants of qRT-PCR Reference Gene Selection during Long-Term Recovery from Brain Trauma  

PubMed Central

Abstract In the present study we examined expression of four real-time quantitative RT-PCR reference genes commonly applied to rodent models of brain injury. Transcripts for ?-actin, cyclophilin A, GAPDH, and 18S rRNA were assessed at 2–15 days post-injury, focusing on the period of synaptic recovery. Diffuse moderate central fluid percussion injury (FPI) was contrasted with unilateral entorhinal cortex lesion (UEC), a model of targeted deafferentation. Expression in UEC hippocampus, as well as in FPI hippocampus and parietotemporal cortex was analyzed by qRT-PCR. Within-group variability of gene expression was assessed and change in expression relative to paired controls was determined. None of the four common reference genes tested was invariant across brain region, survival time, and type of injury. Cyclophilin A appeared appropriate as a reference gene in UEC hippocampus, while ?-actin was most stable for the hippocampus subjected to FPI. However, each gene may fail as a suitable reference with certain test genes whose RNA expression is targeted for measurement. In FPI cortex, all reference genes were significantly altered over time, compromising their utility for time-course studies. Despite such temporal variability, certain genes may be appropriate references if limited to single survival times. These data provide an extended baseline for identification of appropriate reference genes in rodent studies of recovery from brain injury. In this context, we outline additional considerations for selecting a qRT-PCR normalization strategy in such studies. As previously concluded for acute post-injury intervals, we stress the importance of reference gene validation for each brain injury paradigm and each set of experimental conditions. PMID:19505177

Harris, Janna L.; Reeves, Thomas M.

2009-01-01

178

3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer  

PubMed Central

Background Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC). Results Using Brain RNA sample from multiple runs, we demonstrated that the transcript profiles from 3' DGE were highly reproducible between technical and biological replicates from libraries constructed by the same lab and even by different labs, and between two generations of Illumina's Genome Analyzers. Approximately 65% of all sequence reads mapped to mitochondrial genes, ribosomal RNAs, and canonical transcripts. The expression profiles of brain RNA and universal human reference RNA were compared which demonstrated that DGE was also highly quantitative with excellent correlation of differential expression with quantitative real-time PCR. Furthermore, one lane of 3' DGE sequencing, using the current sequencing chemistry and image processing software, had wider dynamic range for transcriptome profiling and was able to detect lower expressed genes which are normally below the detection threshold of microarrays. Conclusion 3' tag DGE profiling with massive parallel sequencing achieved high sensitivity and reproducibility for transcriptome profiling. Although it lacks the ability of detecting alternative splicing events compared to RNA-SEQ, it is much more affordable and clearly out-performed microarrays (Affymetrix) in detecting lower abundant transcripts. PMID:19917133

2009-01-01

179

Reference gene selection for quantitative real-time PCR analysis in virus infected cells: SARS corona virus, Yellow fever virus, Human Herpesvirus-6, Camelpox virus and Cytomegalovirus infections  

PubMed Central

Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, ?-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells. PMID:15705200

Radonic, Aleksandar; Thulke, Stefanie; Bae, Hi-Gung; Muller, Marcel A; Siegert, Wolfgang; Nitsche, Andreas

2005-01-01

180

Identification of reference genes for RT-qPCR in ovine mammary tissue during late pregnancy and lactation and in response to maternal nutritional programming.  

PubMed

The mammary gland is a complex tissue consisting of multiple cell types which, over the lifetime of an animal, go through repeated cycles of development associated with pregnancy, lactation and involution. The mammary gland is also known to be sensitive to maternal programming by environmental stimuli such as nutrition. The molecular basis of these adaptations is of significant interest, but requires robust methods to measure gene expression. Reverse-transcription quantitative PCR (RT-qPCR) is commonly used to measure gene expression, and is currently the method of choice for validating genome-wide expression studies. RT-qPCR requires the selection of reference genes that are stably expressed over physiological states and treatments. In this study we identify suitable reference genes to normalize RT-qPCR data for the ovine mammary gland in two physiological states; late pregnancy and lactation. Biopsies were collected from offspring of ewes that had been subjected to different nutritional paradigms during pregnancy to examine effects of maternal programming on the mammary gland of the offspring. We evaluated eight candidate reference genes and found that two reference genes (PRPF3 and CUL1) are required for normalising RT-qPCR data from pooled RNA samples, but five reference genes are required for analyzing gene expression in individual animals (SENP2, EIF6, MRPL39, ATP1A1, CUL1). Using these stable reference genes, we showed that TET1, a key regulator of DNA methylation, is responsive to maternal programming and physiological state. The identification of these novel reference genes will be of utility to future studies of gene expression in the ovine mammary gland. PMID:24893875

Paten, A M; Pain, S J; Peterson, S W; Blair, H T; Kenyon, P R; Dearden, P K; Duncan, E J

2014-08-01

181

Reverse transcription quantitative real-time polymerase chain reaction reference genes in the spared nerve injury model of neuropathic pain: validation and literature search  

PubMed Central

Background The reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a widely used, highly sensitive laboratory technique to rapidly and easily detect, identify and quantify gene expression. Reliable RT-qPCR data necessitates accurate normalization with validated control genes (reference genes) whose expression is constant in all studied conditions. This stability has to be demonstrated. We performed a literature search for studies using quantitative or semi-quantitative PCR in the rat spared nerve injury (SNI) model of neuropathic pain to verify whether any reference genes had previously been validated. We then analyzed the stability over time of 7 commonly used reference genes in the nervous system – specifically in the spinal cord dorsal horn and the dorsal root ganglion (DRG). These were: Actin beta (Actb), Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal proteins 18S (18S), L13a (RPL13a) and L29 (RPL29), hypoxanthine phosphoribosyltransferase 1 (HPRT1) and hydroxymethylbilane synthase (HMBS). We compared the candidate genes and established a stability ranking using the geNorm algorithm. Finally, we assessed the number of reference genes necessary for accurate normalization in this neuropathic pain model. Results We found GAPDH, HMBS, Actb, HPRT1 and 18S cited as reference genes in literature on studies using the SNI model. Only HPRT1 and 18S had been once previously demonstrated as stable in RT-qPCR arrays. All the genes tested in this study, using the geNorm algorithm, presented gene stability values (M-value) acceptable enough for them to qualify as potential reference genes in both DRG and spinal cord. Using the coefficient of variation, 18S failed the 50% cut-off with a value of 61% in the DRG. The two most stable genes in the dorsal horn were RPL29 and RPL13a; in the DRG they were HPRT1 and Actb. Using a 0.15 cut-off for pairwise variations we found that any pair of stable reference gene was sufficient for the normalization process. Conclusions In the rat SNI model, we validated and ranked Actb, RPL29, RPL13a, HMBS, GAPDH, HPRT1 and 18S as good reference genes in the spinal cord. In the DRG, 18S did not fulfill stability criteria. The combination of any two stable reference genes was sufficient to provide an accurate normalization. PMID:23841944

2013-01-01

182

Using Ribosomal Protein Genes as Reference: A Tale of Lieven Thorrez1,2,5  

E-print Network

, Belgium Abstract Background: Housekeeping genes are needed in every tissue as their expression is required Editor: Sui Huang, Children's Hospital Boston, United States of America Received December 6, 2007-access article distributed under the terms of the Creative Commons Attribution License, which permits

183

Why the need for qPCR publication guidelines?--The case for MIQE.  

PubMed

The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate data analysis methodologies have resulted in the recurrent publication of data that are at best inconsistent and at worst irrelevant and even misleading. Furthermore, there is a lamentable lack of transparency of reporting, with the "Materials and Methods" sections of many publications, especially those with high impact factors, not fit for the purpose of evaluating the quality of any reported qPCR data. This poses a challenge to the integrity of the scientific literature, with serious consequences not just for basic research, but potentially calamitous implications for drug development and disease monitoring. These issues are being addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments ("MIQE"). MIQE aims to restructure to-day's free-for-all qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology. PMID:20025972

Bustin, Stephen A

2010-04-01

184

Identification and validation of reference genes for Populus euphratica gene expression analysis during abiotic stresses by quantitative real-time PCR.  

PubMed

Populus euphratica is the only arboreal species that is established in the world's largest shifting-sand desert in China and is well-adapted to the extreme desert environment, so it is widely considered a model system for researching into abiotic stress resistance of woody plants. However, few P. euphratica reference genes (RGs) have been identified for quantitative real-time polymerase chain reaction (qRT-PCR) until now. Validation of suitable RGs is essential for gene expression normalization research. In this study, we screened 16 endogenous candidate RGs in P. euphratica leaves in six abiotic stress treatments, including abscisic acid (ABA), cold, dehydration, drought, short-duration salt (SS) and long-duration salt (LS) treatments, each with 6 treatment gradients. After calculation of PCR efficiencies, three different software tools, NormFinder, geNorm and BestKeeper, were employed to analyze the qRT-PCR data systematically, and the outputs were merged by means of a non-weighted unsupervised rank aggregation method. The genes selected as optimal for gene expression analysis of the six treatments were RPL17 (ribosomal protein L17) in ABA, EF1? (elongation factor-1 alpha) in cold, HIS (histone superfamily protein H3) in dehydration, GII? in drought and SS, and TUB (tubulin) in LS. The expression of 60S (the 60S ribosomal protein) varied the least during all treatments. To illustrate the suitability of these RGs, the relative quantifications of three stress-inducible genes, PePYL1, PeSCOF-1 and PeSCL7 were investigated with different RGs. The results, calculated using qBasePlus software, showed that compared with the least-appropriate RGs, the expression profiles normalized by the recommended RGs were closer to expectations. Our study provided an important RG application guideline for P. euphratica gene expression characterization. PMID:24720378

Wang, Hou-Ling; Chen, Jinhuan; Tian, Qianqian; Wang, Shu; Xia, Xinli; Yin, Weilun

2014-11-01

185

Validation of a reference gene (BdFIM) for quantifying transgene copy numbers in Brachypodium distachyon by real-time PCR.  

PubMed

Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon. PMID:24497043

Zhu, Hong; Wen, Feng; Li, Peng; Liu, Xiang; Cao, Jianmei; Jiang, Min; Ming, Feng; Chu, Zhaoqing

2014-03-01

186

Reference Gene Selection for Quantitative Real-Time RT-PCR Normalization in Iris. lactea var. chinensis Roots under Cadmium, Lead, and Salt Stress Conditions  

PubMed Central

Quantitative real time PCR (RT-qPCR) has emerged as an accurate and sensitive method to measure the gene expression. However, obtaining reliable result depends on the selection of reference genes which normalize differences among samples. In this study, we assessed the expression stability of seven reference genes, namely, ubiquitin-protein ligase UBC9 (UBC), tubulin alpha-5 (TUBLIN), eukaryotic translation initiation factor (EIF-5A), translation elongation factor EF1A (EF1?), translation elongation factor EF1B (EF1b), actin11 (ACTIN), and histone H3 (HIS), in Iris. lactea var. chinensis (I. lactea var. chinensis) root when the plants were subjected to cadmium (Cd), lead (Pb), and salt stress conditions. All seven reference genes showed a relatively wide range of threshold cycles (Ct) values in different samples. GeNorm and NormFinder algorithms were used to assess the suitable reference genes. The results from the two software units showed that EIF-5A and UBC were the most stable reference genes across all of the tested samples, while TUBLIN was unsuitable as internal controls. I. lactea var. chinensis is tolerant to Cd, Pb, and salt. Our results will benefit future research on gene expression in response to the three abiotic stresses. PMID:24977206

Gu, Chun-Sun; Liu, Liang-qin; Xu, Chen; Zhao, Yan-hai; Zhu, Xu-dong; Huang, Su-Zhen

2014-01-01

187

Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum  

PubMed Central

Hop plant (Humulus lupulus L.), cultivated primarily for its use in the brewing industry, is faced with a variety of diseases, including severe vascular diseases, such as Verticillium wilt, against which no effective protection is available. The understanding of disease resistance with tools such as differentially expressed gene studies is an important objective of plant defense mechanisms. In this study, we evaluated twenty-three reference genes for RT-qPCR expression studies on hop under biotic stress conditions. The candidate genes were validated on susceptible and resistant hop cultivars sampled at three different time points after infection with Verticillium albo-atrum. The stability of expression and the number of genes required for accurate normalization were assessed by three different Excel-based approaches (geNorm v.3.5 software, NormFinder, and RefFinder). High consistency was found among them, identifying the same six best reference genes (YLS8, DRH1, TIP41, CAC, POAC and SAND) and five least stably expressed genes (CYCL, UBQ11, POACT, GAPDH and NADH). The candidate genes in different experimental subsets/conditions resulted in different rankings. A combination of the two best reference genes, YLS8 and DRH1, was used for normalization of RT-qPCR data of the gene of interest (PR-1) implicated in biotic stress of hop. We outlined the differences between normalized and non-normalized values and the importance of RT-qPCR data normalization. The high correlation obtained among data standardized with different sets of reference genes confirms the suitability of the reference genes selected for normalization. Lower correlations between normalized and non-normalized data may reflect different quantity and/or quality of RNA samples used in RT-qPCR analyses. PMID:23874551

Stajner, Natasa; Cregeen, Sara; Javornik, Branka

2013-01-01

188

Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity  

PubMed Central

BACKGROUND Obesity has a complicated metabolic pathology, and defining the underlying mechanisms of obesity requires integrative studies with molecular endpoints. Real time quantitative PCR (RT-qPCR) is a powerful tool that has been widely utilized. However, the importance of using carefully validated reference genes in RT-qPCR seems to be overlooked in obesity-related research. The objective of this study was to select a set of reference genes with stable expressions to be used for RT-qPCR normalization in rats under fasted vs. re-fed and chow vs. high fat diet (HFD) conditions. DESIGN Male Long-Evans rats were treated under four conditions, chow/fasted, chow/re-fed, HFD/fasted, and HFD/re-fed. Expression stabilities of the13 candidate reference genes were evaluated in the rat hypothalamus, duodenum, jejunum, and ileum using ReFinder software program. The optimal number of reference genes needed for RT-qPCR analyses was determined using geNorm. RESULTS Using geNorm analysis, we found that it was sufficient to use the two most stably expressed genes as references in RT-qPCR analyses for each tissue under specific experimental conditions. Unique subsets of reference genes out of the 13 candidate genes were identified, each of which is specific for one type of rat tissue (hypothalamus, duodenum, jejunum, or ileum) under a different combination of diet and feeding condition. CONCLUSIONS Our study demonstrates that gene expression levels of reference genes commonly used in obesity-related studies, such as ACTB, or RPS18, are altered by changes in acute or chronic energy status. These findings underline the importance of using reference genes that are stable in expression across experimental conditions when studying the rat hypothalamus and intestine, because these tissues play an integral role in regulation of energy homeostasis. It is our hope that this study will raise awareness among obesity researchers on the essential need for reference gene validation in gene expression studies. PMID:23736358

Li, B; Matter, EK; Hoppert, HT; Seeley, RJ; Sandoval, DA

2014-01-01

189

Quantification of B16 Melanoma Cells in Lungs Using Triplex Q-PCR - A New Approach to Evaluate Melanoma Cell Metastasis and Tumor Control  

PubMed Central

Skin cancer is the most common type of all cancers. However, it comprises several different types of cancers, one of which is malignant melanoma. Even though melanomas only make up about 5% of skin cancers, they are responsible for the majority of skin cancer deaths due to the poor chance of survival once the tumor has metastasized. In the present study, we have developed a new assay for quantitative analysis of B16 melanoma metastasis in the lungs. We have used a triplex Q-PCR to determine the expression of the melanoma genes GP100/Pmel and tyrosinase-related protein 2 (TRP-2), and found that B16.F10gp cells were detectable in the lungs as early as 2 hours after intravenous challenge with ?104 tumor cells. When investigating the gene expression as a function of time, we observed a gradual decrease from 2–24 hours post tumor challenge followed by an increase of approximately 2 log10 on day 11. The early decrease was accelerated in the presence of activated NK cells. To further evaluate our assay, we also investigated the level of metastasis in the context of vaccination with replication defective adenoviral vectors, Ad-Ii-GP and Ad-GP, previously found to significantly delay the outgrowth of subcutaneous melanomas. Results obtained using Q-PCR were compared to conventional counting of metastatic foci under a dissection microscope. A marked reduction in gene expression was observed in the lungs after vaccination with both vectors; however, Ad-Ii-GP showed the highest protection, and matching results were obtained by enumeration of visible tumor nodules on the lung surfaces. Finally, we could show that inhibition of tumor metastasis required antigen-specific CD8 T cells and IFN?, but not perforin. In conclusion, the presented results validate triplex Q-PCR as a fast, objective, and quantitative method for analysis of melanoma metastasis in the lungs. PMID:24498205

Lindkvist, Annika; Christensen, Jan P.; Thomsen, Allan R.

2014-01-01

190

Stability of reference genes for normalization of reverse transcription quantitative real-time PCR (RT-qPCR) data in bovine blastocysts produced by IVF, ICSI and SCNT.  

PubMed

Summary Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a sensitive and accurate tool for quantitative estimation of gene transcription levels in preimplantation embryos. To control for possible experimental variations, gene expression data must be normalized using internal control genes commonly known as reference genes. However, the stability of reference genes can vary depending on the state of development and/or experimental conditions; hence the assessment of their stability is essential before initiating a gene expression analysis. In the present study, we used RT-qPCR to measure the transcript levels of 10 commonly used reference genes and analyzed their expression stability in bovine blastocysts produced by in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and somatic cell nuclear transfer (SCNT). Using the geNorm program, we found the best combination of genes to normalize gene expression data in bovine embryos at the blastocyst stage produced by IVF (HMBS, SF3A1, and HPRT1), ICSI (H2A, HMBS, and GAPDH), SCNT (ACTB, SF3A1, and SDHA) and/or between blastocysts produced by these methods (GAPDH, HMBS and EEF1A2). We also demonstrated that not only the culture conditions may affect the expression patterns in bovine blastocysts but also the choice of embryo production method may have an important effect. PMID:23731783

Luchsinger, Charlotte; Arias, María Elena; Vargas, Tamara; Paredes, Marcos; Sánchez, Raúl; Felmer, Ricardo

2014-11-01

191

QPCR Determined Fecal Indicator Bacterial Densities in Marine Waters from Two Recreational Beaches  

EPA Science Inventory

The use of real-time qPCR to determine fecal indicator bacteria (FIB) densities is currently being investigated by the U.S. EPA. The present recreational water quality guidelines, based on culturable FIB, prevent same day determinations of water quality whereas results from the ...

192

Effect of Environmental Parameters on the qPCR Signal of Enterococci in Tropical Waters  

EPA Science Inventory

Fecal contamination is the major source of pathogens in recreational waters. The need for quick public notifications has expanded the interest in the use of a rapid, quantitative polymerase chain reaction method (qPCR) to determine enterococci density. However, very little info...

193

Pregnancy Prediction in Single Embryo Transfer Cycles after ICSI Using QPCR: Validation in Oocytes from the Same Cohort  

PubMed Central

Cumulus cell (CC) gene expression is being explored as an additional method to morphological scoring to choose the embryo with the highest chance to pregnancy. In 47 ICSI patients with single embryo transfer (SET), from which individual CC samples had been stored, 12 genes using QPCR were retrospectively analyzed. The CC samples were at the same occasion also used to validate a previously obtained pregnancy prediction model comprising three genes (ephrin-B2 (EFNB2), calcium/calmodulin-dependent protein kinase ID, stanniocalcin 1). Latter validation yielded a correct pregnant/non-pregnant classification in 72% of the samples. Subsequently, 9 new genes were analyzed on the same samples and new prediction models were built. Out of the 12 genes analyzed a combination of the best predictive genes was obtained by stepwise multiple regression. One model retained EFNB2 in combination with glutathione S-transferase alpha 3 and 4, progesterone receptor and glutathione peroxidase 3, resulting in 93% correct predictions when 3 patient and treatment cycle characteristics were included into the model. This large patient group allowed to do an intra-patient analysis for 7 patients, an analysis mimicking the methodology that would ultimately be used in clinical routine. CC related to a SET that did not give pregnancy and CC related to their subsequent frozen/thawed embryos which ended in pregnancy were analyzed. The models obtained in the between-patient analysis were used to rank the oocytes within-patients for their chance to pregnancy and resulted in 86% of correct predictions. In conclusion, prediction models built on selected quantified transcripts in CC might help in the decision making process which is currently only based on subjective embryo morphology scoring. The validity of our current models for routine application still need prospective assessment in a larger and more diverse patient population allowing intra-patient analysis. PMID:23573182

Wathlet, Sandra; Adriaenssens, Tom; Segers, Ingrid; Verheyen, Greta; Van Landuyt, Lisbet; Coucke, Wim; Devroey, Paul; Smitz, Johan

2013-01-01

194

Validation of reference genes in cervical cell samples from human papillomavirus-infected and -uninfected women for quantitative reverse transcription-PCR assays.  

PubMed

Reference genes for quantitative reverse transcription-PCR (qRT-PCR) studies must be validated for the cell type studied and should be stable between the groups that represent the independent variable in an experimental design. We sought to identify the reference genes in cervical cell specimens showing the most stable expression between human papillomavirus (HPV)-infected and -uninfected women without high-grade cervical intraepithelial neoplasia. Using endocervical cells collected by cytology brush and Sybr green-based qRT-PCR, eight candidate genes were screened for amplification efficiency, specificity, and overall stability (by use of geNorm software). The five most stable genes were then further evaluated both for overall stability (geNorm) and intergroup stability (by use of NormFinder software) in specimens from HPV-negative and HPV-positive women. The combination of the glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and RPLP0 was the most stable overall, with a geNorm stability measure of 0.603. The intergroup analysis showed GAPDH to be the most stable single gene and RPLP0 to be second most stable and also showed that these genes represent the most stable two-gene combination, with a NormFinder stability value of 0.130. The fact that these two distinct approaches identified the same pair of genes provides added confidence that, when the focus is on HPV infection, a normalization factor derived from these two genes is likely to be appropriate. PMID:18632922

Daud, Ibrahim I; Scott, Mark E

2008-09-01

195

Parasite load estimation by qPCR differentiates between asymptomatic and symptomatic infection in Indian visceral leishmaniasis.  

PubMed

Using quantitative PCR (qPCR), we differentiated asymptomatic and symptomatic Indian Leishmania donovani infection. qPCR on blood of 40 visceral leishmaniasis, 130 endemic, and 40 non-endemic healthy controls showed 500 times less (P < .0001) parasitemia in asymptomatic compared to the symptomatic ones and threshold of 5 parasite genome/mL for the clinical disease. PMID:25023070

Sudarshan, Medhavi; Sundar, Shyam

2014-09-01

196

Development of a Pacific oyster (Crassostrea gigas) 31,918-feature microarray: identification of reference genes and tissue-enriched expression patterns  

PubMed Central

Background Research using the Pacific oyster Crassostrea gigas as a model organism has experienced rapid growth in recent years due to the development of high-throughput molecular technologies. As many as 56,268 EST sequences have been sequenced to date, representing a genome-wide resource that can be used for transcriptomic investigations. Results In this paper, we developed a Pacific oyster microarray containing oligonucleotides representing 31,918 transcribed sequences selected from the publicly accessible GigasDatabase. This newly designed microarray was used to study the transcriptome of male and female gonads, mantle, gills, posterior adductor muscle, visceral ganglia, hemocytes, labial palps and digestive gland. Statistical analyses identified genes differentially expressed among tissues and clusters of tissue-enriched genes. These genes reflect major tissue-specific functions at the molecular level, such as tissue formation in the mantle, filtering in the gills and labial palps, and reproduction in the gonads. Hierarchical clustering predicted the involvement of unannotated genes in specific functional pathways such as the insulin/NPY pathway, an important pathway under study in our model species. Microarray data also accurately identified reference genes whose mRNA level appeared stable across all the analyzed tissues. Adp-ribosylation factor 1 (arf1) appeared to be the most robust reference for normalizing gene expression data across different tissues and is therefore proposed as a relevant reference gene for further gene expression analysis in the Pacific oyster. Conclusions This study provides a new transcriptomic tool for studies of oyster biology, which will help in the annotation of its genome and which identifies candidate reference genes for gene expression analysis. PMID:21951653

2011-01-01

197

[Investigation of genes within copy number variation regions in pig chromosome 13 and analysis of the genetic law].  

PubMed

Copy number variation (CNV), referring to a genome structure variation, has attracted researchers' great interests. Thirty-two CNV regions (CNV region, CNVR) have been detected on chromosome 13 in our previous work. In order to detect the genes located in these CNVRs, we first obtained the annotated information from Ensembl database, and searched gene functional enrichments using DAVID online tools. In the 32 CNVRs, a total of 236 genes were identified, in which 169 genes were annotated. Gene Ontology (GO) analysis revealed that these genes mainly participate in proteolysis, cell adhesion, and macromolecular catabolic process. To study the genetic law of these CNVs, we chose the RCAN1 (regulators of calcineurin 1) gene as the candidate. We quantified the copy number of RCAN1 gene in 38 Laiwu pigs by using QPCR method, and analyzed the genetic laws in three Laiwu families including 15 pigs. QPCR results showed that both duplication and deletion occurred in RCAN1 gene among Laiwu pigs and the heredity mode corresponds with Mendelian genetic law. PMID:24846980

Liu, Jing; Wang, Yanan; Sun, Yaqi; Wang, Hongyang; Wang, Chao; Peng, Zhongzhen; Liu, Bang

2014-04-01

198

International collaborative study of the endogenous reference gene, sucrose phosphate synthase (SPS), used for qualitative and quantitative analysis of genetically modified rice.  

PubMed

One rice ( Oryza sativa ) gene, sucrose phosphate synthase (SPS), has been proven to be a suitable endogenous reference gene for genetically modified (GM) rice detection in a previous study. Herein are the reported results of an international collaborative ring trial for validation of the SPS gene as an endogenous reference gene and its optimized qualitative and quantitative polymerase chain reaction (PCR) systems. A total of 12 genetically modified organism (GMO) detection laboratories from seven countries participated in the ring trial and returned their results. The validated results confirmed the species specificity of the method through testing 10 plant genomic DNAs, low heterogeneity, and a stable single-copy number of the rice SPS gene among 7 indica varieties and 5 japonica varieties. The SPS qualitative PCR assay was validated with a limit of detection (LOD) of 0.1%, which corresponded to about 230 copies of haploid rice genomic DNA, while the limit of quantification (LOQ) for the quantitative PCR system was about 23 copies of haploid rice genomic DNA, with acceptable PCR efficiency and linearity. Furthermore, the bias between the test and true values of eight blind samples ranged from 5.22 to 26.53%. Thus, we believe that the SPS gene is suitable for use as an endogenous reference gene for the identification and quantification of GM rice and its derivates. PMID:19326953

Jiang, Lingxi; Yang, Litao; Zhang, Haibo; Guo, Jinchao; Mazzara, Marco; Van den Eede, Guy; Zhang, Dabing

2009-05-13

199

A Comprehensive Approach to Identify Reliable Reference Gene Candidates to Investigate the Link between Alcoholism and Endocrinology in Sprague-Dawley Rats  

PubMed Central

Gender and hormonal differences are often correlated with alcohol dependence and related complications like addiction and breast cancer. Estrogen (E2) is an important sex hormone because it serves as a key protein involved in organism level signaling pathways. Alcoholism has been reported to affect estrogen receptor signaling; however, identifying the players involved in such multi-faceted syndrome is complex and requires an interdisciplinary approach. In many situations, preliminary investigations included a straight forward, yet informative biotechniques such as gene expression analyses using quantitative real time PCR (qRT-PCR). The validity of qRT-PCR-based conclusions is affected by the choice of reliable internal controls. With this in mind, we compiled a list of 15 commonly used housekeeping genes (HKGs) as potential reference gene candidates in rat biological models. A comprehensive comparison among 5 statistical approaches (geNorm, dCt method, NormFinder, BestKeeper, and RefFinder) was performed to identify the minimal number as well the most stable reference genes required for reliable normalization in experimental rat groups that comprised sham operated (SO), ovariectomized rats in the absence (OVX) or presence of E2 (OVXE2). These rat groups were subdivided into subgroups that received alcohol in liquid diet or isocalroic control liquid diet for 12 weeks. Our results showed that U87, 5S rRNA, GAPDH, and U5a were the most reliable gene candidates for reference genes in heart and brain tissue. However, different gene stability ranking was specific for each tissue input combination. The present preliminary findings highlight the variability in reference gene rankings across different experimental conditions and analytic methods and constitute a fundamental step for gene expression assays. PMID:24824616

Taki, Faten A.; Abdel-Rahman, Abdel A.; Zhang, Baohong

2014-01-01

200

Sample-ready multiplex qPCR assay for detection of malaria  

PubMed Central

Background Microscopy and antigen detecting rapid diagnostic tests are the diagnostic tests of choice in management of clinical malaria. However, due to their limitations, the need to utilize more sensitive methods such as real-time PCR (qPCR) is evident as more studies are now utilizing molecular methods in detection of malaria. Some of the challenges that continue to limit the widespread utilization of qPCR include lack of assay standardization, assay variability, risk of contamination, and the need for cold-chain. Lyophilization of molecular assays can overcome some of these limitations and potentially enable widespread qPCR utilization. Methods A recently published multiplex malaria qPCR assay was lyophilized by freezing drying into Sample-Ready™ format (MMSR). MMSR assay contained all the required reagents for qPCR including primers and probes, requiring only the addition of water and sample to perform qPCR. The performance of the MMSR assay was compared to the non-freeze dried, “wet” assay. Stability studies were done by maintaining the MMSR assays at four different ambient temperatures of 4°C, room temperature (RT), 37°C and 42°C over a period of 42 days, tested at seven-day intervals. Plasmodium falciparum and Plasmodium vivax DNAs were used for analysis of the MMSR assay either as single or mixed parasites, at two different concentrations. The CT values and the standard deviations (SD) were used in the analysis of the assay performance. Results The limit of detection for the MMSR assay was 0.244 parasites/?L for Plasmodium spp. (PLU) and P. falciparum (FAL) assay targets compared to “wet” assay which was 0.39 and 3.13 parasites/?L for PLU and FAL assay targets, respectively. The MMSR assay performed with high efficiencies similar to those of the “wet” assay and was stable at 37°C for 42 days, with estimated shelf-life of 5 months. When used to analyse field clinical samples, MMSR assay performed with 100% sensitivity and specificity compared to the “wet” assay. Conclusion The MMSR assay has the same robust performance characteristics as the “wet” assay and is highly stable. Availability of MMSR assay allows flexibility and provides an option in choosing assay for malaria diagnostics depending on the application, needs and budget. PMID:24767409

2014-01-01

201

A rapid qPCR method for genetic sex identification of Salmo salar and Salmo trutta including simultaneous elucidation of interspecies hybrid paternity by high-resolution melt analysis.  

PubMed

This study presents an improved duplex quantitative polymerase chain reaction (qPCR) method using the master sex-determining gene sdY as a marker for simultaneous genetic sex identification of salmonids of the Salmo genus and paternity elucidation for Salmo salar × Salmo trutta hybrids. This method will provide a new, simple and economical molecular tool for ecological studies of these species as well as for aquaculture purposes. PMID:24814478

Anglès d'Auriac, M B; Urke, H A; Kristensen, T

2014-06-01

202

Circumventing qPCR inhibition to amplify miRNAs in plasma  

PubMed Central

Background Circulating microRNAs (c-miRNAs) have be identified in saliva, urine and blood, which has led to increasing interest in their development as biomarkers for diverse diseases including cancers. One of the key advantages of c-miRNAs over other biomarkers is the ability to be amplified and quantified by quantitative PCR (qPCR). However, at phlebotomy when whole blood is dispensed into heparinized tubes, residual levels of the anti-coagulant lithium heparin may remain in the plasma and hence with RNA isolated from the plasma. This can confound the detection of c-miRNAs by qPCR because it inhibits reverse transcriptase (RT). Here we present a procedure, modified from earlier techniques, to detect c-miRNAs in plasma that improves sensitivity and streamlines performance. Findings Treatment of total RNA isolated from human blood plasma with Bacteroides heparinase I during reverse transcription at 37°C for one hour improved sensitivity and performance of the qPCR. This is in comparison to no treatment or treatment of the RNA prior to RT, which is the current suggested method and exposes plasma to Flavobacterium heparinum heparinase I for up to 2 hours before RT. This modest alteration improved qPCR performance and resulted in lowered threshold cycles (Ct) for detection of the target sequence, candidate c-miRNA biomarkers, and controls. It also reduced the expense and number of processing steps, shortening the duration of the assay and minimizing exposure of RNA to elevated temperatures. Conclusion Incorporating Bacteroides heparinase I treatment into conventional RT protocols targeting c-miRNA in plasma can be expected to expedite the discovery of biomarkers. PMID:25075309

2014-01-01

203

The need for transparency and good practices in the qPCR literature.  

PubMed

Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not. PMID:24173381

Bustin, Stephen A; Benes, Vladimir; Garson, Jeremy; Hellemans, Jan; Huggett, Jim; Kubista, Mikael; Mueller, Reinhold; Nolan, Tania; Pfaffl, Michael W; Shipley, Gregory; Wittwer, Carl T; Schjerling, Peter; Day, Philip J; Abreu, Mónica; Aguado, Begoña; Beaulieu, Jean-François; Beckers, Anneleen; Bogaert, Sara; Browne, John A; Carrasco-Ramiro, Fernando; Ceelen, Liesbeth; Ciborowski, Kate; Cornillie, Pieter; Coulon, Stephanie; Cuypers, Ann; De Brouwer, Sara; De Ceuninck, Leentje; De Craene, Jurgen; De Naeyer, Hélène; De Spiegelaere, Ward; Deckers, Kato; Dheedene, Annelies; Durinck, Kaat; Ferreira-Teixeira, Margarida; Fieuw, Annelies; Gallup, Jack M; Gonzalo-Flores, Sandra; Goossens, Karen; Heindryckx, Femke; Herring, Elizabeth; Hoenicka, Hans; Icardi, Laura; Jaggi, Rolf; Javad, Farzad; Karampelias, Michael; Kibenge, Frederick; Kibenge, Molly; Kumps, Candy; Lambertz, Irina; Lammens, Tim; Markey, Amelia; Messiaen, Peter; Mets, Evelien; Morais, Sofia; Mudarra-Rubio, Alberto; Nakiwala, Justine; Nelis, Hilde; Olsvik, Pal A; Pérez-Novo, Claudina; Plusquin, Michelle; Remans, Tony; Rihani, Ali; Rodrigues-Santos, Paulo; Rondou, Pieter; Sanders, Rebecca; Schmidt-Bleek, Katharina; Skovgaard, Kerstin; Smeets, Karen; Tabera, Laura; Toegel, Stefan; Van Acker, Tim; Van den Broeck, Wim; Van der Meulen, Joni; Van Gele, Mireille; Van Peer, Gert; Van Poucke, Mario; Van Roy, Nadine; Vergult, Sarah; Wauman, Joris; Tshuikina-Wiklander, Marina; Willems, Erik; Zaccara, Sara; Zeka, Fjoralba; Vandesompele, Jo

2013-11-01

204

Estimation of the Relative Sensitivity of qPCR Analysis Using Pooled Samples  

PubMed Central

The high sensitivity of qPCR makes it a desirable diagnostic method in epidemiological surveillance programs. However, due to high costs, the use of pooling has been suggested. In this paper, an algorithm based on the Montecarlo method has been designed and implemented. The algorithm had been tested in many different situations, and finally it was validated with a real dataset. Moreover, based on the results obtained and depending on pooling conditions, a drastic decrease of sensitivity is observed. PMID:24722485

Muniesa, Ana; Ferreira, Chelo; Fuertes, Hector; Halaihel, Nabil; de Blas, Ignacio

2014-01-01

205

Interlaboratory comparison of three microbial source tracking quantitative polymerase chain reaction (qPCR) assays from fecal-source and environmental samples  

USGS Publications Warehouse

During summer and early fall 2010, 15 river samples and 6 fecal-source samples were collected in West Virginia. These samples were analyzed by three laboratories for three microbial source tracking (MST) markers: AllBac, a general fecal indicator; BacHum, a human-associated fecal indicator; and BoBac, a ruminant-associated fecal indicator. MST markers were analyzed by means of the quantitative polymerase chain reaction (qPCR) method. The aim was to assess interlaboratory precision when the three laboratories used the same MST marker and shared deoxyribonucleic acid (DNA) extracts of the samples, but different equipment, reagents, and analyst experience levels. The term assay refers to both the markers and the procedure differences listed above. Interlaboratory precision was best for all three MST assays when using the geometric mean absolute relative percent difference (ARPD) and Friedman's statistical test as a measure of interlaboratory precision. Adjustment factors (one for each MST assay) were calculated using results from fecal-source samples analyzed by all three laboratories and applied retrospectively to sample concentrations to account for differences in qPCR results among labs using different standards and procedures. Following the application of adjustment factors to qPCR results, ARPDs were lower; however, statistically significant differences between labs were still observed for the BacHum and BoBac assays. This was a small study and two of the MST assays had 52 percent of samples with concentrations at or below the limit of accurate quantification; hence, more testing could be done to determine if the adjustment factors would work better if the majority of sample concentrations were above the quantification limit.

Stelzer, Erin A.; Strickler, Kriston M.; Schill, William B.

2012-01-01

206

Selection of reference genes for quantitative RT-PCR studies in Rhipicephalus (Boophilus) microplus and Rhipicephalus appendiculatus ticks and determination of the expression profile of Bm86  

PubMed Central

Background For accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is essential. In most studies on ticks where (semi-)quantitative RT-PCR is employed, normalization occurs with a single reference gene, usually ?-actin, without validation of its presumed expression stability. The first goal of this study was to evaluate the expression stability of commonly used reference genes in Rhipicephalus appendiculatus and Rhipicephalus (Boophilus) microplus ticks. To demonstrate the usefulness of these results, an unresolved issue in tick vaccine development was examined. Commercial vaccines against R. microplus were developed based on the recombinant antigen Bm86, but despite a high degree of sequence homology, these vaccines are not effective against R. appendiculatus. In fact, Bm86-based vaccines give better protection against some tick species with lower Bm86 sequence homology. One possible explanation is the variation in Bm86 expression levels between R. microplus and R. appendiculatus. The most stable reference genes were therefore used for normalization of the Bm86 expression profile in all life stages of both species to examine whether antigen abundance plays a role in Bm86 vaccine susceptibility. Results The transcription levels of nine potential reference genes: ?-actin (ACTB), ?-tubulin (BTUB), elongation factor 1? (ELF1A), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), glutathione S-transferase (GST), H3 histone family 3A (H3F3A), cyclophilin (PPIA), ribosomal protein L4 (RPL4) and TATA box binding protein (TBP) were measured in all life stages of R. microplus and R. appendiculatus. ELF1A was found to be the most stable expressed gene in both species following analysis by both geNorm and Normfinder software applications, GST showed the least stability. The expression profile of Bm86 in R. appendiculatus and R. microplus revealed a more continuous Bm86 antigen abundance in R. microplus throughout its one-host life cycle compared to the three-host tick R. appendiculatus where large variations were observed between different life stages. Conclusion Based on these results, ELF1A can be proposed as an initial reference gene for normalization of quantitative RT-PCR data in whole R. microplus and R. appendiculatus ticks. The observed differences in Bm86 expression profile between the two species alone can not adequately explain the lack of a Bm86 vaccination effect in R. appendiculatus. PMID:20040102

2009-01-01

207

Validation of an rpoB gene PCR assay for detection of Tropheryma whipplei: 10 years' experience in a National Reference Laboratory.  

PubMed

The performance of a real-time PCR assay targeting the Tropheryma whipplei rpoB gene was evaluated using test strains and 1,236 clinical specimens in a national reference laboratory. The novel rpoB-PCR assay proved to be specific, revealed improved analytical sensitivity, and substantially accelerated detection of T. whipplei DNA in clinical specimens. PMID:23966507

Moter, Annette; Schmiedel, Dinah; Petrich, Annett; Wiessner, Alexandra; Kikhney, Judith; Schneider, Thomas; Moos, Verena; Göbel, Ulf B; Reischl, Udo

2013-11-01

208

Development of TaqMan-based qPCR method for detection of caprine arthritis-encephalitis virus (CAEV) infection.  

PubMed

A specific and sensitive two-step TaqMan real-time PCR has been developed for rapid diagnosis of caprine arthritis-encephalitis virus (CAEV) infection by using a set of specific primers and a TaqMan probe targeting a highly conserved region within the gene encoding the viral capsid protein (CA). The assay successfully detected CAEV proviral DNA in total DNA extracts originating from cell culture, whole blood samples and isolated PBMCs, with a lower detection limit of 10(2) copies and a linear dynamic range of 10(5) to 10(10) copies/ml. There was no cross-reaction with other animal viruses (e.g., goat pox virus, bovine leukemia virus, bovine mucosal disease virus, swine influenza virus and Nipah virus). When applied in parallel with serological AGID and conventional PCR for detection of CAEV in field samples, this assay exhibited a higher sensitivity than these traditional methods, and 7.8 % of the 308 specimens collected in the Shanxi and Tianjin regions of China from 1993 to 2011 were found to be positive. Thus, the TaqMan qPCR assay provides a fast, specific and sensitive means for detecting CAEV proviral DNA in goat specimens and should be useful for large-scale detection in eradication programs and epidemiological studies. PMID:23670072

Li, Yi; Zhou, Fengjuan; Li, Xia; Wang, Jianhua; Zhao, Xiangping; Huang, Jinhai

2013-10-01

209

Construction of Genetically Engineered Streptococcus gordonii Strains to Provide Control in QPCR Assays for Assessing Microbiological Quality in Recreational Water.  

EPA Science Inventory

Quantitative PCR (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for...

210

Validation of reference genes for the determination of platelet transcript level in healthy individuals and in patients with the history of myocardial infarction.  

PubMed

RT-qPCR is the standard method for studying changes in relative transcript level in different experimental and clinical conditions and in different tissues. No validated reference genes have been reported for the normalization of transcript level in platelets. The very low level of platelet RNA and the elimination of leukocyte contamination represented special methodological difficulties. Our aims were to apply a simple technique to separate platelets for transcript level studies, and select the most stable reference genes for platelets from healthy individuals and from patients with the history of myocardial infarction. We developed a simple, straightforward method of platelet separation for RNA isolation. Platelet activation was inhibited by using acid-citrate-dextrose for anticoagulation and by prostaglandin E1. Leukocyte contamination was eliminated by three consecutive centrifugations. Samples prepared by this method were free of leukocytes, showed no inhibition in PCR reaction and no RNA degradation. The assay demands low blood volume, which complies with the requirements of everyday laboratory routine. Seventeen potential reference genes were investigated, but eight of them were excluded during optimization. The stability of the remaining genes, EEF2, EAR, ACTB, GAPDH, ANAPC5, OAZ1, HDGF, GNAS, and CFL1, were determined by four different descriptive statistics. GAPDH, GNAS, and ACTB were shown to be the most stable genes in platelets of healthy individuals, while HDGF, GNAS, and ACTB were the most stable in platelets of patients with the history of myocardial infarction. The results confirm that data normalization needs assessment of appropriate reference genes for a particular sample set. PMID:23389042

Zsóri, Katalin S; Muszbek, László; Csiki, Zoltán; Shemirani, Amir H

2013-01-01

211

Gene transcription in sea otters (Enhydra lutris); development of a diagnostic tool for sea otter and ecosystem health  

USGS Publications Warehouse

Gene transcription analysis for diagnosing or monitoring wildlife health requires the ability to distinguish pathophysiological change from natural variation. Herein, we describe methodology for the development of quantitative real-time polymerase chain reaction (qPCR) assays to measure differential transcript levels of multiple immune function genes in the sea otter (Enhydra lutris); sea otter-specific qPCR primer sequences for the genes of interest are defined. We establish a ‘reference’ range of transcripts for each gene in a group of clinically healthy captive and free-ranging sea otters. The 10 genes of interest represent multiple physiological systems that play a role in immuno-modulation, inflammation, cell protection, tumour suppression, cellular stress response, xenobiotic metabolizing enzymes, antioxidant enzymes and cell–cell adhesion. The cycle threshold (CT) measures for most genes were normally distributed; the complement cytolysis inhibitor was the exception. The relative enumeration of multiple gene transcripts in simple peripheral blood samples expands the diagnostic capability currently available to assess the health of sea otters in situ and provides a better understanding of the state of their environment.

Bowen, Lizabeth; Miles, A. Keith; Murray, Michael; Haulena, Martin; Tuttle, Judy; van Bonn, William; Adams, Lance; Bodkin, James L.; Ballachey, Brenda; Estes, James; Tinker, M. Tim; Keister, Robin; Stott, Jeffrey L.

2012-01-01

212

A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.  

PubMed

The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean. PMID:25078400

Venturelli, Gustavo L; Brod, Fábio C A; Rossi, Gabriela B; Zimmermann, Naíra F; Oliveira, Jaison P; Faria, Josias C; Arisi, Ana C M

2014-11-01

213

Comparative evaluation of rumen metagenome community using qPCR and MG-RAST  

PubMed Central

Microbial profiling of metagenome communities have been studied extensively using MG-RAST and other related metagenome annotation databases. Although, database based taxonomic profiling provides snapshots of the metagenome architecture, their reliability needs to be validated through more accurate methods. Here, we performed qPCR based absolute quantitation of selected rumen microbes in the liquid and solid fraction of the rumen fluid of river buffalo adapted to varying proportion of concentrate to green or dry roughages and compared with the MG-RAST based annotation of the metagenomes sequences of 16S r-DNA amplicons and high throughput shotgun sequencing. Animals were adapted to roughage-to-concentrate ratio in the proportion of 50:50, 75:25 and 100:00, respectively for six weeks. At the end of each treatment, rumen fluid was collected at 3 h post feeding. qPCR revealed that the relative abundance of Prevotella bryantii was higher, followed by the two cellulolytic bacteria Fibrobacter succinogens and Ruminococcus flavefaciens that accounted up to 1.33% and 0.78% of the total rumen bacteria, respectively. While, Selenomonas ruminantium and archaea Methanomicrobiales were lower in microbial population in the rumen of buffalo. There was no statistically significant difference between the enumerations shown by qPCR and analysis of the shotgun sequencing data by MG-RAST except for Prevotella. These results indicate the variations in abundance of different microbial species in buffalo rumen under varied feeding regimes as well as in different fractions of rumen liquor, i.e. solid and the liquid. The results also present the reliability of shotgun sequencing to describe metagenome and analysis/annotation by MG-RAST. PMID:24025701

2013-01-01

214

Expressed Repeat Elements Improve RT-qPCR Normalization across a Wide Range of Zebrafish Gene Expression Studies  

PubMed Central

The selection and validation of stably expressed reference genes is a critical issue for proper RT-qPCR data normalization. In zebrafish expression studies, many commonly used reference genes are not generally applicable given their variability in expression levels under a variety of experimental conditions. Inappropriate use of these reference genes may lead to false interpretation of expression data and unreliable conclusions. In this study, we evaluated a novel normalization method in zebrafish using expressed repetitive elements (ERE) as reference targets, instead of specific protein coding mRNA targets. We assessed and compared the expression stability of a number of EREs to that of commonly used zebrafish reference genes in a diverse set of experimental conditions including a developmental time series, a set of different organs from adult fish and different treatments of zebrafish embryos including morpholino injections and administration of chemicals. Using geNorm and rank aggregation analysis we demonstrated that EREs have a higher overall expression stability compared to the commonly used reference genes. Moreover, we propose a limited set of ERE reference targets (hatn10, dna15ta1 and loopern4), that show stable expression throughout the wide range of experiments in this study, as strong candidates for inclusion as reference targets for qPCR normalization in future zebrafish expression studies. Our applied strategy to find and evaluate candidate expressed repeat elements for RT-qPCR data normalization has high potential to be used also for other species. PMID:25310091

Vanhauwaert, Suzanne; Van Peer, Gert; Rihani, Ali; Janssens, Els; Rondou, Pieter; Lefever, Steve; De Paepe, Anne; Coucke, Paul J.; Speleman, Frank; Vandesompele, Jo; Willaert, Andy

2014-01-01

215

Novel reference gene, High-mobility-group protein I/Y, used in qualitative and real-time quantitative polymerase chain reaction detection of transgenic rapeseed cultivars.  

PubMed

With the development of transgenic crops, regulations to label the genetically modified organisms (GMOs) and their derived products have been issued in many countries. Polymerase chain reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods are generally needed to amplify the transgene and compare the amplified results with that of a corresponding reference gene to get the reliable results. Specific primers were developed for the rapeseed (Brassica napus), high-mobility-group protein I/Y(HMG-I/Y) single-copy gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 15 different rapeseed varieties, and identical amplified products were obtained with all of them. No amplification was observed when templates were the DNA samples from the other species of Brassica genus or other species, such as broccoli, stem mustard, cauliflower, Chinese cabbage, cabbage, sprouts, Arabidopsis thaliana, carrot, tobacco, soybean, mung bean, tomato, pepper, eggplant, plum, wheat, maize, barley, rice, lupine, and sunflower. This system was specific for rapeseed. Limits of detection and quantitation in qualitative and quantitative PCR systems were about 13 pg DNA (about 10 haploid genomes) and about 1.3 pg DNA (about 1 haploid genome), respectively. To further test the feasibility of this HMG-I/Y gene as an endogenous reference gene, samples containing transgenic rapeseed GT73 with the inserted glyphosate oxidoreductase (GOX) gene were quantitated. These demonstrated that the endogenous PCR detection systems were applicable to the qualitative and quantitative detection of transgenic rapeseed. PMID:15859086

Weng, Haibo; Yang, Litao; Liu, Zhili; Ding, Jiayu; Pan, Aihu; Zhang, Dabing

2005-01-01

216

Selection and validation of reference genes for real-time RT-PCR studies in the non-model species Delomys sublineatus, an endemic Brazilian rodent.  

PubMed

Quantitative real-time RT-PCR (qRT-PCR) is a sensitive technique for gene expression analysis. A critical factor for creating reliable data in relative quantification is the normalization of the expression data of genes of interest. Therefore the needed normalization factor is calculated out of the expression data of co-amplified genes that are stable expressed in the certain sample material, the so-called reference genes. In this study, we demonstrate the important process of validating potential reference genes using a non-model species. As there are almost no sequences known of the Pallid Atlantic Forest Rat (Delomys sublineatus), a rodent used as indicator species in conservation studies of the endangered Brazilian rainforest, suitable primer sets are more problematic to find than in model species. Out of nine tested primer sets designed for the fully sequenced Mus musculus, five could be used for the establishment of a proper running SYBR-Green assay and validation of their constant expression. qRT-PCR results of 12 cDNAs of Delomys livers were analyzed with three different validation software programs: BestKeeper, NormFinder and geNorm. Our approach showed that out of the five (Sdha, Canx, Pgk1, Actb and Actg1) potential reference genes, the first four should be used for accurate normalization in further relative quantification analyses. Transferring data from close-by model organisms makes high sensitive real-time RT-PCR applicable even to free-ranging non-model organisms. Our approach might be suitable for other non-model organisms. PMID:20059981

Weyrich, Alexandra; Axtner, Jan; Sommer, Simone

2010-02-01

217

Global Genome Transcription Profiling of Bifidobacterium bifidum PRL2010 under In Vitro Conditions and Identification of Reference Genes for Quantitative Real-Time PCR?†  

PubMed Central

Bifidobacteria have attracted significant scientific attention due to their perceived role as health-promoting microorganisms, although the genetics of the bacterial group is still underexplored. In this study, we investigated the transcriptome of Bifidobacterium bifidum PRL2010 during in vitro growth by microarray technology. When B. bifidum PRL2010 was grown in liquid broth, 425 of the 1,644 PRL2010 genes represented on the array were expressed in at least one of the three investigated growth phases, i.e., the lag, exponential, and stationary phases. These transcriptional analyses identified a core in vitro transcriptome encompassing 150 genes that are expressed in all phases. A proportion of these genes were further investigated as potential reference genes by quantitative real-time reverse transcription-PCR (qRT-PCR) assays. Their expression stability was evaluated under different growth conditions, which included cultivation on different carbon sources, exposure to environmental stresses (thermal, acidic, and osmotic), and growth phases. Our analyses validated six reference genes suitable for normalizing mRNA expression levels in qRT-PCR experiments applied to bifidobacteria. PMID:22003014

Turroni, Francesca; Foroni, Elena; Montanini, Barbara; Viappiani, Alice; Strati, Francesco; Duranti, Sabrina; Ferrarini, Alberto; Delledonne, Massimo; van Sinderen, Douwe; Ventura, Marco

2011-01-01

218

Evaluation of quantitative real-time PCR workflow modifications on 16S rRNA and tetA gene quantification in environmental samples.  

PubMed

The study examined the variability in 16S ribosomal RNA (16S rRNA) and tetracycline resistance tetA gene quantification from environmental samples in relation to modifications in quantitative polymerase chain reaction (qPCR) workflow and subsequent data evaluation and analysis. We analysed three types of soil samples using two DNA extraction methods, two qPCR chemistries (SYBR green, LUX™), and qPCR reaction kits from different manufacturers. To improve data quality, we employed a three-step amplification outlier removal approach prior to gene quantification calculations. We compared three variants of target gene enumerations and four variants of functional tetA gene normalisations against 16S rRNA genes. Results reveal that modifications in qPCR workflow steps significantly influence the gene quantification results from environmental samples. Primary factors affecting qPCR amplification efficiency included the variability of the target amplicon and the qPCR chemistry; the quality of the resulting datasets also had an impact. Although LUX™ qPCR has shown promise for environmental samples, SYBR green qPCR yielded considerably better-quality datasets and higher, more stable amplification efficiency values. Gene enumeration data of outlier-removed and unmodified sample sets showed minor differences for good-quality datasets (i.e., amplifications with SYBR green), but differed by up to 40% among lower-quality datasets. Different DNA extraction methods yielded varying amounts and purities of extracted microbial community DNA from environmental samples, with as much as an order of magnitude variation in gene copy numbers. Target gene normalisations yielded stable results on good-quality data, regardless of the DNA extraction method or qPCR chemistry used. Even though qPCR is regarded as a precise method with low detection limit, technical variability in the qPCR workflow tends to overestimate or effectively mask minute changes in community. PMID:22521102

Nõlvak, Hiie; Truu, Marika; Truu, Jaak

2012-06-01

219

Multiplex qPCR for reliable detection and differentiation of Burkholderia mallei and Burkholderia pseudomallei  

PubMed Central

Background Burkholderia mallei and B. pseudomallei are two closely related species of highly virulent bacteria that can be difficult to detect. Pathogenic Burkholderia are endemic in many regions worldwide and cases of infection, sometimes brought by travelers from unsuspected regions, also occur elsewhere. Rapid, sensitive methods for identification of B. mallei and B. pseudomallei are urgently needed in the interests of patient treatment and epidemiological surveillance. Methods Signature sequences for sensitive, specific detection of pathogenic Burkholderia based on published genomes were identified and a qPCR assay was designed and validated. Results A single-reaction quadruplex qPCR assay for the detection of pathogenic Burkholderia, which includes a marker for internal control of DNA extraction and amplification, was developed. The assay permits differentiation of B. mallei and B. pseudomallei strains, and probit analysis showed a very low detection limit. Use of a multicopy signature sequence permits detection of less than 1 genome equivalent per reaction. Conclusions The new assay permits rapid detection of pathogenic Burkholderia and combines enhanced sensitivity, species differentiation, and inclusion of an internal control for both DNA extraction and PCR amplification. PMID:23409683

2013-01-01

220

Optimization of RNA Extraction for PCR Quantification of Aromatic Compound Degradation Genes? †  

PubMed Central

Seven different bacterial strains and primer sets and a mixed community were used to evaluate the use of reverse transcriptase quantitative PCR (Q-PCR) and Q-PCR of oxygenase genes to assess various approaches for monitoring the bioremediation of polluted sites. Differences in maximum activity were seen when different RNA extraction kits were compared. PMID:20023086

Kong, Weidong; Nakatsu, Cindy H.

2010-01-01

221

Assessment and validation of a suite of reverse transcription-quantitative PCR reference genes for analyses of density-dependent behavioural plasticity in the Australian plague locust  

PubMed Central

Background The Australian plague locust, Chortoicetes terminifera, is among the most promising species to unravel the suites of genes underling the density-dependent shift from shy and cryptic solitarious behaviour to the highly active and aggregating gregarious behaviour that is characteristic of locusts. This is because it lacks many of the major phenotypic changes in colour and morphology that accompany phase change in other locust species. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most sensitive method available for determining changes in gene expression. However, to accurately monitor the expression of target genes, it is essential to select an appropriate normalization strategy to control for non-specific variation between samples. Here we identify eight potential reference genes and examine their expression stability at different rearing density treatments in neural tissue of the Australian plague locust. Results Taking advantage of the new orthologous DNA sequences available in locusts, we developed primers for genes encoding 18SrRNA, ribosomal protein L32 (RpL32), armadillo (Arm), actin 5C (Actin), succinate dehydrogenase (SDHa), glyceraldehyde-3P-dehydrogenase (GAPDH), elongation factor 1 alpha (EF1a) and annexin IX (AnnIX). The relative transcription levels of these eight genes were then analyzed in three treatment groups differing in rearing density (isolated, short- and long-term crowded), each made up of five pools of four neural tissue samples from 5th instar nymphs. SDHa and GAPDH, which are both involved in metabolic pathways, were identified as the least stable in expression levels, challenging their usefulness in normalization. Based on calculations performed with the geNorm and NormFinder programs, the best combination of two genes for normalization of gene expression data following crowding in the Australian plague locust was EF1a and Arm. We applied their use to studying a target gene that encodes a Ca2+ binding glycoprotein, SPARC, which was previously found to be up-regulated in brains of gregarious desert locusts, Schistocerca gregaria. Interestingly, expression of this gene did not vary with rearing density in the same way in brains of the two locust species. Unlike S. gregaria, there was no effect of any crowding treatment in the Australian plague locust. Conclusion Arm and EF1a is the most stably expressed combination of two reference genes of the eight examined for reliable normalization of RT-qPCR assays studying density-dependent behavioural change in the Australian plague locust. Such normalization allowed us to show that C. terminifera crowding did not change the neuronal expression of the SPARC gene, a gregarious phase-specific gene identified in brains of the desert locust, S. gregaria. Such comparative results on density-dependent gene regulation provide insights into the evolution of gregarious behaviour and mass migration of locusts. The eight identified genes we evaluated are also candidates as normalization genes for use in experiments involving other Oedipodinae species, but the rank order of gene stability must necessarily be determined on a case-by-case basis. PMID:21324174

2011-01-01

222

Characterization of EvaGreen and the implication of its physicochemical properties for qPCR applications  

PubMed Central

Background EvaGreen (EG) is a newly developed DNA-binding dye that has recently been used in quantitative real-time PCR (qPCR), post-PCR DNA melt curve analysis and several other applications. However, very little is known about the physicochemical properties of the dye and their relevance to the applications, particularly to qPCR and post PCR DNA melt curve analysis. In this paper, we characterized EG along with a widely used qPCR dye, SYBR Green I (SG), for their DNA-binding properties and stability, and compared their performance in qPCR under a variety of conditions. Results This study systematically compared the DNA binding profiles of the two dyes under different conditions and had these findings: a) EG had a lower binding affinity for both double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA) than SG; b) EG showed no apparent preference for either GC- or AT-rich sequence while SG had a slight preference for AT-rich sequence; c) both dyes showed substantially lower affinity toward ssDNA than toward dsDNA and even lower affinity toward shorter ssDNA fragments except that this trend was more pronounced for EG. Our results also demonstrated that EG was stable both under PCR condition and during routine storage and handling. In the comparative qPCR study, both EG and SG exhibited PCR interference when used at high dye concentration, as evident from delayed Ct and/or nonspecific product formation. The problem worsened when the chain extension time was shortened or when the amplicon size was relatively long (>500 bp). However, qPCR using EG tolerated a significantly higher dye concentration, thus permitting a more robust PCR signal as well as a sharper and stronger DNA melt peak. These differences in qPCR performance between the two dyes are believed to be attributable to their differences in DNA binding profiles. Conclusion These findings suggest that an ideal qPCR dye should possess several DNA-binding characteristics, including a "just right" affinity for dsDNA and low or no affinity for ssDNA and short DNA fragments. The favorable DNA-binding profile of EG, coupled with its good stability and instrument-compatibility, should make EG a promising dye for qPCR and related applications. PMID:17996102

Mao, Fei; Leung, Wai-Yee; Xin, Xing

2007-01-01

223

Comparisons of statistical models to predict fecal indicator bacteria concentrations enumerated by qPCR- and culture-based methods.  

PubMed

Recently, the United States Environmental Protection Agency (USEPA) revised their recreational water quality criteria, in which adjustments were made by approving enterococci (ENT) quantitative PCR (qPCR) as an alternative, rapid method and advocating the use of predictive models for water quality management. The implementation of qPCR-based methods and prediction models are meant to decrease the time between sample collection and public advisories and notifications. To date, few studies have compared qPCR-based models to culture-based prediction models and none of these studies have been conducted in coastal estuarine systems. In this study, we created prediction models using qPCR-based fecal indicator bacteria (FIB) data in dual-use recreational and shellfish harvesting waters and compared them to published ENT and Escherichia coli (EC) culture-based prediction models in eastern North Carolina estuaries. Furthermore, an empirical statistical model was created to predict qPCR inhibition levels so that proper remediation techniques can be applied when it is a problem. Predictor variable selection in both qPCR- and culture-based ENT models was very similar; both models included 14-day rain total, dissolved oxygen, and salinity/conductivity, with 89 and 90% of qPCR and culture data described, respectively. Using ENT management action thresholds, qPCR- and culture-based models showed high accuracy in management decisions. The qPCR model had 92 and 96% accuracy using the 110 and 1000 cell equivalents (CE)/100 ml thresholds, respectively, and the culture model had 90% accuracy in management decisions with the 110 MPN/100 ml threshold. EC models for qPCR- and culture-based concentrations used similar independent variables (14-day humidity, salinity/conductivity, a rain/storm variable, and a measure of air temperature), with each model explaining 26 and 55% of the data variation, respectively. When using different thresholds that were logs apart for management decisions, the two EC models accurately predicted management decisions; qPCR models correctly predicted management decisions 96 and 77% of the time (using 31 and 320 CE/100 ml, respectively) while culture models correctly predicted management decisions 96 and 88% percent of the time (with 31 and 320 MPN/100 ml, respectively). Equivalency between models was shown in our non-point source impacted estuaries, with ENT models performing slightly better than EC models. In addition, inhibition of the qPCR was a major issue that had to be addressed. An inhibition model was created with easily obtained meteorological data and accounted for a high level of data variability (adjusted R(2) = 0.82). PMID:24139103

Gonzalez, Raul A; Noble, Rachel T

2014-01-01

224

Analysis of Natural and Induced Variation in Tomato Glandular Trichome Flavonoids Identifies a Gene Not Present in the Reference Genome[W][OPEN  

PubMed Central

Flavonoids are ubiquitous plant aromatic specialized metabolites found in a variety of cell types and organs. Methylated flavonoids are detected in secreting glandular trichomes of various Solanum species, including the cultivated tomato (Solanum lycopersicum). Inspection of the sequenced S. lycopersicum Heinz 1706 reference genome revealed a close homolog of Solanum habrochaites MOMT1 3?/5? myricetin O-methyltransferase gene, but this gene (Solyc06g083450) is missing the first exon, raising the question of whether cultivated tomato has a distinct 3? or 3?/5? O-methyltransferase. A combination of mining genome and cDNA sequences from wild tomato species and S. lycopersicum cultivar M82 led to the identification of Sl-MOMT4 as a 3? O-methyltransferase. In parallel, three independent ethyl methanesulfonate mutants in the S. lycopersicum cultivar M82 background were identified as having reduced amounts of di- and trimethylated myricetins and increased monomethylated myricetin. Consistent with the hypothesis that Sl-MOMT4 is a 3? O-methyltransferase gene, all three myricetin methylation defective mutants were found to have defects in MOMT4 sequence, transcript accumulation, or 3?-O-methyltransferase enzyme activity. Surprisingly, no MOMT4 sequence is found in the Heinz 1706 reference genome sequence, and this cultivar accumulates 3-methyl myricetin and is deficient in 3?-methyl myricetins, demonstrating variation in this gene among cultivated tomato varieties. PMID:25128240

Kim, Jeongwoon; Matsuba, Yuki; Ning, Jing; Schilmiller, Anthony L.; Hammar, Dagan; Jones, A. Daniel; Pichersky, Eran; Last, Robert L.

2014-01-01

225

Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans  

PubMed Central

Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R2?=?0.60; p<0.0001) and patients (R2?=?0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R2?=?0.35; p<0.0001) and low correlation for patients (R2?=?0.20; p?=?0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R2?=?0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R2?=?0.1, p?=?0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; p?=?0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; p?=?0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes. PMID:25409313

Gutierrez-Rodrigues, Fernanda; Santana-Lemos, Bárbara A.; Scheucher, Priscila S.; Alves-Paiva, Raquel M.; Calado, Rodrigo T.

2014-01-01

226

Direct Comparison of Flow-FISH and qPCR as Diagnostic Tests for Telomere Length Measurement in Humans.  

PubMed

Telomere length measurement is an essential test for the diagnosis of telomeropathies, which are caused by excessive telomere erosion. Commonly used methods are terminal restriction fragment (TRF) analysis by Southern blot, fluorescence in situ hybridization coupled with flow cytometry (flow-FISH), and quantitative PCR (qPCR). Although these methods have been used in the clinic, they have not been comprehensively compared. Here, we directly compared the performance of flow-FISH and qPCR to measure leukocytes' telomere length of healthy individuals and patients evaluated for telomeropathies, using TRF as standard. TRF and flow-FISH showed good agreement and correlation in the analysis of healthy subjects (R2?=?0.60; p<0.0001) and patients (R2?=?0.51; p<0.0001). In contrast, the comparison between TRF and qPCR yielded modest correlation for the analysis of samples of healthy individuals (R2?=?0.35; p<0.0001) and low correlation for patients (R2?=?0.20; p?=?0.001); Bland-Altman analysis showed poor agreement between the two methods for both patients and controls. Quantitative PCR and flow-FISH modestly correlated in the analysis of healthy individuals (R2?=?0.33; p<0.0001) and did not correlate in the comparison of patients' samples (R2?=?0.1, p?=?0.08). Intra-assay coefficient of variation (CV) was similar for flow-FISH (10.8±7.1%) and qPCR (9.5±7.4%; p?=?0.35), but the inter-assay CV was lower for flow-FISH (9.6±7.6% vs. 16±19.5%; p?=?0.02). Bland-Altman analysis indicated that flow-FISH was more precise and reproducible than qPCR. Flow-FISH and qPCR were sensitive (both 100%) and specific (93% and 89%, respectively) to distinguish very short telomeres. However, qPCR sensitivity (40%) and specificity (63%) to detect telomeres below the tenth percentile were lower compared to flow-FISH (80% sensitivity and 85% specificity). In the clinical setting, flow-FISH was more accurate, reproducible, sensitive, and specific in the measurement of human leukocyte's telomere length in comparison to qPCR. In conclusion, flow-FISH appears to be a more appropriate method for diagnostic purposes. PMID:25409313

Gutierrez-Rodrigues, Fernanda; Santana-Lemos, Bárbara A; Scheucher, Priscila S; Alves-Paiva, Raquel M; Calado, Rodrigo T

2014-01-01

227

Seasonal dynamics of harmful algae in outer Oslofjorden monitored by microarray, qPCR, and microscopy.  

PubMed

Monitoring of marine microalgae is important to predict and manage harmful algal blooms. Microarray Detection of Toxic ALgae (MIDTAL) is an FP7-funded EU project aiming to establish a multi-species microarray as a tool to aid monitoring agencies. We tested the suitability of different prototype versions of the MIDTAL microarray for the monthly monitoring of a sampling station in outer Oslofjorden during a 1-year period. Microarray data from two different versions of the MIDTAL chip were compared to results from cell counts (several species) and quantitative real-time PCR (qPCR; only Pseudochattonella spp.). While results from generation 2.5 microarrays exhibited a high number of false positive signals, generation 3.3 microarray data generally correlated with microscopy and qPCR data, with three important limitations: (1) Pseudo-nitzschia cells were not reliably detected, possibly because cells were not sufficiently retained during filtration or lysed during the extraction, and because of low sensitivity of the probes; (2) in the case of samples with high concentrations of non-target species, the sensitivity of the arrays was decreased; (3) one occurrence of Alexandrium pseudogonyaulax was not detected due to a 1-bp mismatch with the genus probe represented on the microarray. In spite of these shortcomings our data demonstrate the overall progress made and the potential of the MIDTAL array. The case of Pseudochattonella - where two morphologically similar species impossible to separate by light microscopy were distinguished - in particular, underlines the added value of molecular methods such as microarrays in routine phytoplankton monitoring. PMID:23325054

Dittami, Simon M; Hostyeva, Vladyslava; Egge, Elianne Sirnæs; Kegel, Jessica U; Eikrem, Wenche; Edvardsen, Bente

2013-10-01

228

Quantification of Cryptosporidium parvum in natural soil matrices and soil solutions using qPCR.  

PubMed

Traditional microscopy methods for the detection and quantification of Cryptosporidium parvum in soil matrices are time-consuming, labor-intensive, and lack sensitivity and specificity. This research focused on developing a qPCR protocol for the sensitive and specific detection and quantification of C. parvum in natural soil matrices and soil-water extracts. The physico-chemical parameters - lysis media, number of thermal shocks and thawing temperatures - controlling DNA extraction efficiency were investigated. Experimental results identified oocyst age as a critical parameter affecting oocyst disruption and quantification. The most efficient oocyst disruption method for C. parvum oocysts regardless of their age was established as 5 thermal shocks with thawing at 65°C in Tris-EDTA (TE) buffer. In addition to the purification columns used to remove PCR inhibitors present in environmental matrices, a combination of 3mM MgCl(2) and 600ng/?l BSA yielded the highest amplicon yield for both young and aged oocysts. Sucrose flotation was determined to be a better oocyst isolation method than two-phase flotation. The optimized parameters for DNA extraction and the qPCR assay resulted in very specific and sensitive detection of C. parvum. Minimum detection limits were 0.667 for young C. parvum oocysts and 6.67 for aged C. parvum oocysts per PCR reaction. The accuracy of the detections and quantifications was 0.999. Protocol performance was tested in contrasting soil samples and soil-water extract samples on the basis of percentage of recovery (PR) values. Depending on the number of oocysts used to inoculate the samples, the average PR values ranged from 7.2 to 43.5%, 29.3-52.5%, and 11.5-60.8% for Trenton, Greenson, and Sparta soil-water extracts, respectively, and 12.1-77% for DI water. PR values ranged from 4.3% to 107.8% for Trenton, Greenson and Sparta soil samples. PMID:23201484

Koken, Emre; Darnault, Christophe J G; Jacobson, Astrid R; Powelson, David; Hendrickson, William

2013-02-15

229

Differentiation and Phylogenetic Relationships in Mycobacterium spp with Special Reference to the RNase P RNA Gene rnpB.  

PubMed

The rnpB gene encodes for the RNA subunit of the catalytic ribonuclease RNase P and is present in all bacteria and has both conserved and highly variable sequence regions. Determination of rnpB in 35 Mycobacterium spp. showed species specific sequences for all species except the Mycobacterium tuberculosis complex (four species). High sequence variation was seen in the P3, P15 and P19 regions of suggested secondary structures of the corresponding RNase P RNA molecules. Phylogenetic analysis showed that rnpB gave similar tree topologies as 16S rRNA and hsp65 genes. A combined analysis of the three genes increased the number of nodes with significant support from 10 to 19. The results indicate that rnpB is useful for phylogenetic studies and is a possible target for identification and detection of Mycobacterium spp. PMID:24962595

Herrmann, Björn; Stolt, Pelle; Abdeldaim, Guma; Rubin, Carl-Johan; Kirsebom, Leif A; Thollesson, Mikael

2014-11-01

230

Generalizability of Gene Expression Programming-based approaches for estimating daily reference evapotranspiration in coastal stations of Iran  

NASA Astrophysics Data System (ADS)

We used Genetic Programming (GP) to model daily reference evapotranspiration.Generalizability assessment of GP model was performed.Results confirmed the capability of GP model through k-fold test.Externally trained GP may be good alternative for locally trained GP.

Shiri, Jalal; Sadraddini, Ali Ashraf; Nazemi, Amir Hossein; Kisi, Ozgur; Landeras, Gorka; Fakheri Fard, Ahmad; Marti, Pau

2014-01-01

231

Establishment of a quantitative PCR system for discriminating chitinase-like proteins: catalytically inactive breast regression protein-39 and Ym1 are constitutive genes in mouse lung  

PubMed Central

Background Mice and humans produce chitinase-like proteins (CLPs), which are highly homologous to chitinases but lack chitinolytic activity. Mice express primarily three CLPs, including breast regression protein-39 (BRP-39) [chitinase 3-like-1 (Chi3l1) or 38-kDa glycoprotein (gp38k)], Ym1 (Chi3l3) and Ym2 (Chi3l4). Recently, CLPs have attracted considerable attention due to their increased expression in a number of pathological conditions, including asthma, allergies, rheumatoid arthritis and malignant tumors. Although the exact functions of CLPs are largely unknown, the significance of their increased expression levels during pathophysiological states needs to be determined. The quantification of BRP-39, Ym1 and Ym2 is an important step in gaining insight into the in vivo regulation of the CLPs. Methods We constructed a standard DNA for quantitative real-time PCR (qPCR) by containing three CLPs target fragments and five reference genes cDNA in a one-to-one ratio. We evaluated this system by analyzing the eight target cDNA sequences. Tissue cDNAs obtained by reverse transcription from total RNA from four embryonic stages and eight adult tissues were analyzed using the qPCR system with the standard DNA. Results We established a qPCR system detecting CLPs and comparing their expression levels with those of five reference genes using the same scale in mouse tissues. We found that BRP-39 and Ym1 were abundant in the mouse lung, whereas Ym2 mRNA was abundant in the stomach, followed by lung. The expression levels of BRP-39 and Ym1 in the mouse lung were higher than those of two active chitinases and were comparable to glyceraldehyde-3-phosphate dehydrogenase, a housekeeping gene which is constitutively expressed in all tissues. Conclusion Our results indicate that catalytically inactive BRP-39 and Ym1 are constitutive genes in normal mouse lung. PMID:25294623

2014-01-01

232

Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods.  

PubMed

The U.S. EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 recreational water quality criteria (RWQC). The CCE quantification unit stems from the calibration model used to estimate enterococci densities in recreational beach waters in the EPA National Epidemiological and Environmental Assessment of Recreational (NEEAR) Water Study and directly informed the derivation of the RWQC recommendations. Recent studies have demonstrated that CCE estimates from the method can vary when using different cultured Enterococcus cell preparations in calibrator samples. These differences have been attributed to differences in the quantities of targeted gene copies (target sequences) that are recovered per nominal calibrator cell by DNA extraction. Standardization of results from the calibration model will require the estimation of target sequence recoveries from the calibrator and water samples. In addition, comparisons of water sample results with the RWQC values will require a knowledge of target sequence recoveries from the NEEAR study calibrator samples. In this study recoveries of target sequences and the mean target sequence/cell ratio for the NEEAR study calibrator samples were retrospectively estimated with a corroborated standard curve. A modification of the calibration model was then used to estimate enterococci target sequence quantities in water samples from eight midwestern U.S. rivers. CCE estimates were obtained by dividing these target sequence quantities by the mean NEEAR study target sequence/cell ratio. This target sequence-based quantification approach resulted in a high degree of agreement in beach action decisions (determinations of whether bacterial fecal indicator densities are above or below RWQC-recommended values) from CCE results of the qPCR method and from culture dependent enumeration of both enterococci and Eschericia coli in the corresponding water samples. PMID:25038459

Haugland, Richard A; Siefring, Shawn D; Varma, Manju; Dufour, Alfred P; Brenner, Kristen P; Wade, Timothy J; Sams, Elizabeth; Cochran, Stacey; Braun, Steve; Sivaganensan, Mano

2014-10-01

233

Determination of an effective housekeeping gene for the quantification of mRNA for forensic applications.  

PubMed

The potential application of mRNA for the identification of biological fluids using molecular techniques has been a recent development in forensic serology. Constitutively expressed housekeeping genes can assess the amount of mRNA recovered from a sample, establish its suitability for downstream applications, and provide a reference point to corroborate the identity of the fluid. qPCR was utilized to compare the expression levels of housekeeping genes from forensic-like body fluid stains to establish the most appropriate assessment of human mRNA quantity prior to profiling. Although variability was observed between fluids and individuals, results indicated that beta-2 microglobulin exhibited the highest expression for all body fluids examined and across donors. A one-way analysis of variance was performed for housekeeping gene variability between donors (at the ?, 0.05, significance level), and the results indicated significant differences for semen, vaginal secretions, and menstrual blood. PMID:22309221

Moreno, Lilliana I; Tate, Courtney M; Knott, Erika L; McDaniel, Jade E; Rogers, Stephanie S; Koons, Barbara W; Kavlick, Mark F; Craig, Rhonda L; Robertson, James M

2012-07-01

234

Detection, identification and quantification of Campylobacter jejuni, coli and lari in food matrices all at once using multiplex qPCR  

PubMed Central

Background Thermotolerant Campylobacter jejuni, coli and lari are recognized as leading food-borne pathogens causing an acute bacterial enteritis worldwide. Due to narrow spectrum of their biochemical activity, it is very complicated to distinguish between individual species. For reliable risk assessment, proper incidence evaluation or swift sample analysis regarding individual species, a demand for simple and rapid method for their distinguishing is reasonable. In this study, we evaluated a reliable and simple approach for their simultaneous detection, species identification and quantification using multiplex qPCR. Results Species specific primers and hydrolysis probes are directed to hippuricase gene of C. jejuni, serine hydroxymethyltransferase gene of C. coli and peptidase T gene of C. lari. Efficiencies of reactions were 90.85% for C. jejuni, 96.97% for C. coli and 92.89% for C. lari. At 95.00% confidence level and when cut off is set to 38 cycles, limits of detection are in all cases under 10 genome copies per reaction which is very appreciated since it is known that infectious doses are very low. Conclusions Proposed assay was positively validated on different food matrices (chicken wing rinses, chicken juice and homogenized fried chicken strips). No inhibition of PCR reaction occurred. Assay was evaluated in accordance with MIQE handbook. PMID:25057300

2014-01-01

235

Comparison of Enterococcus qPCR analysis results from fresh and marine water samples on two real-time instruments -  

EPA Science Inventory

EPA is currently considering a quantitative polymerase chain reaction (qPCR) method, targeting Enterococcus spp., for beach monitoring. Improvements in the method?s cost-effectiveness may be realized by the use of newer instrumentation such as the Applied Biosystems StepOneTM a...

236

FECAL INDICATOR BACTERIA MEASUREMENTS BY QUANTITATIVE POLYMERASE CHAIN REACTION (QPCR) ANALYSIS IN FRESH ARCHIVED DNA EXTRACT OF WATER SAMPLE FILTRATES  

EPA Science Inventory

The U.S. EPA has initiated a new recreational water study to evaluate the correlation between illness rates in swimmers and Enterococcus concentrations determined by the mEI agar membrane filter (MF) method and several new technologies including QPCR analysis. Results of this stu...

237

Comparison of Enterococcus qPCR analysis results from fresh and marine waters on two real-tme instruments  

EPA Science Inventory

The U.S. Environmental Protection Agency (EPA) will be recommending a quantitativ e polymerase chain reaction (qPCR) method targeting Enterococcus spp. as an option for monitoring recreational beach water quality. A practical consideration for widespread implementation of this o...

238

Quantification of Staphylococcus aureus in white cheese by the improved DNA extraction strategy combined with TaqMan and LNA probe-based qPCR.  

PubMed

Four different bacterial DNA extraction strategies and two different qPCR probe chemistries were studied for detection of Stapylococcus aureus from white cheeses. Method employing trypsin treatment followed by a commercial kit application and TaqMan probe-based qPCR was the most sensitive one detecting higher counts than standards in naturally contaminated samples. PMID:25016130

Kadiro?lu, P?nar; Korel, Figen; Ceylan, Cagatay

2014-10-01

239

Selection of reference genes for quantitative real-time PCR expression studies of microdissected reproductive tissues in apomictic and sexual Boechera  

PubMed Central

Background Apomixis, a natural form of asexual seed production in plants, is considered to have great biotechnological potential for agriculture. It has been hypothesised that de-regulation of the sexual developmental pathway could trigger apomictic reproduction. The genus Boechera represents an interesting model system for understanding apomixis, having both sexual and apomictic genotypes at the diploid level. Quantitative qRT-PCR is the most extensively used method for validating genome-wide gene expression analyses, but in order to obtain reliable results, suitable reference genes are necessary. In this work we have evaluated six potential reference genes isolated from a 454 (FLX) derived cDNA library of Boechera. RNA from live microdissected ovules and anthers at different developmental stages, as well as vegetative tissues of apomictic and sexual Boechera, were used to validate the candidates. Results Based on homologies with Arabidopsis, six genes were selected from a 454 cDNA library of Boechera: RPS18 (Ribosomal sub protein 18), Efalpha1 (Elongation factor 1 alpha), ACT 2 (Actin2), UBQ (polyubiquitin), PEX4 (Peroxisomal ubiquitin conjugating enzyme) and At1g09770.1 (Arabidopsis thaliana cell division cycle 5). Total RNA was extracted from 17 different tissues, qRT-PCRs were performed, and raw Ct values were analyzed for primer efficiencies and gene ratios. The geNorm and normFinder applications were used for selecting the most stable genes among all tissues and specific tissue groups (ovule, anthers and vegetative tissues) in both apomictic and sexual plants separately. Our results show that BoechRPS18, BoechEf?1, BoechACT2 and BoechUBQ were the most stable genes. Based on geNorm, the combinations of BoechRPS18 and BoechEf?1 or BoechUBQ and BoechEf?1 were the most stable in the apomictic plant, while BoechRPS18 and BoechACT2 or BoechUBQ and BoechACT2 performed best in the sexual plant. When subgroups of tissue samples were analyzed, different optimal combinations were identified in sexual ovules (BoechUBQ and BoechEf?1), in anthers from both reproductive systems (BoechACT2 and BoechEf?1), in apomictic vegetative tissues (BoechEf?1 and BoechACT2) and sexual vegetative tissues (BoechRPS18 and BoechEf?1). NormFinder ranked BoechACT2 as the most stable in the apomictic plant, while BoechRPS18 was the best in the sexual plant. The subgroups analysis identified the best gene for both apomictic and sexual ovules (BoechRPS18), for anthers from both reproductive system (BoechEf?1) and for apomictic and vegetative tissues (BoechACT2 and BoechRPS18 respectively) Conclusions From a total of six tested genes, BoechRPS18, BoechEf?1, BoechACT2 and BoechUBQ showed the best stability values. We furthermore provide detailed information for the accurate normalization of specific tissue gene expression analyses of apomictic and sexual Boechera. PMID:21851639

2011-01-01

240

Molecular Identification of Unusual Pathogenic Yeast Isolates by Large Ribosomal Subunit Gene Sequencing: 2 Years of Experience at the United Kingdom Mycology Reference Laboratory?  

PubMed Central

Rapid identification of yeast isolates from clinical samples is particularly important given their innately variable antifungal susceptibility profiles. We present here an analysis of the utility of PCR amplification and sequence analysis of the hypervariable D1/D2 region of the 26S rRNA gene for the identification of yeast species submitted to the United Kingdom Mycology Reference Laboratory over a 2-year period. A total of 3,033 clinical isolates were received from 2004 to 2006 encompassing 50 different yeast species. While more than 90% of the isolates, corresponding to the most common Candida species, could be identified by using the AUXACOLOR2 yeast identification kit, 153 isolates (5%), comprised of 47 species, could not be identified by using this system and were subjected to molecular identification via 26S rRNA gene sequencing. These isolates included some common species that exhibited atypical biochemical and phenotypic profiles and also many rarer yeast species that are infrequently encountered in the clinical setting. All 47 species requiring molecular identification were unambiguously identified on the basis of D1/D2 sequences, and the molecular identities correlated well with the observed biochemical profiles of the various organisms. Together, our data underscore the utility of molecular techniques as a reference adjunct to conventional methods of yeast identification. Further, we show that PCR amplification and sequencing of the D1/D2 region reliably identifies more than 45 species of clinically significant yeasts and can also potentially identify new pathogenic yeast species. PMID:17251397

Linton, Christopher J.; Borman, Andrew M.; Cheung, Grace; Holmes, Ann D.; Szekely, Adrien; Palmer, Michael D.; Bridge, Paul D.; Campbell, Colin K.; Johnson, Elizabeth M.

2007-01-01

241

CopyRighter: a rapid tool for improving the accuracy of microbial community profiles through lineage-specific gene copy number correction  

PubMed Central

Background Culture-independent molecular surveys targeting conserved marker genes, most notably 16S rRNA, to assess microbial diversity remain semi-quantitative due to variations in the number of gene copies between species. Results Based on 2,900 sequenced reference genomes, we show that 16S rRNA gene copy number (GCN) is strongly linked to microbial phylogenetic taxonomy, potentially under-representing Archaea in amplicon microbial profiles. Using this relationship, we inferred the GCN of all bacterial and archaeal lineages in the Greengenes database within a phylogenetic framework. We created CopyRighter, new software which uses these estimates to correct 16S rRNA amplicon microbial profiles and associated quantitative (q)PCR total abundance. CopyRighter parses microbial profiles and, because GCN estimates are pre-computed for all taxa in the reference taxonomy, rapidly corrects GCN bias. Software validation with in silico and in vitro mock communities indicated that GCN correction results in more accurate estimates of microbial relative abundance and improves the agreement between metagenomic and amplicon profiles. Analyses of human-associated and anaerobic digester microbiomes illustrate that correction makes tangible changes to estimates of qPCR total abundance, ? and ? diversity, and can significantly change biological interpretation. For example, human gut microbiomes from twins were reclassified into three rather than two enterotypes after GCN correction. Conclusions The CopyRighter bioinformatic tools permits rapid correction of GCN in microbial surveys, resulting in improved estimates of microbial abundance, ? and ? diversity. PMID:24708850

2014-01-01

242

Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR)  

PubMed Central

Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR) assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5?-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix. PMID:23105972

Felder, Eva; Mossbrugger, Ilona; Lange, Mirko; Wolfel, Roman

2012-01-01

243

Microarray detection and qPCR screening of potential biomarkers of Folsomia candida (Collembola: Isotomidae) exposed to Bt proteins (Cry1Ab and Cry1Ac).  

PubMed

The impact of Bt proteins on non-target arthropods is less understood than their effects on target organisms where the mechanism of toxic action is known. Here, we report the effects of two Bt proteins, Cry1Ab and Cry1Ac, on gene expression in the non-target collembolan, Folsomia candida. A customized microarray was used to study gene expression in F. candida specimens that were exposed to Cry1Ab and Cry1Ac. All selected transcripts were subsequently confirmed by qPCR. Eleven transcripts were finally verified, and three of them were annotated. The responses of all eleven transcripts were tested in specimens for both Cry1Ab and Cry1Ac at a series of concentrations. These transcripts were separated into two and three groups for Cry1Ab and Cry1Ac, respectively, depend on their expression levels. However, those eleven transcripts did not respond to the Bt proteins in Bt-rice residues. PMID:24056072

Yuan, Yiyang; Krogh, Paul Henning; Bai, Xue; Roelofs, Dick; Chen, Fajun; Zhu-Salzman, Keyan; Liang, Yuyong; Sun, Yucheng; Ge, Feng

2014-01-01

244

Development and qualification of a high sensitivity, high throughput Q-PCR assay for quantitation of residual host cell DNA in purification process intermediate and drug substance samples.  

PubMed

Methods of high sensitivity, accuracy and throughput are needed for quantitation of low level residual host cell DNA in purification process intermediates and drug substances of therapeutic proteins. In this study, we designed primer/probe sets targeting repetitive Alu repeats or Alu-equivalent sequences in the human, Chinese hamster and murine genomes. When used in quantitative polymerase chain reactions (Q-PCRs), these primer/probe sets showed high species specificity and gave significantly higher sensitivity compared to those targeting the low copy number GAPDH gene. This allowed for detection of residual host cell DNA of much lower concentrations and, for some samples, eliminated the need for DNA extraction. By combining the high sensitivity Alu Q-PCR with high throughput automated DNA extraction using an automated MagMAX magnetic particle processor, we successfully developed and qualified a highly accurate, specific, sensitive and efficient method for the quantitation of residual host cell DNA in process intermediates and drug substances of multiple therapeutic proteins purified from cells of multiple species. Compared to the previous method using manual DNA extraction and primer/probe sets targeting the GAPDH gene, this new method increased our DNA extraction throughput by over sevenfold, and lowered the lower limit of quantitation by up to eightfold. PMID:25165010

Zhang, Wei; Wu, Meng; Menesale, Emily; Lu, Tongjun; Magliola, Aeona; Bergelson, Svetlana

2014-11-01

245

Molecular cloning, tissue expression of gene Muc2 in blunt snout bream Megalobrama amblycephala and regulation after re-feeding  

NASA Astrophysics Data System (ADS)

Mucins are important components of mucus, which form a natural, physical, biochemical and semipermeable mucosal layer on the epidermis of fish gills, skin, and the gastrointestinal tract. As the first step towards characterizing the function of Muc2, we cloned a partial Megalobrama amblycephala Muc2 cDNA of 2 175 bp, and analyzed its tissue-specific expression pattern by quantitative real-time PCR (qPCR). The obtained sequence comprised 41 bp 5'-untranslated region (5'-UTR), 2 134 bp open reading frame encoding a protein of 711 amino acids. BLAST searching and phylogenetic analysis showed that the predicted protein contained several common secreted mucin-module domains (VWD-C8-TIL-VWD-C8) and had high homology with mucins from other vertebrates. Among four candidate reference genes (?-Actin, RPI13?, RPII, 18S) for the qPCR, RPII was chosen as an appropriate reference gene because of its lowest variation in different tissues. M. amblycephala Muc2 was mainly expressed in the intestine, in the order (highest to lowest) middle-intestine > fore-intestine > hind-intestine. Muc2 was expressed relatively poorly in other organs (brain, liver, kidney, spleen, skin and gill). Furthermore, after 20-days of starvation, M. amblycephala Muc2 expressions after refeeding for 0 h, 3 h, 16 h, 3 d, and 10 d were significantly decreased in the three intestinal segments (P <0.05) at 16 h, and were then upregulated to near the initial level at 10 d.

Xue, Chunyu; Xi, Bingwen; Ren, Mingchun; Dong, Jingjing; Xie, Jun; Xu, Pao

2014-10-01

246

Gene expression profiling in Helicobacter-induced MALT lymphoma with reference to antigen drive and protective immunization.  

PubMed

We have previously shown that long-term infection of BALB/c mice with gastric Helicobacter species results in the development of histopathological lesions that resemble those seen in patients diagnosed with gastric mucosa associated lymphoid tissue (MALT) lymphoma. This paper describes analysis of this disease at the molecular level through the use of microarray technology and immunohistochemical staining. We were able to monitor the genetic changes in the gastric mucosa characterized by distinct transcriptional signatures and correlate these with histological changes as the infection progressed from a chronic inflammatory infiltrate through to MALT lymphoma. This model system also enabled us to further dissect the role of antigen presentation and prophylactic immunization in the disease process. Antimicrobial therapy to eradicate the antigen correlated with significant reduction in pathology and major changes in the gene expression profile. Subsequent reintroduction of the antigen resulted in rapid tumor development which correlated with an increase in aggressively proliferating cells and changes in the cellular composition of the tumor. The response in vaccinated animals showed that the protected animals exhibited a strikingly different transcriptional profile compared to those of non-protected or control mice, indicating that the vaccination targeted the appropriate site leaving a long-lasting signature. The genes which were most significantly up-regulated included a number of adipocyte-specific factors, such as fat-cell specific cytokines and adipocyte surface markers. This study allowed for us to highlight the significance of antigen presentation in this disease and to hypothesis mechanisms associated with protective immunity. PMID:19120889

O'Rourke, Jani L

2008-12-01

247

Genetics Home Reference: Paralysis  

MedlinePLUS

... Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Paralysis Related topics on Genetics Home Reference: alternating hemiplegia of childhood hyperkalemic periodic paralysis hypokalemic periodic paralysis infantile-onset ascending hereditary spastic ...

248

Genetics Home Reference: Anemia  

MedlinePLUS

... Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Anemia Related topics on Genetics Home Reference: acute promyelocytic ... syndrome beta thalassemia Coats plus syndrome congenital dyserythropoietic anemia Diamond-Blackfan anemia Fanconi anemia Ghosal hematodiaphyseal dysplasia ...

249

Genetics Home Reference: Dystonia  

MedlinePLUS

... U.S. National Library of Medicine® Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Dystonia Related topics on Genetics Home Reference: dopa-responsive dystonia dystonia 6 early-onset primary dystonia GM1 gangliosidosis hypermanganesemia with ...

250

Genetics Home Reference: Dementia  

MedlinePLUS

... Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Dementia Related topics on Genetics Home Reference: adult polyglucosan body disease Alzheimer disease cerebral autosomal dominant arteriopathy with subcortical infarcts and ...

251

Genetics Home Reference: Epilepsy  

MedlinePLUS

... Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Epilepsy Related topics on Genetics Home Reference: Aicardi syndrome autosomal dominant nocturnal frontal lobe epilepsy autosomal dominant partial epilepsy with auditory features benign ...

252

Genetics Home Reference: Seizures  

MedlinePLUS

... Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions > Seizures Related topics on Genetics Home Reference: 15q13.3 ... deficiency aspartylglucosaminuria ataxia neuropathy spectrum benign familial ... idiopathic basal ganglia calcification fucosidosis juvenile myoclonic ...

253

Twenty-five years of quantitative PCR for gene expression analysis.  

PubMed

Following its invention 25 years ago, PCR has been adapted for numerous molecular biology applications. Gene expression analysis by reverse-transcription quantitative PCR (RT-qPCR) has been a key enabling technology of the post-genome era. Since the founding of BioTechniques, this journal has been a resource for the improvements in qPCR technology, experimental design, and data analysis. qPCR and, more specifically, real-time qPCR has become a routine and robust approach for measuring the expression of genes of interest, validating microarray experiments, and monitoring biomarkers. The use of real-time qPCR has nearly supplanted other approaches (e.g., Northern blotting, RNase protection assays). This review examines the current state of qPCR for gene expression analysis now that the method has reached a mature stage of development and implementation. Specifically, the different fluorescent reporter technologies of real-time qPCR are discussed as well as the selection of endogenous controls. The conceptual framework for data analysis methods is also presented to demystify these analysis techniques. The future of qPCR remains bright as the technology becomes more rapid, cost-effective, easier to use, and capable of higher throughput. PMID:18474036

VanGuilder, Heather D; Vrana, Kent E; Freeman, Willard M

2008-04-01

254

Real-Time PCR for Detection of NDM-1 Carbapenemase Genes from Spiked Stool Samples?  

PubMed Central

An in-house quantitative real-time PCR (qPCR) assay using TaqMan chemistry has been developed to detect NDM-1 carbapenemase genes from bacterial isolates and directly from stool samples. The qPCR amplification of blaNDM-1 DNA was linear over 10 log dilutions (r2 = 0.99), and the amplification efficiency was 1.03. The qPCR detection limit was reproducibly 1 CFU, or 10 plasmid molecules, and there was no cross-reaction with DNA extracted from several multidrug-resistant bacteria harboring other ?-lactam resistance genes. Feces spiked with decreasing amounts of enterobacterial isolates producing NDM-1 were spread on ChromID ESBL and on CHROMagar KPC media and were subjected to the qPCR. The limits of carbapenem-resistant bacterial detection from stools was reproducibly 1 × 101 to 3 × 101 CFU/100 mg feces with ChromID ESBL medium. The CHROMagar KPC culture medium had higher limits of detection (1 × 101 to 4 × 103 CFU/ml), especially with bacterial isolates having low carbapenem MICs. The limits of detection with the qPCR assay were reproducibly below 1 × 101 CFU/100 mg of feces by qPCR assay. Samples spiked with NDM-1-negative bacteria were negative by qPCR. The sensitivity and specificity of the blaNDM-1 qPCR assay on spiked samples were 100% in both cases. Using an automated DNA extraction system (QIAcube system), the qPCR assay was reproducible. The use of qPCR is likely to shorten the time for blaNDM-1 detection from 48 h to 4 h and will be a valuable tool for outbreak follow-up in order to rapidly isolate colonized patients and assign them to cohorts. PMID:21690281

Naas, Thierry; Ergani, Ayla; Carrer, Amelie; Nordmann, Patrice

2011-01-01

255

[Comparison of nucleic acid extraction efficiency using different commercial kits and qPCR. Effect of inhibitors].  

PubMed

The detection of specific nucleic acid (NA) sequences by PCR has revolutionized the biological and medical sciences. Real-time PCR (qPCR) opened up the possibility of obtaining quantitative results. NA extraction is a decisive step prior to qPCR since it may produce either the removal or co-extraction of inhibitory substances of the enzymatic reaction, which in turn affects the amplification efficiency. In the present work we compared the commercial NA extraction kits from Qiagen, Invitrogen and Macherey-Nagel, which were used to extract DNA from mice blood artificially infected with Trypanosoma cruzi and PP7 RNA, Pseudomonas aeruginosa bacteriophage, in spiked aqueous matrices. NA recovery efficiency in samples without inhibitors was similar for the three extraction kits. However, the Invitrogen kit was the only one that remained unaffected in the presence of inhibitors in the samples. PMID:23102460

Poma, Hugo R; Davies, Carolina; Gutiérrez Cacciabue, Dolores; Mora, María C; Basombrío, Miguel Á; Rajal, Verónica B

2012-01-01

256

qPCR standard operating procedure for measuring microorganisms in dust from dwellings in large cohort studies.  

PubMed

The aim of the present study was to assess performance, feasibility and relevance of a Standard Operational Procedure (SOP) for large-scale use in the microbial analysis of children's indoor environments. We analyzed dust settled on Electrostatic Dust Fall Collectors (EDCs) by using qPCR which targeted 6 molds, 3 bacteria and 1 mite, chosen for their involvement in allergic or inflammatory processes. Six types of commercialized electrostatic wipes were tested for their releasing capacity of fungal DNA from fungal spores captured by the wipes. Specificity, repeatability and detection limits of the qPCR procedure were tested using calibrated microbial suspensions. The feasibility and relevance of this sampling and analysis method were assessed in a 75-home pilot study. Our result showed that one specific make of wipe was more effective than the others in releasing fungal DNA. qPCR procedure showed good repeatability. The quantification limit was about 5 fg DNA/?L for all species except Penicillium chrysogenum (0.5 fg DNA/?L) and Dermatophagoïdes pteronyssinus (10 fg DNA/?L). No cross-reactivity was observed. DNA concentrations in the 53/75 homes participating in the pilot study were between 0 and 24 625, 0 and 69 738 equivalent cells per cm(2) for the fungi and bacteria, and between 0 and 1 equivalent mites per cm(2) for D. pteronyssinus. Using the SOP described, we were able to classify the 53 dwellings from the least to the most contaminated according to the quantity of DNA measured for each species. Our SOP measured fungi, bacteria and mites using a cost-efficient, discreet and well-accepted sampling method with just one qPCR tool. The whole procedure can be used for microbial analysis in large cohort studies such as the ELFE study ("Etude Longitudinale Française depuis l'Enfance") and could help improve our understanding of the interactions between the environment, allergic diseases and child development. PMID:23973537

Scherer, Emeline; Rocchi, Steffi; Reboux, Gabriel; Vandentorren, Stéphanie; Roussel, Sandrine; Vacheyrou, Mallory; Raherison, Chantal; Millon, Laurence

2014-01-01

257

Clinical Evaluation of the Cartridge-Based GeneXpert Human Papillomavirus Assay in Women Referred for Colposcopy  

PubMed Central

High-risk human papillomavirus (hrHPV) testing is now being introduced as a potential primary screening test for improved detection of cervical precancer and cancer. Current U.S. Food and Drug Administration-approved tests are batch tests that take several hours to complete. A rapid, non-batch test might permit point-of-care (POC) testing, which can facilitate same-day screen and management strategies. For a non-batch, random-access platform (GeneXpert; Cepheid, Sunnyvale, CA), a prototype hrHPV assay (Xpert) has been developed where testing for 14 hrHPV types can be completed in 1 h. In the first clinical evaluation, Xpert was compared to two validated hrHPV tests, the cobas HPV test (cobas, Roche Molecular Systems) and Hybrid Capture 2 (hc2, Qiagen), and to histologic outcomes using specimens from colposcopy referral populations at 7 clinical sites in the United States (n = 697). The sensitivity of Xpert for cervical intraepithelial neoplasia grade 2 or more severe diagnoses (CIN2+) (n = 141) was equal to that of cobas (90.8% versus 90.8%, P = 1) and greater than that of hc2 (90.8% versus 81.6%, P = 0.004). Xpert was more specific than cobas (42.6% versus 39.6%, P = 0.02) and less specific than hc2 (42.6% versus 47.7%, P < 0.001). Similar results were observed for cervical intraepithelial neoplasia grade 3 or higher (CIN3+) (n = 91). HPV16 detection by Xpert identified 41.8% of the CIN2+ specimens with a positive predictive value (PPV) of 54.6%. By comparison, HPV16 detection by cobas identified 42.6% of the CIN2+ specimens with a PPV of 55.0%. hrHPV detection by the Xpert demonstrated excellent clinical performance for identifying women with CIN2+ and CIN3+ that was comparable to that of currently available clinically validated tests. PMID:24719440

Smith, Katherine M.; Davis, Thomas E.; Schmeler, Kathleen M.; Ferris, Daron G.; Savage, Ashlyn H.; Gray, Jermaine E.; Stoler, Mark H.; Wright, Thomas C.; Ferenczy, Alex; Castle, Philip E.

2014-01-01

258

Genetic analysis of chromosome 11p13 and the PAX6 gene in a series of 125 cases referred with aniridia.  

PubMed

A series of 125 patients referred primarily with aniridia classified as either sporadic (74), familial (24), or in association with WAGR syndrome (14) or other malformations (13) was analysed for mutations, initially by karyotyping and targeted FISH analysis of chromosome 11p13. These methods identified mutations in a significant proportion of patients, 34/125 (27%). Two cases had chromosome rearrangements involving 11p13, 16 cases had visible deletions, and 16 cases had cryptic deletions identified by FISH. The frequency of cryptic deletions in familial aniridia was 27% and in sporadic isolated aniridia was 22%. Of the 14 cases referred with WAGR syndrome, 10 (71%) had chromosomal deletions, 2 cryptic and 8 visible. Of the 13 cases with aniridia and other malformations, 5 (38%) had a chromosomal rearrangement or deletion. In 37 cases with no karyotypic or cryptic chromosome abnormality, sequence analysis of the PAX6 gene was performed. Mutations were identified in 33 cases; 22 with sporadic aniridia, 10 with familial aniridia and 1 with aniridia and other non-WAGR syndrome associated anomalies. Overall, 67 of 71 cases (94%) undergoing full mutation analysis had a mutation in the PAX6 genomic region. PMID:18241071

Robinson, David O; Howarth, Rachel J; Williamson, Kathleen A; van Heyningen, Veronica; Beal, Sarah J; Crolla, John A

2008-03-01

259

Single nucleotide polymorphism in the genes of mce1 and mce4 operons of Mycobacterium tuberculosis: analysis of clinical isolates and standard reference strains  

PubMed Central

Background The presence of four mammalian cell entry (mce) operons in Mycobacterium tuberculosis suggests the essentiality of the functions of the genes in these operons. The differential expression of the four mce operons in different phases of in vitro growth and in infected animals reported earlier from our laboratory further justifies the apparent redundancy for these genes in the genome. Here we investigate the extent of polymorphism in eight genes in the mce1 and mce4 operons of M. tuberculosis from four standard reference strains (H37Rv, H37Ra, LVS (Low Virulent Strain) and BCG) and 112 clinical isolates varying in their drug susceptibility profile, analysed by direct sequencing and Sequenom MassARRAY platform. Results We discovered 20 single nucleotide polymorphisms (SNPs) in the two operons. The comparative analysis of the genes of mce1 and mce4 operons revealed that yrbE1A [Rv0167] was most polymorphic in mce1 operon while yrbE4A [Rv3501c] and lprN [Rv3495c] had the highest number of SNPs in the mce4 operon. Of 20 SNPs, 12 were found to be nonsynonymous and were further analysed for their pathological relevance to M. tuberculosis using web servers PolyPhen and PMut, which predicted five deleterious nonsynonymous SNPs. A mutation from proline to serine at position 359 of the native Mce1A protein was most deleterious as predicted by both PolyPhen and PMut servers. Energy minimization of the structure of native Mce1A protein and mutated protein was performed using InsightII. The mutated Mce1A protein showed structural changes that could account for the effects of this mutation. Conclusions Our results show that SNPs in the coding sequences of mce1 and mce4 operons in clinical isolates can be significantly high. Moreover, mce4 operon is significantly more polymorphic than mce1 operon (p < 0.001). However, the frequency of nonsynonymous substitutions is higher in mce1 operon and synonymous substitutions are more in mce4 operon. In silico modeling predict that nonsynonymous SNP at mce1A [Rv0169], a virulence gene could play a pivotal role in causing functional changes in M. tuberculosis that may reflect upon the biology of the bacteria. PMID:21345183

2011-01-01

260

Integration of high volume portable aerosol-to-hydrosol sampling and qPCR in monitoring bioaerosols.  

PubMed

In this study, the integration of a high volume, portable aerosol-to-hydrosol sampling technique and quantitative polymerase chain reaction (qPCR) was investigated for bioaerosol monitoring by adapting the RCS High Flow to sample air with mineral-oil-strips. Bacillus subtilis var niger and Pseudomonas fluorescens were aerosolized and collected by the RCS High Flow loaded with mineral-oil-strips for 1, 2 and 5 min. In addition, the adapted aerosol-to-hydrosol sampler was also tested for sampling environmental bacterial aerosols in four different environments (a back yard, a student dorm, a dining hall, and a play ground). The performances of the RCS High Flow with mineral-oil-strips were compared with the use of agar strips under similar conditions in all experiments. Air samples collected by the RCS High Flow were cultured, and in addition those collected with mineral-oil-strips were also quantified using qPCR. When sampling B. subtilis var niger aerosols, the use of mineral-oil-strips was shown to report significantly higher culturable concentrations than those obtained by agar strips regardless of the sampling time tested (p-value = 0.04). In contrast, the differences between the two methods when sampling P. fluorescens aerosols were not statistically significant (p-value = 0.5). When coupled with qPCR, the RCS High Flow loaded with mineral-oil-strips obtained significantly higher bacterial aerosol concentrations than those detected by the culturing method. The sampling time was observed to have negligible effects on the efficiency of the technology developed here. When sampling in different environments, the use of mineral-oil-strip was observed to yield significantly higher, about 4-12 times, culturable bacterial aerosol concentration levels compared to the use of agar. This study demonstrated a high volume (100 L min?¹) portable aerosol-to-hydrosol sampling technique, holding broad promise in monitoring airborne biological threats when coupled with qPCR technology. Yet, caution should be taken in relating the bioaerosol concentrations to health risks as qPCR detects both culturable and non-culturable cells including inactivated ones. PMID:21258725

He, Qishuang; Yao, Maosheng

2011-03-01

261

Determination of parasitic load in different tissues of murine toxoplasmosis after immunization by excretory-secretory antigens using Real time QPCR.  

PubMed

Excretory-secretory antigens (ESAs) of Toxoplasma gondii are one of the candidates for immunization against toxoplasmosis. For evaluation of immunization, we determined the kinetics of the distribution of Toxoplasma and parasite load in different tissues of mice immunized by ESAs. In this experimental study, 36 mice in case (n=18) and control (n=18) groups were immunized with ESAs and PBS, respectively. After 2weeks, mice were challenged intraperitoneally with Toxoplasma virulent RH strain. Blood and different tissues (brain, spleen, liver, heart, kidney, and muscle) were collected daily after challenge (1, 2, 3 and last day before death). Parasite load was calculated using Real time QPCR targeted at the B1 gene. ESAs as vaccine in different tissues showed various effects. However, infected mice which received the vaccine in comparison with control group, displayed a drastically decreasing in parasite burden, in their blood and tissues (P=0.000). These results indicated that ESAs with reduction of parasite load in different tissues of host could be evaluable candidate for the development of immunization strategies against toxoplasmosis. PMID:24852216

Daryani, Ahmad; Sharif, Mehdi; Dadimoghaddam, Yousef; Souteh, Mohammad Bagher Hashemi; Ahmadpour, Ehsan; Khalilian, Alireza; Sarvi, Shahabeddin; Farazmand, Touraj; Kalani, Hamed; Rasouli, Mehdi

2014-08-01

262

Improved detection of the KIT D816V mutation in patients with systemic mastocytosis using a quantitative and highly sensitive real-time qPCR assay.  

PubMed

The vast majority of patients with systemic mastocytosis (SM) carry the somatic D816V mutation in the KIT gene. The KIT D816V mutation is one of the minor criteria for a diagnosis of SM according to the 2008 World Health Organization classification of myeloproliferative neoplasms. In the present study, we present a real-time qPCR assay that allows quantification of as little as 0.003% KIT D816V mutation-positive cells. A total of 61 samples from 31 cases of SM were included in the study. We detected the mutation in skin or bone marrow in 95% of the cases of SM. We demonstrate the clinical relevance of the assay by identifying as little as 0.03% mutation-positive cells in bone marrow aspirates from SM patients and calculate the analytical sensitivity of negative samples to determine the reliability of the result. We further demonstrate that this method also detects the KIT D816V mutation in peripheral blood in 81% of the mutation-positive cases with SM. The method also allows comparison of mutation-positive and mast cell fractions to determine whether the mutation is present in non-mast cells, a parameter that has recently been reported to be of prognostic importance in patients with indolent SM. Finally, the assay is suitable for use in prospective studies of the KIT D816V allele burden as a treatment endpoint in SM. PMID:21354053

Kristensen, Thomas; Vestergaard, Hanne; Møller, Michael Boe

2011-03-01

263

Monitoring Subsurface Microbial Biomass, Community Composition and Physiological Status during Biological Uranium Reduction with Acetate Addition using Lipid Analysis, DNA Arrays and q-PCR  

NASA Astrophysics Data System (ADS)

Our objectives for this effort were to investigate microbial community dynamics during each of the distinct terminal electron accepting phases that occur during long-term acetate addition for the immobilization of Uranium. Groundwater was collected from four wells (one up gradient and three down gradient) at three different depths and at four different times (pre-acetate injection, peak iron reduction, iron/sulfate reduction transition and during heavy sulfate reduction). Phospholipid fatty acid analysis (PLFA) results from ground water showed that microbial biomass was highest during Iron reduction and then lower during the transition from Iron reduction to Sulfate reduction and lowest during Sulfate reduction. Microbial community composition parameters as measured by PLFA showed distinct differences with terminal electron accepting status. Monounsaturated PLFA that have been shown to correspond with Gram-negative bacteria and Geobacteracea increased markedly with Iron reduction and then decreased with the onset of sulfate reduction. Bacterial physiological stress levels as measured by PLFA fluctuated with terminal electron acceptor status. Low bacterial stress levels coincided with pre-donor addition and Iron reduction but were much higher during Iron to Sulfate transition and during Sulfate reduction. Microarray results showed the expected progression of microbial signatures from Iron to Sulfate -reducers with changes in acetate amendment and in situ field conditions. The microarray response for Geobacter was highly correlated with qPCR for the same target gene (R2 = 0.84). Probes targeting Desulfobacter and Desulfitobacterium were the most reactive during the Iron to Sulfate transition and into Sulfate reduction, with a consistent Desulfotomaculum signature throughout the field experiment and a general decrease in Geobacter signal to noise ratios during the onset of Sulfate reducing conditions. Nitrate reducers represented by Dechloromonas and Dechlorosoma signatures were consistently detected throughout the field experiment. The intensity of the microarray signature(s) were correlated with depth, where the 12- and 15-ft intervals showed a more pronounced response than the 20-ft interval. In general the q-PCR, PLFA and array data from the experiment were complimentary and have provided a very complete picture of microbial dynamics during donor addition. Results are being compiled with specific attention to the microbial community dynamics during the onset of sulfate reduction and the relationship between ground water and sediment associated communities.

Peacock, A. D.; Long, P. E.; N'Guessan, L.; Williams, K. H.; Chandler, D.

2011-12-01

264

Genes  

NSDL National Science Digital Library

Illustration of the placement of genes in a chromosome. A gene can be defined as a region of DNA that controls a hereditary characteristic. It usually corresponds to a sequence used in the production of a specific protein or RNA. A gene carries biological information in a form that must be copied and transmitted from each cell to all its progeny. This includes the entire functional unit: coding DNA sequences, non-coding regulatory DNA sequences, and introns. Genes can be as short as 1000 base pairs or as long as several hundred thousand base pairs. It can even be carried by more than one chromosome. The estimate for the number of genes in humans has decreased as our knowledge has increased. As of 2001, humans are thought to have between 30,000 and 40,000 genes.

Excellence, Access

2005-03-12

265

Rapid and sensitive detection of Plesiomonas shigelloides by loop-mediated isothermal amplification of the hugA gene.  

PubMed

Plesiomonas shigelloides is one of the causative agents of human gastroenteritis, with increasing number of reports describing such infections in recent years. In this study, the hugA gene was chosen as the target to design loop-mediated isothermal amplification (LAMP) assays for the rapid, specific, and sensitive detection of P. shigelloides. The performance of the assay with reference plasmids and spiked human stools as samples was evaluated and compared with those of quantitative PCR (qPCR). No false-positive results were observed for the 32 non-P. shigelloides strains used to evaluate assay specificity. The limit of detection for P. shigelloides was approximately 20 copies per reaction in reference plasmids and 5×10(3) CFU per gram in spiked human stool, which were more sensitive than the results of qPCR. When applied in human stool samples spiked with 2 low levels of P. shigelloides, the LAMP assays achieved accurate detection after 6-h enrichment. In conclusion, the LAMP assay developed in this study is a valuable method for rapid, cost-effective, and simple detection of P. shigelloides in basic clinical and field laboratories in the rural areas of China. PMID:23077478

Meng, Shuang; Xu, Jianguo; Xiong, Yanwen; Ye, Changyun

2012-01-01

266

Cross-Species Comparison of Genes Related to Nutrient Sensing Mechanisms Expressed along the Intestine  

PubMed Central

Introduction Intestinal chemosensory receptors and transporters are able to detect food-derived molecules and are involved in the modulation of gut hormone release. Gut hormones play an important role in the regulation of food intake and the control of gastrointestinal functioning. This mechanism is often referred to as “nutrient sensing”. Knowledge of the distribution of chemosensors along the intestinal tract is important to gain insight in nutrient detection and sensing, both pivotal processes for the regulation of food intake. However, most knowledge is derived from rodents, whereas studies in man and pig are limited, and cross-species comparisons are lacking. Aim To characterize and compare intestinal expression patterns of genes related to nutrient sensing in mice, pigs and humans. Methods Mucosal biopsy samples taken at six locations in human intestine (n?=?40) were analyzed by qPCR. Intestinal scrapings from 14 locations in pigs (n?=?6) and from 10 locations in mice (n?=?4) were analyzed by qPCR and microarray, respectively. The gene expression of glucagon, cholecystokinin, peptide YY, glucagon-like peptide-1 receptor, taste receptor T1R3, sodium/glucose cotransporter, peptide transporter-1, GPR120, taste receptor T1R1, GPR119 and GPR93 was investigated. Partial least squares (PLS) modeling was used to compare the intestinal expression pattern between the three species. Results and conclusion The studied genes were found to display specific expression patterns along the intestinal tract. PLS analysis showed a high similarity between human, pig and mouse in the expression of genes related to nutrient sensing in the distal ileum, and between human and pig in the colon. The gene expression pattern was most deviating between the species in the proximal intestine. Our results give new insights in interspecies similarities and provide new leads for translational research and models aiming to modulate food intake processes in man. PMID:25216051

van der Wielen, Nikkie; van Avesaat, Mark; de Wit, Nicole J. W.; Vogels, Jack T. W. E.; Troost, Freddy; Masclee, Ad; Koopmans, Sietse-Jan; van der Meulen, Jan; Boekschoten, Mark V.; Muller, Michael; Hendriks, Henk F. J.; Witkamp, Renger F.; Meijerink, Jocelijn

2014-01-01

267

Limitations of Using Propidium Monoazide with qPCR to Discriminate between Live and Dead Legionella in Biofilm Samples  

PubMed Central

Accurately quantifying Legionella for regulatory purposes to protect public health is essential. Real-time PCR (qPCR) has been proposed as a better method for detecting and enumerating Legionella in samples than conventional culture method. However, since qPCR amplifies any target DNA in the sample, the technique’s inability to discriminate between live and dead cells means that counts are generally significantly overestimated. Propidium monoazide (PMA) has been used successfully in qPCR to aid live/dead discrimination. We tested PMA use as a method to count only live Legionella cells in samples collected from a modified chemostat that generates environmentally comparable samples. Counts from PMA-treated samples that were pretreated with either heat or three types of disinfectants (to kill the cells) were highly variable, with the only consistent trend being the relationship between biofilm mass and numbers of Legionella cells. Two possibilities explain this result: 1. PMA treatment worked and the subsequent muted response of Legionella to disinfection treatment is a factor of biofilm/microbiological effects; although this does not account for the relationship between the amount of biofilm sampled and the viable Legionella count as determined by PMA-qPCR; or 2. PMA treatment did not work, and any measured decrease or increase in detectable Legionella is because of other factors affecting the method. This is the most likely explanation for our results, suggesting that higher concentrations of PMA might be needed to compensate for the presence of other compounds in an environmental sample or that lower amounts of biofilm need to be sampled. As PMA becomes increasingly toxic at higher concentrations and is very expensive, augmenting the method to include higher PMA concentrations is both counterproductive and cost prohibitive. Conversely, if smaller volumes of biofilm are used, the reproducibility of the method is reduced. Our results suggest that using PMA is not an appropriate method for discriminating between live and dead cells to enumerate Legionella for regulatory purposes. PMID:25288885

Taylor, Michael J; Bentham, Richard H; Ross, Kirstin E

2014-01-01

268

Influence of qPCR workflow on target gene enumeration from environmental samples in the case of bioremediation potential estimation.  

E-print Network

??Keskkonna reostumine erinevate saasteainetega (nt. naftasaadused, kloororgaanilised ühendid) on muutunud kriitiliseks probleemiks üle maailma kahjustades inimtervist, kahandades puhta joogivee varusid ning mõjutades terveid ökosüsteeme. Et… (more)

Nõlvak, Hiie

2012-01-01

269

Comparison of two poultry litter qPCR assays targeting the 16S rRNA gene of Brevibacterium sp  

EPA Science Inventory

Chicken feces are vectors of human pathogens and are also important sources of fecal pollution in environmental waters. Consequently, methods that can detect chicken fecal pollution are needed in public health and environmental monitoring studies. In this study, we compared a pre...

270

Quantifying Genes and Transcripts To Assess the In Situ Physiology of \\  

Microsoft Academic Search

Quantitative PCR (qPCR) was coupled with reverse transcription (RT) to analyze both gene copy numbers and transcripts of the 16S rRNA gene and three reductive dehalogenase (RDase) genes (tceA, vcrA, and bvcA) as biomarkers of \\

Patrick K. H. Lee; Tamzen W. Macbeth; Kent S. Sorenson; Rula A. Deeb; Lisa Alvarez-Cohen

2008-01-01

271

Determination of specific types and relative levels of QPCR inhibitors in environmental water samples using excitation-emission matrix spectroscopy and PARAFAC.  

PubMed

Assays that utilize PCR offer powerful tools to detect pathogens and other microorganisms in environmental samples. However, PCR inhibitors present in nucleic acid extractions can increase a sample's limit of detection, skew calculated marker concentrations, or cause false-negative results. It would be advantageous to predict which samples contain various types and levels of PCR inhibitors, especially the humic and fulvic acids that are frequently cited as PCR inhibitors in natural water samples. This study investigated the relationships between quantitative PCR (qPCR) inhibition and the humic and fulvic content of dissolved organic matter (DOM), as well as several other measures of DOM quantity and quality, in water samples. QPCR inhibition was also compared to water quality parameters, precipitation levels, and land use adjacent to the sampling location. Results indicate that qPCR inhibition in the tested water samples was correlated to several humic substance-like, DOM components, most notably terrestrially-derived, humic-like DOM and microbially-derived, fulvic-like DOM. No correlation was found between qPCR inhibition and water quality parameters or land use, but a relationship was noted between inhibition and antecedent rainfall. This study suggests that certain fractions of humic substances are responsible for PCR inhibition from temperate, freshwater systems. PARAFAC modeling of excitation-emission matrix spectroscopy provides insight on the components of the DOM pool that impact qPCR success and may be useful in evaluating methods to remove PCR inhibitors present in samples. PMID:23601829

Gentry-Shields, Jennifer; Wang, Angela; Cory, Rose M; Stewart, Jill R

2013-06-15

272

Reference Assessment  

ERIC Educational Resources Information Center

This article presents interesting articles that explore several different areas of reference assessment, including practical case studies and theoretical articles that address a range of issues such as librarian behavior, patron satisfaction, virtual reference, or evaluation design. They include: (1) "Evaluating the Quality of a Chat Service"…

Bivens-Tatum, Wayne

2006-01-01

273

Effect of platform, reference material, and quantification model on enumeration of Enterococcus by quantitative PCR methods.  

PubMed

Quantitative polymerase chain reaction (qPCR) is increasingly being used for the quantitative detection of fecal indicator bacteria in beach water. QPCR allows for same-day health warnings, and its application is being considered as an option for recreational water quality testing in the United States (USEPA, 2011. EPA-OW-2011-0466, FRL-9609-3, Notice of Availability of Draft Recreational Water Quality Criteria and Request for Scientific Views). However, transition of qPCR from a research tool to routine water quality testing requires information on how various method variations affect target enumeration. Here we compared qPCR performance and enumeration of enterococci in spiked and environmental water samples using three qPCR platforms (Applied Biosystem StepOnePlus™, the BioRad iQ™5 and the Cepheid SmartCycler(®) II), two reference materials (lyophilized cells and frozen cells on filters) and two comparative CT quantification models (?CT and ??CT). Reference materials exerted the biggest influence, consistently affecting results by approximately 0.5 log(10) unit. Platform had the smallest effect, generally exerting <0.1 log(10) unit difference in final results. Quantification model led to small differences (0.04-0.2 log(10) unit) in this study with relatively uninhibited samples, but has the potential to cause as much as 8-fold (0.9 log(10) unit) difference in potentially inhibitory samples. Our findings indicate the need for a certified and centralized source of reference materials and additional studies to assess applicability of the quantification models in analyses of PCR inhibitory samples. PMID:23123048

Cao, Yiping; Sivaganesan, Mano; Kinzelman, Julie; Blackwood, A Denene; Noble, Rachel T; Haugland, Richard A; Griffith, John F; Weisberg, Stephen B

2013-01-01

274

A mixture model with a reference-based automatic selection of components for disease classification from protein and/or gene expression levels  

PubMed Central

Background Bioinformatics data analysis is often using linear mixture model representing samples as additive mixture of components. Properly constrained blind matrix factorization methods extract those components using mixture samples only. However, automatic selection of extracted components to be retained for classification analysis remains an open issue. Results The method proposed here is applied to well-studied protein and genomic datasets of ovarian, prostate and colon cancers to extract components for disease prediction. It achieves average sensitivities of: 96.2 (sd = 2.7%), 97.6% (sd = 2.8%) and 90.8% (sd = 5.5%) and average specificities of: 93.6% (sd = 4.1%), 99% (sd = 2.2%) and 79.4% (sd = 9.8%) in 100 independent two-fold cross-validations. Conclusions We propose an additive mixture model of a sample for feature extraction using, in principle, sparseness constrained factorization on a sample-by-sample basis. As opposed to that, existing methods factorize complete dataset simultaneously. The sample model is composed of a reference sample representing control and/or case (disease) groups and a test sample. Each sample is decomposed into two or more components that are selected automatically (without using label information) as control specific, case specific and not differentially expressed (neutral). The number of components is determined by cross-validation. Automatic assignment of features (m/z ratios or genes) to particular component is based on thresholds estimated from each sample directly. Due to the locality of decomposition, the strength of the expression of each feature across the samples can vary. Yet, they will still be allocated to the related disease and/or control specific component. Since label information is not used in the selection process, case and control specific components can be used for classification. That is not the case with standard factorization methods. Moreover, the component selected by proposed method as disease specific can be interpreted as a sub-mode and retained for further analysis to identify potential biomarkers. As opposed to standard matrix factorization methods this can be achieved on a sample (experiment)-by-sample basis. Postulating one or more components with indifferent features enables their removal from disease and control specific components on a sample-by-sample basis. This yields selected components with reduced complexity and generally, it increases prediction accuracy. PMID:22208882

2011-01-01

275

Overexpression and Unique Rearrangement of VH2 Transcripts in Immunoglobulin Variable Heavy Chain Genes in Ankylosing Spondylitis Patients  

Microsoft Academic Search

\\u000a To identify immunoglobulin (Ig) variable heavy chain (VH) gene usages in Korean ankylosing spondylitis (AS) patients, expression\\u000a level of VH2 genes from peripheral blood mononuclear cells (PBMCs) of 8 AS patients and 9 healthy donors was analysed by quantitative\\u000a real-time PCR (Q-PCR). Q-PCR results demonstrated VH2 genes were overexpressed in AS patients (Relative amount of mRNA of\\u000a VH2 genes to

Yeon Joo Kim; Nayoung Kim; Min-Kyung Lee; Hyo-Jin Choi; Han Joo Baek; Chang-Hoon Nam

276

Genetics Home Reference: Genetic Conditions  

MedlinePLUS

... the U.S. National Library of Medicine® Home Conditions Genes Chromosomes Handbook Glossary Resources Conditions Find a specific ... Act Copyright Privacy Accessibility Indicates a page outside Genetics Home Reference. Links to web sites outside the ...

277

DNA barcoding of freshwater fishes and the development of a quantitative qPCR assay for the species-specific detection and quantification of fish larvae from plankton samples.  

PubMed

The barcoding of mitochondrial cytochrome c oxidase subunit 1 (coI) gene was amplified and sequenced from 16 species of freshwater fishes found in Lake Wivenhoe (south-eastern Queensland, Australia) to support monitoring of reservoir fish populations, ecosystem function and water health. In this study, 630-650 bp sequences of the coI barcoding gene from 100 specimens representing 15 genera, 13 families and two subclasses of fishes allowed 14 of the 16 species to be identified and differentiated. The mean ± s.e. Kimura 2 parameter divergence within and between species was 0.52 ± 0.10 and 23.8 ± 2.20% respectively, indicating that barcodes can be used to discriminate most of the fish species accurately. The two terapontids, Amniataba percoides and Leiopotherapon unicolor, however, shared coI DNA sequences and could not be differentiated using this gene. A barcoding database was established and a qPCR assay was developed using coI sequences to identify and quantify proportional abundances of fish species in ichthyoplankton samples from Lake Wivenhoe. These methods provide a viable alternative to the time-consuming process of manually enumerating and identifying ichthyoplankton samples. PMID:24963726

Loh, W K W; Bond, P; Ashton, K J; Roberts, D T; Tibbetts, I R

2014-08-01

278

Standardization of enterococci density estimates by EPA qPCR methods and comparison of beach action value exceedances in river waters with culture methods  

EPA Science Inventory

The U.S.EPA has published recommendations for calibrator cell equivalent (CCE) densities of enterococci in recreational waters determined by a qPCR method in its 2012 Recreational Water Quality Criteria (RWQC). The CCE quantification unit stems from the calibration model used to ...

279

Influences of sample interference and interference controls on quantification of enterococci fecal indicator bacteria in surface water samples by the qPCR method  

EPA Science Inventory

A quantitative polymerase chain reaction (qPCR) method for the detection of entercocci fecal indicator bacteria has been shown to be generally applicable for the analysis of temperate fresh (Great Lakes) and marine coastal waters and for providing risk-based determinations of wat...

280

Laboratory Evaluations of the Enterococcus qPCR Method for Recreational Water Quality Testing: Method Performance and Sources of Uncertainty in Quantitative Measurements  

EPA Science Inventory

The BEACH Act of 2000 directed the U.S. EPA to establish more expeditious methods for the detection of pathogen indicators in coastal waters, as well as new water quality criteria based on these methods. Progress has been made in developing a quantitative PCR (qPCR) method for en...

281

Evaluation and validation of the suitable control genes for quantitative PCR studies in plasma DNA for non?invasive prenatal diagnosis.  

PubMed

Quantitative polymerase chain reaction (qPCR) is widely used in quantitation of plasma DNA for non?invasive prenatal diagnosis (NIPD). Control genes are indispensable as standard normalizers in qPCR analysis, and there is increasing evidence indicating that the content levels of commonly used control genes vary significantly in different independent experiments. The commonly used control genes for DNA quantitation using qPCR in plasma DNA analysis are frequently chosen without any preliminary evaluation of their suitability. The present study aimed to examine a panel of six common control genes (HBB, TERT, GAPDH, ALB, ACTB and TRG) in order to evaluate and validate the most reliable control genes for qPCR studies in the quantitation of plasma DNA from pregnant and non?pregnant females for NIPD. Plasma DNA was extracted from the peripheral blood of 18 pregnant females and 18 non?pregnant females by the QIAamp DNA mini kit. qPCR followed by geNorm, NormFinder and BestKeeper based analysis was conducted to evaluate the DNA content stabilities of the six candidate control genes. DSCR3 was used to validate the result. The study recommended TERT and the combination of ACTB and TERT as the optimal control genes for qPCR studies on pregnant/non?pregnant plasma DNA quantitation. Thus, the study reveals that the DNA content stability of widely used control genes varies significantly in pregnant and non?pregnant plasma DNA. PMID:25269617

Yang, Qiwei; Ali, Hassan Abdellah Ahmed; Yu, Shan; Zhang, Lin; Li, Xiuying; Du, Zhenwu; Zhang, Guizhen

2014-12-01

282

Validation of Candidate Reference Genes for the Accurate Normalization of Real-Time Quantitative RT-PCR Data in Rice During Seed Development  

Microsoft Academic Search

Rice seed, a natural storage organ for starch and protein, is also an ideal bioreactor for the production of valuable proteins.\\u000a Increasingly, studies focused on rice have tried to determine the functions of its genes and also to improve its yield and\\u000a quality. Real-time RT-PCR is the best available choice at present for gene expression analysis due to its accuracy,

Qian-Feng Li; Samuel S. M. Sun; Ding-Yang Yuan; Heng-Xiu Yu; Ming-Hong Gu; Qiao-Quan Liu

2010-01-01

283

Comparison of quantitative PCR assays for Escherichia coli targeting ribosomal RNA and single copy genes  

EPA Science Inventory

Aims: Compare specificity and sensitivity of quantitative PCR (qPCR) assays targeting single and multi-copy gene regions of Escherichia coli. Methods and Results: A previously reported assay targeting the uidA gene (uidA405) was used as the basis for comparing the taxono...

284

Transients in chloroplast gene transcription Sujith Puthiyaveetil, John F. Allen *  

E-print Network

Inc. All rights reserved. Keywords: Photosynthesis; Redox control; Plastoquinone; Plastid genome; Gene plants and algae contain chloroplasts, which perform photosynthesis [2] and also contain a functional important when working with young plants and the small leaves of Arabidopsis. Our results with qPCR confirm

Allen, John F.

285

Reference Roundup.  

ERIC Educational Resources Information Center

Briefly describes the nature and availability of reference books for children and adolescents and then reviews some recent publications of this type, including works of a general nature and works on social science, science, the arts, language, history and geography, and biography. (JL)

Silver, Linda; And Others

1982-01-01

286

A simple screening method for detection of Klinefelter syndrome and other X-chromosome aneuploidies based on copy number of the androgen receptor gene.  

PubMed

Due to the high prevalence and variable phenotype of patients with Klinefelter syndrome, there is a need for a robust and rapid screening method allowing early diagnosis. Here, we report on the development and detailed clinical validation of a quantitative real-time PCR (qPCR)-based method of the copy number assessment of the androgen receptor (AR) gene, located to Xq11.2-q12. We analysed samples from 50 individuals, including a healthy male and female controls and patients with Klinefelter syndrome (47,XXY; 48,XXXY) (n = 28), mosaicisms (46,XX/47,XXY/48XXYY; 45,X/46,XY) (n = 3), other sex chromosome abnormalities (46,XX males; 47,XYY)(n = 4) and normal karyotypes (46,XY) (n = 13). The reference range for the AR-copy number was established as 0.8-1.2 for one copy and 1.7-2.3 for two copies. The qPCR results were within the reference range in 17/18 samples (94%) or 30/31 (97%) samples with one or two copies of the AR gene, respectively. None of the Klinefelter patients were misdiagnosed as having a karyotype with only one X-chromosome, and in none of the 46,XY males were two copies demonstrated. We systematically compared qPCR results with those obtained with another PCR-based method, the XIST-gene expression. The XIST-expression based assay was correct in only 29/36 samples (81%). Our findings demonstrated that the AR-qPCR technique is a simple and reliable screening method for diagnosis of patients with Klinefelter syndrome or other chromosomal disorders involving an aberrant number of X-chromosomes. PMID:17720778

Ottesen, A M; Garn, I D; Aksglaede, L; Juul, A; Rajpert-De Meyts, E

2007-10-01

287

Genetics Home Reference: Gray platelet syndrome  

MedlinePLUS

Gray platelet syndrome Related Gene(s) References Quick links to this topic MedlinePlus Health information Genetic and Rare Diseases Information Center Information about genetic conditions and rare diseases ...

288

Genetics Home Reference: Phosphoglycerate dehydrogenase deficiency  

MedlinePLUS

Phosphoglycerate dehydrogenase deficiency Related Gene(s) References Quick links to this topic MedlinePlus Health information Additional NIH Resources National Institutes of Health Educational resources Information pages Patient support ...

289

Genetics Home Reference: Alpers-Huttenlocher syndrome  

MedlinePLUS

Alpers-Huttenlocher syndrome Related Gene(s) References Quick links to this topic MedlinePlus Health information Genetic and Rare Diseases Information Center Information about genetic conditions and rare diseases ...

290

Genetics Home Reference: Mucopolysaccharidosis type IV  

MedlinePLUS

Mucopolysaccharidosis type IV Related Gene(s) References Quick links to this topic MedlinePlus Health information Additional NIH Resources National Institutes of Health Educational resources Information pages Patient support ...

291

Simplex and duplex polymerase chain reaction analysis of Herculex RW (59122) maize based on one reference molecule including separated fragments of 5' integration site and endogenous gene.  

PubMed

Reference molecules, as positive controls and calibrators, have been recently developed in genetically modified organism analysis as a potential substitute for reference materials derived from plant raw materials. In this study, a novel reference molecule p59122, including the revealed 5' integration sequence of maize Herculex RW (59122), was constructed that was suitable for simplex and duplex event-specific qualitative and quantitative PCR detections. The LOD values were 10 copies both for simplex and duplex qualitative PCR when p59122 was used as the calibrator. These values were comparable to those of using genomic DNA samples with 0.01 and 0.05%, approximately 5 and 25 hyploid genomic DNA copies, respectively. The absolute LOD and LOQ values were confirmed to be as low as 10 and 25 copies of p59122 DNA both in simplex and duplex quantitative systems. Furthermore, ideal quantification data with low bias, SD and RSD values were obtained from the practical samples analyses in simplex and duplex real-time PCR systems using the reference molecule p59122 as a calibrator. All these results suggested that the developed reference molecule p59122 and the qualitative and quantitative PCR detection methods are suitable for identification and quantification of GM maize 59122 and its derived products. PMID:19916386

Li, Xiang; Yang, Litao; Zhang, Jianzhong; Wang, Shu; Shen, Kailin; Pan, Liangwen; Zhang, Dabing

2009-01-01

292

Evaluation of viable Mycobacterium avium subsp. paratuberculosis in milk using peptide-mediated separation and Propidium Monoazide qPCR.  

PubMed

The causative agent of paratuberculosis in ruminants, Mycobacterium avium subsp. paratuberculosis (MAP), although still a matter of debate, has been linked with Crohn's and other human diseases. The availability of rapid methods for assessing the viability of MAP cells in food, in particular milk, could be of great use for risk management in food safety. MAP viability is generally assessed using culture techniques that require prolonged incubation periods for the growth of MAP. To differentiate between viable and nonviable MAP cells in milk samples, this study explores the combination of two already described techniques: peptide magnetic bead separation followed by Propidium Monoazide qPCR. Using an Ordinal Multinomial Logistic Regression model to analyze the results obtained after spiking milk samples with mixtures containing different percentages of viable/dead cells, we were able to assess the probability of the viability status of MAP found in milk. This model was applied to contaminated pasteurized milk to ascertain the efficacy of heat treatment in MAP killing. The method reported herein can potentially be used for direct detection of MAP viability in milk. PMID:24860938

Ricchi, Matteo; De Cicco, Caterina; Kralik, Petr; Babak, Vladimir; Boniotti, Maria B; Savi, Roberto; Cerutti, Giulia; Cammi, Giuliana; Garbarino, Chiara; Arrigoni, Norma

2014-07-01

293

Highly aggressive behavior of malignant rhabdoid tumor: a special reference to SMARCB1\\/INI1 gene alterations using molecular genetic analysis including quantitative real-time PCR  

Microsoft Academic Search

Purpose  \\u000a SMARCB1\\/INI1, which negatively regulates cell cycle progression from G0\\/G1 into the S-phase via the p16INK4a-RB-E2F pathway, has been\\u000a reported to be inactivated homozygously by deletion and\\/or mutations in malignant rhabdoid tumor (MRT). In the current study,\\u000a we investigated the alteration of the SMARCB1\\/INI1 gene using simple methods, and its gene product at the protein level. Moreover, we investigated the

Kenichi Kohashi; Yoshinao Oda; Hidetaka Yamamoto; Sadafumi Tamiya; Teiyu Izumi; Shigeru Ohta; Tomoaki Taguchi; Sachiyo Suita; Masazumi Tsuneyoshi

2007-01-01

294

VH gene analysis in sporadic Burkitt's lymphoma: somatic mutation and intraclonal diversity with special reference to the tumor cells involving germinal center.  

PubMed

We analyzed the immunoglobulin heavy chain variable region (V(H)) gene in seven cases of sporadic Burkitt's lymphoma (sBL) to elucidate their cell of origin. In particular, we focused on the V(H) gene status of tumor cells involving adjacent germinal center (GC) by microdissecting histological sections. Among the seven V(H) genes V(H)1 family was found in two, V(H)3 in four, and V(H)4 in one. All rearranged V(H) genes demonstrated somatic mutations at percentages ranging from 1.4 to 7.5% (mean, 4.2%), which is a similar level to that seen in IgM-only B cells. Three out of four V(H) genes with more than 2% sequence difference from their corresponding germline counterpart showed evidence of antigen selection in their framework region 3. Three cases demonstrated signs of intraclonal diversity with a mutational frequency of 0.47-0.98%, which was 13.5-28.8 times as great as the Taq infidelity in our experimental conditions. However the level of somatic mutation and the effect of antigen selection on V(H) gene were diverse in these three cases, and the relationship between V(H) gene somatic mutation status and intraclonal diversity was unclear in sBL. In the analysis of microdissected tissues, all 20 tumor clones in the adjacent GCs showed additional replacement mutations in complementarity determining region 3, suggesting a role of antigen in tumor progression. This finding resembles the phenomenon that memory B-cells reenter into GC to undergo further affinity maturation. In contrast, 7/11 V(H) gene sequences irrelevant to GC were identical to those of the major tumor clone. Thus our findings suggested that sBL is derived from memory B-cells rather than GC B-cells, and that antigen stimulation is involved in the clonal expansion of a proportion of sBL. PMID:11908722

Isobe, Kouichi; Tamaru, Jun-Ichi; Nakamura, Shigeo; Harigaya, Kenichi; Mikata, Atsuo; Ito, Hisao

2002-01-01

295

Molecular Identification and Real-time Quantitative PCR (qPCR) for Rapid Detection of Thelohanellus kitauei, a Myxozoan Parasite Causing Intestinal Giant Cystic Disease in the Israel Carp  

PubMed Central

Intestinal giant-cystic disease (IGCD) of the Israel carp (Cyprinus carpio nudus) has been recognized as one of the most serious diseases afflicting inland farmed fish in the Republic of Korea, and Thelohanellus kitauei has been identified as the causative agent of the disease. Until now, studies concerning IGCD caused by T. kitauei in the Israel carp have been limited to morphological and histopathological examinations. However, these types of diagnostic examinations are relatively time-consuming, and the infection frequently cannot be detected in its early stages. In this study, we cloned the full-length 18S rRNA gene of T. kitauei isolated from diseased Israel carps, and carried out molecular identification by comparing the sequence with those of other myxosporeans. Moreover, conventional PCR and real-time quantitative PCR (qPCR) using oligonucleotide primers for the amplification of 18S rRNA gene fragment were established for further use as methods for rapid diagnosis of IGCD. Our results demonstrated that both the conventional PCR and real-time quantitative PCR systems applied herein are effective for rapid detection of T. kitauei spores in fish tissues and environmental water. PMID:22711920

Seo, Jung Soo; Jeon, Eun Ji; Kim, Moo Sang; Woo, Sung Ho; Kim, Jin Do; Jung, Sung Hee; Park, Myoung Ae; Jee, Bo Young; Kim, Jin Woo; Kim, Yi-Cheong

2012-01-01

296

PhD Studentship Outline. (Reference: DCJ002) Title: Defining the Biomass Objective of CHO cells for Recombinant Gene Design and  

E-print Network

the new generation of recombinant DNA derived therapeutic proteins (e.g. cancer medicines for Recombinant Gene Design and Metabolic Engineering Supervisor: Professor David James, Department of Chemical a synthetic DNA genetic "code" to manufacture the complex protein product. This is a cornerstone of modern

Dixon, Peter

297

Reference gene selection for quantitative reverse transcription-polymerase chain reaction normalization during in vitro adventitious rooting in Eucalyptus globulus Labill  

Microsoft Academic Search

BACKGROUND: Eucalyptus globulus and its hybrids are very important for the cellulose and paper industry mainly due to their low lignin content and frost resistance. However, rooting of cuttings of this species is recalcitrant and exogenous auxin application is often necessary for good root development. To date one of the most accurate methods available for gene expression analysis is quantitative

Márcia R de Almeida; Carolina M Ruedell; Felipe K Ricachenevsky; Raul A Sperotto; Giancarlo Pasquali; Arthur G Fett-Neto

2010-01-01

298

Age-related changes in the expression of schizophrenia susceptibility genes in the human prefrontal cortex  

Microsoft Academic Search

The molecular basis of complex neuropsychiatric disorders most likely involves many genes. In recent years, specific genetic\\u000a variations influencing risk for schizophrenia and other neuropsychiatric disorders have been reported. We have used custom\\u000a DNA microarrays and qPCR to investigate the expression of putative schizophrenia susceptibility genes and related genes of\\u000a interest in the normal human brain. Expression of 31 genes

Carlo Colantuoni; Thomas M. Hyde; Shruti Mitkus; Andrew Joseph; Leah Sartorius; Claudia Aguirre; Johanna Creswell; Elizabeth Johnson; Amy Deep-Soboslay; Mary M. Herman; Barbara K. Lipska; Daniel R. Weinberger; Joel E. Kleinman

2008-01-01

299

The multivariate detection limit for Mycoplasma pneumoniae as determined by nanorod array-surface enhanced Raman spectroscopy and comparison with limit of detection by qPCR.  

PubMed

Mycoplasma pneumoniae is a cell wall-less bacterial pathogen of the human respiratory tract that accounts for up to 20% of community-acquired pneumonia. At present, the standard for detection and genotyping is quantitative polymerase chain reaction (qPCR), which can exhibit excellent sensitivity but lacks standardization and has limited practicality for widespread, point-of-care use. We previously described a Ag nanorod array-surface enhanced Raman spectroscopy (NA-SERS) biosensing platform capable of detecting M. pneumoniae in simulated and true clinical throat swab samples with statistically significant specificity and sensitivity. We report here that differences in sample preparation influence the integrity of mycoplasma cells for NA-SERS analysis, which in turn impacts the resulting spectra. We have established a multivariate detection limit (MDL) using NA-SERS for M. pneumoniae intact-cell sample preparations. Using an adaptation of International Union of Pure and Applied Chemistry (IUPAC)-recommended methods for analyzing multivariate data sets, we found that qPCR had roughly 10× better detection limits than NA-SERS when expressed in CFU ml(-1) and DNA concentration (fg). However, the NA-SERS MDL for intact M. pneumoniae was 5.3 ± 1.0 genome equivalents (cells per ?l). By comparison, qPCR of a parallel set of samples yielded a limit of detection of 2.5 ± 0.25 cells per ?l. Therefore, for certain standard metrics NA-SERS provides a multivariate detection limit for M. pneumoniae that is essentially identical to that determined via qPCR. PMID:25335653

Henderson, Kelley C; Sheppard, Edward S; Rivera-Betancourt, Omar E; Choi, Joo-Young; Dluhy, Richard A; Thurman, Kathleen A; Winchell, Jonas M; Krause, Duncan C

2014-11-10

300

Advantageous Direct Quantification of Viable Closely Related Probiotics in Petit-Suisse Cheeses under In Vitro Gastrointestinal Conditions by Propidium Monoazide - qPCR  

PubMed Central

Species-specific Quantitative Real Time PCR (qPCR) alone and combined with the use of propidium monoazide (PMA) were used along with the plate count method to evaluate the survival of the probiotic strains Lactobacillus acidophilus La-5 and Bifidobacterium animalis subsp. lactis Bb-12, and the bacteriocinogenic and potentially probiotic strain Lactobacillus sakei subsp. sakei 2a in synbiotic (F1) and probiotic (F2) petit-suisse cheeses exposed throughout shelf-life to in vitro simulated gastrointestinal tract conditions. The three strains studied showed a reduction in their viability after the 6 h assay. Bb-12 displayed the highest survival capacity, above 72.6 and 74.6% of the initial populations, respectively, by plate count and PMA-qPCR, maintaining population levels in the range or above 6 log CFU/g. The prebiotic mix of inulin and FOS did not offer any additional protection for the strains against the simulated gastrointestinal environment. The microorganisms' populations were comparable among the three methods at the initial time of the assay, confirming the presence of mainly viable and culturable cells. However, with the intensification of the stress induced throughout the various stages of the in vitro test, the differences among the methods increased. The qPCR was not a reliable enumeration method for the quantification of intact bacterial populations, mixed with large numbers of injured and dead bacteria, as confirmed by the scanning electron microscopy results. Furthermore, bacteria plate counts were much lower (P<0.05) than with the PMA-qPCR method, suggesting the accumulation of stressed or dead microorganisms unable to form colonies. The use of PMA overcame the qPCR inability to differentiate between dead and alive cells. The combination of PMA and species-specific qPCR in this study allowed a quick and unequivocal way of enumeration of viable closely related species incorporated into probiotic and synbiotic petit-suisse cheeses and under stress conditions. PMID:24358142

Villarreal, Martha Lissete Morales; Padilha, Marina; Vieira, Antonio Diogo Silva; Franco, Bernadette Dora Gombossy de Melo; Martinez, Rafael Chacon Ruiz; Saad, Susana Marta Isay

2013-01-01

301

[Change of expression pattern of CD3 genes in peripheral blood T-cells from CML patients].  

PubMed

Our previous finding showed that down-regulation of CD3? gene was detected in patients with chronic myeloid leukemia (CML). In order to further elucidate the feature of T cell immune status in the signal transduction in CML patients, the expression patterns of all 4 CD3 genes were characterized in peripheral blood of patients, the expression levels of CD3?, ?, ? and ? chain genes were detected by real time qPCR with SYBR Green I staining in peripheral blood mononuclear cells (PBMNCs) from 17 cases of de novo CML patients in chronic phase and 17 cases of healthy individuals, the ß?-microglobulin gene was used as an internal reference, and the mRNA expression level of each CD3 gene was evaluated by the 2(-?Ct) x 100% method. The results showed that the median expression levels of CD3?, ? and ? genes (2.344%, 0.515% and 3.516%) in CML patients were not significantly different from healthy individuals (p = 0.072, p = 0.190, p = 0.615, respectively), while the expression level of CD3? gene in PBMNCs from CML patients (0.395%) was lower than that from healthy individuals (1.538%) (p < 0.001). The expression patterns of 4 CD3 genes in proper order were CD3? > CD3? > CD3? > CD3? in CML group, in contrast, the expression patterns were presented as CD3? > CD3? > CD3? > CD3? in healthy group. It is concluded that the present study characterized the expression pattern of CD3?, ?, ? and ? chain genes in CML patients, lower expression of CD3? is the feature of TCR signal transduction immunodeficiency and the expression patterns of 4 CD3 genes are changed in CML patients. PMID:20723304

Yang, Li-Jian; Chen, Shao-Hua; Wang, Liang; Chen, Si; Yu, Zhi; Lu, Yu-Hong; Li, Yang-Qiu

2010-08-01

302

A theoretical introduction to "combinatory SYBRGreen qPCR screening", a matrix-based approach for the detection of materials derived from genetically modified plants.  

PubMed

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. "Combinatory qPCR SYBRGreen screening" (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBRGREEN qPCR analysis based on four values: the C(t)- and T(m) values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, "Prime number tracing", matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBRGREEN qPCR methods and through application of a newly developed "prime number"-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product. PMID:19960341

Van den Bulcke, Marc; Lievens, Antoon; Barbau-Piednoir, Elodie; MbongoloMbella, Guillaume; Roosens, Nancy; Sneyers, Myriam; Casi, Amaya Leunda

2010-03-01

303

DEVELOPMENT OF DATA QUALITY OBJECTIVES AND USE OF TWO VARIATIONS OF GENETICALLY-MODIFIED STREPTOCOCCUS GORDONIL AS LYSIS CONTROLS IN A QPCR ASSAY FOR ASSESSING SANITARY QUALITY OF WATER  

EPA Science Inventory

Joseph B. James and Fred J. Genthner United States Environmental Protection Agency, Gulf Breeze, FL Background: Methods using rapid cycle, real-time, quantitative (QPCR) are being developed for detecting and quantifying Enterococcus spp. as well as other aquatic b...

304

Comparison of Five Major Trichome Regulatory Genes in Brassica villosa with Orthologues within the Brassicaceae  

PubMed Central

Coding sequences for major trichome regulatory genes, including the positive regulators GLABRA 1(GL1), GLABRA 2 (GL2), ENHANCER OF GLABRA 3 (EGL3), and TRANSPARENT TESTA GLABRA 1 (TTG1) and the negative regulator TRIPTYCHON (TRY), were cloned from wild Brassica villosa, which is characterized by dense trichome coverage over most of the plant. Transcript (FPKM) levels from RNA sequencing indicated much higher expression of the GL2 and TTG1 regulatory genes in B. villosa leaves compared with expression levels of GL1 and EGL3 genes in either B. villosa or the reference genome species, glabrous B. oleracea; however, cotyledon TTG1 expression was high in both species. RNA sequencing and Q-PCR also revealed an unusual expression pattern for the negative regulators TRY and CPC, which were much more highly expressed in trichome-rich B. villosa leaves than in glabrous B. oleracea leaves and in glabrous cotyledons from both species. The B. villosa TRY expression pattern also contrasted with TRY expression patterns in two diploid Brassica species, and with the Arabidopsis model for expression of negative regulators of trichome development. Further unique sequence polymorphisms, protein characteristics, and gene evolution studies highlighted specific amino acids in GL1 and GL2 coding sequences that distinguished glabrous species from hairy species and several variants that were specific for each B. villosa gene. Positive selection was observed for GL1 between hairy and non-hairy plants, and as expected the origin of the four expressed positive trichome regulatory genes in B. villosa was predicted to be from B. oleracea. In particular the unpredicted expression patterns for TRY and CPC in B. villosa suggest additional characterization is needed to determine the function of the expanded families of trichome regulatory genes in more complex polyploid species within the Brassicaceae. PMID:24755905

Nayidu, Naghabushana K.; Kagale, Sateesh; Taheri, Ali; Withana-Gamage, Thushan S.; Parkin, Isobel A. P.; Sharpe, Andrew G.; Gruber, Margaret Y.

2014-01-01

305

Validation of ST-LS1 as an Endogenous Reference Gene for Detection of AmA1 and cry1Ab Genes in Genetically Modified Potatoes using Multiplex and Real Time PCR  

Microsoft Academic Search

Solanum tuberosum L. belonging to family Solanaceae being the most important tuberous vegetable crop, the development of genetically modified\\u000a (GM) potato with improved traits is the need of the hour. PCR (polymerase chain reaction) assays are being widely used in\\u000a GM detection to meet the regulatory and legislative requirements. Detection of target sequences along with plant species specific\\u000a endogenous reference

Gurinder Jit Randhawa; Monika Singh; Ruchi Sharma

2009-01-01

306

Fluorescence in situ hybridization and qPCR to detect Merkel cell polyomavirus physical status and load in Merkel cell carcinomas.  

PubMed

The Merkel cell polyomavirus (MCPyV) is detected in 80% of Merkel cell carcinomas (MCC). Clonal integration and tumor-specific mutations in the large T antigen are strong arguments that MCPyV is a human tumor virus. However, the relationship between viral presence and cancer induction remains discussed controversially. Since almost all studies on virus prevalence are based on PCR techniques, we performed MCPyV fluorescence in situ hybridization (FISH) on MCC to gain information about the quality of the viral presence on the single cell level. MCPyV-FISH was performed on tissue microarrays containing 62 formalin-fixed and paraffin-embedded tissue samples including all tumor grades of 42 patients. The hybridization patterns were correlated to the qPCR data determined on corresponding whole tissue sections. Indeed, MCPyV-FISH and qPCR data were highly correlated, i.e. 83% for FISH-positive and 93% for FISH-negative cores. Accordingly, the mean of the qPCR values of all MCPyV-positive cores differed significantly from the mean of the negative cores (p = 0.0076). Importantly, two hybridization patterns were definable in the MCPyV-FISH: a punctate pattern (85%) indicating viral integration, which correlated with a moderate viral abundance and a combination of the punctate with a diffuse pattern (15%), suggesting a possible coexistence of integrated and episomal virus which was associated with very high viral load and VP1 expression. Thus, MCPyV-FISH adds important information on the single cell level within the histomorphological context and could therefore be an important tool to further elucidate MCPyV related carcinogenesis. PMID:24771111

Haugg, Anke M; Rennspiess, Dorit; Hausen, Axel Zur; Speel, Ernst-Jan M; Cathomas, Gieri; Becker, Jürgen C; Schrama, David

2014-12-15

307

16S rRNA gene pyrosequencing of reference and clinical samples and investigation of the temperature stability of microbiome profiles  

PubMed Central

Background Sample storage conditions, extraction methods, PCR primers, and parameters are major factors that affect metagenomics analysis based on microbial 16S rRNA gene sequencing. Most published studies were limited to the comparison of only one or two types of these factors. Systematic multi-factor explorations are needed to evaluate the conditions that may impact validity of a microbiome analysis. This study was aimed to improve methodological options to facilitate the best technical approaches in the design of a microbiome study. Three readily available mock bacterial community materials and two commercial extraction techniques, Qiagen DNeasy and MO BIO PowerSoil DNA purification methods, were used to assess procedures for 16S ribosomal DNA amplification and pyrosequencing-based analysis. Primers were chosen for 16S rDNA quantitative PCR and amplification of region V3 to V1. Swabs spiked with mock bacterial community cells and clinical oropharyngeal swabs were incubated at respective temperatures of -80°C, -20°C, 4°C, and 37°C for 4 weeks, then extracted with the two methods, and subjected to pyrosequencing and taxonomic and statistical analyses to investigate microbiome profile stability. Results The bacterial compositions for the mock community DNA samples determined in this study were consistent with the projected levels and agreed with the literature. The quantitation accuracy of abundances for several genera was improved with changes made to the standard Human Microbiome Project (HMP) procedure. The data for the samples purified with DNeasy and PowerSoil methods were statistically distinct; however, both results were reproducible and in good agreement with each other. The temperature effect on storage stability was investigated by using mock community cells and showed that the microbial community profiles were altered with the increase in incubation temperature. However, this phenomenon was not detected when clinical oropharyngeal swabs were used in the experiment. Conclusions Mock community materials originated from the HMP study are valuable controls in developing 16S metagenomics analysis procedures. Long-term exposure to a high temperature may introduce variation into analysis for oropharyngeal swabs, suggestive of storage at 4°C or lower. The observed variations due to sample storage temperature are in a similar range as the intrapersonal variability among different clinical oropharyngeal swab samples. PMID:25228989

2014-01-01

308

Quantitative PCR analysis of genes expressed during biofilm development of methicillin resistant Staphylococcus aureus (MRSA).  

PubMed

Staphylococcus aureus biofilm associated infections remains a major clinical concern in patients with indwelling devices. Quantitative real-time PCR (qPCR) can be used to investigate the pathogenic role of such biofilms. We describe qPCRs for 12 adhesion and biofilm-related genes of four S. aureus isolates which were applied during in vitro biofilm development. An endogenous control (16S rRNA) was used for signal normalization. We compared the qPCR results with structural analysis using scanning electron microscopy (SEM). The SEM studies showed different cellular products surrounding the aggregated cells at different times of biofilm formation. Using qPCR, we found that expression levels of the gene encoding fibronectin binding protein A and B and clumping factor B (fnbA/B and clfB), which involves in primary adherence of S. aureus, were significantly increased at 24h and decreased slightly and variably at 48 h when all 4 isolates were considered. The elastin binding protein (ebps) RNA expression level was significantly enhanced more than 6-fold at 24 and 48 h compared to 12h. Similar results were obtained for the intercellular adhesion biofilm required genes type C (icaC). In addition, qPCR revealed a fluctuation in expression levels at different time points of biofilm growth of other genes, indicating that different parameter modes of growth processes are operating at different times. PMID:23669446

Atshan, Salman Sahab; Shamsudin, Mariana Nor; Karunanidhi, Arunkumar; van Belkum, Alex; Lung, Leslie Than Thian; Sekawi, Zamberi; Nathan, Jayakayatri Jeevajothi; Ling, King Hwa; Seng, Johnson Shueh Chong; Ali, Alreshidi Mateg; Abduljaleel, Salwa A; Hamat, Rukman Awang

2013-08-01

309

A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants  

Microsoft Academic Search

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process\\u000a invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample.\\u000a “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials\\u000a in products. The CoSYPS decision-support system (DSS) interprets the

Marc Van den Bulcke; Antoon Lievens; Elodie Barbau-Piednoir; Guillaume MbongoloMbella; Nancy Roosens; Myriam Sneyers; Amaya Leunda Casi

2010-01-01

310

qPCR is a sensitive and rapid method for detection of cytomegaloviral DNA in formalin-fixed, paraffin-embedded biopsy tissue.  

PubMed

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies. PMID:25046572

McCoy, Morgan H; Post, Kristin; Sen, Joyashree D; Chang, Hsim Y; Zhao, Zijin; Fan, Rong; Chen, Shaoxiong; Leland, Diane; Cheng, Liang; Lin, Jingmei

2014-01-01

311

Screening biological stains with qPCR versus lateral flow immunochromatographic test strips: a quantitative comparison using analytical figures of merit.  

PubMed

Biological fluid identification is an important facet of evidence examination in forensic laboratories worldwide. While identifying bodily fluids may provide insight into which downstream DNA methods to employ, these screening techniques consume a vital portion of the available evidence, are usually qualitative, and rely on visual interpretation. In contrast, qPCR yields information regarding the amount and proportion of amplifiable genetic material. In this study, dilution series of either semen or male saliva were prepared in either buffer or female blood. The samples were subjected to both lateral flow immunochromatographic test strips and qPCR analysis. Analytical figures of merit-including sensitivity, minimum distinguishable signal (MDS) and limit of detection (LOD)-were calculated and compared between methods. By applying the theory of the propagation of random errors, LODs were determined to be 0.05 ?L of saliva for the RSID™ Saliva cards, 0.03 ?L of saliva for Quantifiler(®) Duo, and 0.001 ?L of semen for Quantifiler(®) Duo. In conclusion, quantitative PCR was deemed a viable and effective screening method for subsequent DNA profiling due to its stability in different matrices, sensitivity, and low limits of detection. PMID:24117798

Oechsle, Crystal Simson; Haddad, Sandra; Sgueglia, Joanne B; Grgicak, Catherine M

2014-01-01

312

Transients in chloroplast gene transcription  

SciTech Connect

Transcriptional regulation of chloroplast genes is demonstrated by Quantitative Polymerase Chain Reaction (qPCR). These genes encode apoproteins of the reaction centres of photosystem I and photosystem II. Their transcription is regulated by changes in wavelength of light selectively absorbed by photosystem I and photosystem II, and therefore by the redox state of an electron carrier located between the two photosystems. Chloroplast transcriptional redox regulation is shown to have greater amplitude, and the kinetics of transcriptional changes are more complex, than suggested by previous experiments using only DNA probes in Northern blot experiments. Redox effects on chloroplast transcription appear to be superimposed on an endogenous rhythm of mRNA abundance. The functional significance of these transients in chloroplast gene transcription is discussed.

Puthiyaveetil, Sujith [School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS (United Kingdom); Allen, John F. [School of Biological and Chemical Sciences, Queen Mary, University of London, Mile End Road, London E1 4NS (United Kingdom)], E-mail: j.f.allen@qmul.ac.uk

2008-04-18

313

Seasonal changes in hepatic gene expression reveal modulation of multiple processes in rainbow smelt (Osmerus mordax).  

PubMed

Rainbow smelt (Osmerus mordax) are freeze-resistant fish that accumulate glycerol and produce an antifreeze protein during winter. Quantitative reverse transcription PCR (qPCR) and subtractive hybridization studies have previously revealed five genes in rainbow smelt liver to be differentially regulated in winter in comparison with the fall when water temperatures are warmer. In order to further define the suite of processes that are regulated seasonally, we undertook a large-scale analysis of gene expression by hybridization of smelt cDNA to the salmonid 16K cGRASP microarray. In total, 69 genes were identified as up-regulated and 14 genes as down-regulated under winter conditions. A subset of these genes was examined for differential regulation by qPCR in the individual cDNA samples that were pooled for microarray analysis. Ten of the 15 genes tested showed significant change in the same direction as microarray results, whereas one showed significant change in the opposite direction. Fructose-bisphosphate aldolase B and the cytosolic NAD-dependent glycerol-3-phosphate dehydrogenase were among the most highly up-regulated genes, a result supporting a metabolic focus on glycerol synthesis during winter. Modulation of other processes, including endoplasmic reticulum stress, lipid metabolism and transport, and protein synthesis, was also suggested by the qPCR analysis of array-identified genes. The 15 genes were subsequently examined by qPCR for seasonal variation in expression over five sampling times between October and March, and ten showed significant variation in expression over the sampling period. Taken together, these results provide new understanding of the biochemical adaptations of vertebrates to an extremely low seasonal temperature. PMID:20107851

Richards, Robert C; Short, Connie E; Driedzic, William R; Ewart, K Vanya

2010-11-01

314

Gene expression underlying adaptive variation in Heliconius wing patterns: non-modular regulation of overlapping cinnabar and vermilion prepatterns  

Microsoft Academic Search

Geographical variation in the mimetic wing patterns of the butterfly Heliconius erato is a textbook example of adaptive polymorphism; however, little is known about how this variation is controlled developmentally. Using microarrays and qPCR, we identified and compared expression of candidate genes potentially involved with a red\\/yellow forewing band polymorphism in H. erato. We found that transcripts encoding the pigment

Robert D. Reed; W. Owen McMillan; Lisa M. Nagy

2008-01-01

315

Comparison of gull-specific assays targeting 16S rRNA gene of Catellicoccus marimammalium and Streptococcus spp.  

EPA Science Inventory

Gulls have been implicated as a source of fecal contamination in inland and coastal waters. Only one gull-specific assay is currently available (i.e., gull2 qPCR assay). This assay is based on the 16S rRNA gene of Catellicocclls marimammalium and has showed a high level of host-s...

316

A high-throughput open-array qPCR gene panel to identify, virulotype, and subtype O157 and non-O157 enterohemorrhagic Escherichia coli  

Microsoft Academic Search

Enterohemorrhagic Escherichia coli (EHEC), including O157 and non-O157 serotypes are significant foodborne pathogens that require sensitive and discriminatory methods for detection and characterization. There are numerous PCR-based methods for the detection of EHEC virulence factors, but the time and cost involved with large-scale screening efforts and population level analyses have limited the size and scope of studies. Recent technological advancements

Tina K. Gonzales; Megan Kulow; Dong-Jin Park; Charles W. Kaspar; Kelly S. Anklam; Kelly M. Pertzborn; Kristen D. Kerrish; Renata Ivanek; Dörte Döpfer

2011-01-01

317

Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems  

PubMed Central

Background There is a paucity of data regarding molecular markers that identify the phenotype of the tendon cell. This study aims to quantify gene expression markers that distinguish between tendon fibroblasts and other mesenchymal cells which may be used to investigate tenogenesis. Methods Expression levels for 12 genes representative of musculoskeletal tissues, including the proposed tendon progenitor marker scleraxis, relative to validated reference genes, were evaluated in matched samples of equine tendon (harvested from the superficial digital flexor tendon), cartilage and bone using quantitative PCR (qPCR). Expression levels of genes associated with tendon phenotype were then evaluated in healthy, including developmental, and diseased equine tendon tissue and in tendon fibroblasts maintained in both monolayer culture and in three dimensional (3D) collagen gels. Results Significantly increased expression of scleraxis was found in tendon compared with bone (P = 0.002) but not compared to cartilage. High levels of COL1A2 and scleraxis and low levels of tenascin-C were found to be most representative of adult tensional tendon phenotype. While, relative expression of scleraxis in developing mid-gestational tendon or in acute or chronically diseased tendon did not differ significantly from normal adult tendon, tenascin-C message was significantly upregulated in acutely injured equine tendon (P = 0.001). Relative scleraxis gene expression levels in tendon cell monolayer and 3D cultures were significantly lower than in normal adult tendon (P = 0.002, P = 0.02 respectively). Conclusion The findings of this study indicate that high expression of both COL1A2 and scleraxis, and low expression of tenascin-C is representative of a tensional tendon phenotype. The in vitro culture methods used in these experiments however, may not recapitulate the phenotype of normal tensional tendon fibroblasts in tissues as evidenced by gene expression. PMID:19245707

Taylor, Sarah E; Vaughan-Thomas, Anne; Clements, Dylan N; Pinchbeck, Gina; Macrory, Lisa C; Smith, Roger KW; Clegg, Peter D

2009-01-01

318

Genetics Home Reference: Core binding factor acute myeloid leukemia  

MedlinePLUS

Core binding factor acute myeloid leukemia Related Chromosome(s) Related Gene(s) References Quick links to this topic MedlinePlus Health information Additional NIH Resources National Institutes of Health Educational resources Information pages Patient ...

319

Genetics Home Reference: 46,XX testicular disorder of sex development  

MedlinePLUS

46,XX testicular disorder of sex development Related Chromosome(s) Related Gene(s) References Quick links to this topic ... a condition in which individuals with two X chromosomes in each cell, the pattern normally found in ...

320

Genetics Home Reference: 22q11.2 deletion syndrome  

MedlinePLUS

22q11.2 deletion syndrome Related Chromosome(s) Related Gene(s) Related Condition(s) References Quick links to this topic MedlinePlus Health information Genetic and Rare Diseases Information Center Information about ...

321

Genetics Home Reference: 22q13.3 deletion syndrome  

MedlinePLUS

22q13.3 deletion syndrome Related Chromosome(s) Related Gene(s) References Quick links to this topic MedlinePlus Health information Genetic and Rare Diseases Information Center Information about genetic conditions ...

322

Genetics Home Reference: Hereditary sensory and autonomic neuropathy type II  

MedlinePLUS

HSAN2 Related Gene(s) Related Condition(s) References Quick links to this topic MedlinePlus Health information Additional NIH Resources National Institutes of Health Educational resources Information pages Patient support ...

323

Genetics Home Reference: Hypochondroplasia  

MedlinePLUS

... people worldwide have been diagnosed with hypochondroplasia. What genes are related to hypochondroplasia? About 70 percent of ... hypochondroplasia are caused by mutations in the FGFR3 gene. This gene provides instructions for making a protein ...

324

Reach for Reference. Four Recent Reference Books  

ERIC Educational Resources Information Center

This article provides descriptions of four new science and technology encyclopedias that are appropriate for inclusion in upper elementary and/or middle school reference collections. "The Macmillan Encyclopedia of Weather" (Stern, Macmillan Reference/Gale), a one-volume encyclopedia for upper elementary and middle level students, is a…

Safford, Barbara Ripp

2004-01-01

325

Fundamentals of Reference  

ERIC Educational Resources Information Center

The all-in-one "Reference reference" you've been waiting for, this invaluable book offers a concise introduction to reference sources and services for a variety of readers, from library staff members who are asked to work in the reference department to managers and others who wish to familiarize themselves with this important area of…

Mulac, Carolyn M.

2012-01-01

326

Statistical Reference Datasets  

National Institute of Standards and Technology Data Gateway

Statistical Reference Datasets (Web, free access)   The Statistical Reference Datasets is also supported by the Standard Reference Data Program. The purpose of this project is to improve the accuracy of statistical software by providing reference datasets with certified computational results that enable the objective evaluation of statistical software.

327

Live, Digital Reference.  

ERIC Educational Resources Information Center

Discusses digital reference services, also known as virtual reference, chat reference, or online reference, based on a round table discussion at the 2002 American Library Association annual conference in Atlanta. Topics include numbers and marketing; sustainability; competition and models; evaluation methods; outsourcing; staffing and training;…

Kenney, Brian

2002-01-01

328

Genetics Home Reference: SADDAN  

MedlinePLUS

... genes are related to SADDAN? Mutations in the FGFR3 gene cause SADDAN. The FGFR3 gene provides instructions for making a protein that ... A mutation in this gene may cause the FGFR3 protein to be overly active, which leads to ...

329

nanosheets for gene therapy  

NASA Astrophysics Data System (ADS)

A new class of two-dimensional (2D) nanomaterial, transition metal dichalcogenides (TMDCs) such as MoS2, MoSe2, WS2, and WSe2 which have fantastic physical and chemical properties, has drawn tremendous attention in different fields recently. Herein, we for the first time take advantage of the great potential of MoS2 with well-engineered surface as a novel type of 2D nanocarriers for gene delivery and therapy of cancer. In our system, positively charged MoS2-PEG-PEI is synthesized with lipoic acid-modified polyethylene glycol (LA-PEG) and branched polyethylenimine (PEI). The amino end of positively charged nanomaterials can bind to the negatively charged small interfering RNA (siRNA). After detection of physical and chemical characteristics of the nanomaterial, cell toxicity was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Polo-like kinase 1 (PLK1) was investigated as a well-known oncogene, which was a critical regulator of cell cycle transmission at multiple levels. Through knockdown of PLK1 with siRNA carried by novel nanovector, qPCR and Western blot were used to measure the interfering efficiency; apoptosis assay was used to detect the transfection effect of PLK1. All results showed that the novel nanocarrier revealed good biocompatibility, reduced cytotoxicity, as well as high gene-carrying ability without serum interference, thus would have great potential for gene delivery and therapy.

Kou, Zhongyang; Wang, Xin; Yuan, Renshun; Chen, Huabin; Zhi, Qiaoming; Gao, Ling; Wang, Bin; Guo, Zhaoji; Xue, Xiaofeng; Cao, Wei; Guo, Liang

2014-10-01

330

Microarray Analysis of the Global Alterations in the Gene Expression in the Placentas From Cigarette-smoking Mothers  

Microsoft Academic Search

The effects of maternal cigarette smoking on the transcriptome of human full-term placentas were investigated by a microarray analysis. QPCR was performed for a selected set of metabolizing genes. Differentially expressed genes were selected by fold change (±1.5-fold) and analysis of variance (P<0.05) between the control and smoker groups. The expression of 174 probe sets was affected significantly. Chronic cigarette

P Huuskonen; M Storvik; M Reinisalo; P Honkakoski; J Rysä; J Hakkola; M Pasanen

2008-01-01

331

Genetics Home Reference  

MedlinePLUS

... Bar Home Current Issue Past Issues Genetics Home Reference Past Issues / Spring 2007 Table of Contents For ... page please turn Javascript on. The Genetics Home Reference (GHR) Web site — ghr.nlm.nih.gov — is ...

332

Quick Reference Contents  

Cancer.gov

Skip to Main Content CCR Home | About CCR | CCR Intranet Main Navigation Home Profiles Research Newsworthy References Special Interest Groups Training Main Links Psycho-Oncology Home Profiles Research Publications Newsworthy/Resources References Special

333

QuickReferenceContents  

Cancer.gov

Skip to Main Content CCR Home | About CCR | CCR Intranet Main Navigation Home Profiles Research Newsworthy References Special Interest Groups Training Main Links Psycho-Oncology Home Profiles Research Publications Newsworthy/Resources References Special

334

Chemistry Reference Sheets  

NSDL National Science Digital Library

This reference sheet, presented by the National Nanotechnology Infrastructure Network provides a valuable chemistry reference sheet for high school students. Definition of terms, diagrams, abbreviations, mathematical notations, the periodic table, and other useful information is provided in an easy to use format. Included in this lesson are the front and back sides of this reference sheet.

2014-07-25

335

Physical Science Reference Sheets  

NSDL National Science Digital Library

This reference sheet, presented by the National Nanotechnology Infrastructure Network provides a valuable physical science reference sheet for high school students. Definition of terms, diagrams, abbreviations, mathematical notations, the periodic table, and other useful information is provided in an easy to use format. Included in this lesson are the front and back sides of this reference sheet.

2014-07-11

336

Biology Reference Sheets  

NSDL National Science Digital Library

This reference sheet, presented by the National Nanotechnology Infrastructure Network provides a valuable list biology reference material for high school students in the life sciences. Definition of terms, diagrams, abbreviations, mathematical notations, and other useful information is provided in an easy to use format. Included in this lesson are the front and back sides of this reference sheet.

2014-07-25

337

Genetics Home Reference: Galactosemia  

MedlinePLUS

... mutations in a particular gene, and affect different enzymes involved in breaking down galactose. Classic galactosemia, also ... GALK1 , and GALT genes provide instructions for making enzymes that are essential for processing galactose obtained from ...

338

References for marine science  

NASA Astrophysics Data System (ADS)

Standard and Reference Materials for Marine Science, National Oceanic and Atmospheric Administration Technical Memo OMA-51 (2nd edition, 434 pp.), by A. Y. Cantillo, is now available. This compilation of reference materials was prepared at the request of the Group of Experts on Standards and Reference Materials and was printed by NOAA. GESREM is sponsored by the International Atomic Energy Agency, the Intergovernmental Oceanographic Commission, and the United Nations Program.Reference materials are included on ashes, gases, instrument performance materials, oils, physical properties, rocks, sediments, sludges, tissues and waters. For each reference material, source, description and preparation, analyses and values, cost, references, and comments are given. Indices are included for elements, isotopes and organic compounds. Cross references to Chemical Abstracts Service registry numbers and alternate names and chemical structures of organic compounds are also provided.

1990-06-01

339

Testing an aflatoxin B1 gene signature in rat archival tissues.  

PubMed

Archival tissues from laboratory studies represent a unique opportunity to explore the relationship between genomic changes and agent-induced disease. In this study, we evaluated the applicability of qPCR for detecting genomic changes in formalin-fixed, paraffin-embedded (FFPE) tissues by determining if a subset of 14 genes from a 90-gene signature derived from microarray data and associated with eventual tumor development could be detected in archival liver, kidney, and lung of rats exposed to aflatoxin B1 (AFB1) for 90 days in feed at 1 ppm. These tissues originated from the same rats used in the microarray study. The 14 genes evaluated were Adam8, Cdh13, Ddit4l, Mybl2, Akr7a3, Akr7a2, Fhit, Wwox, Abcb1b, Abcc3, Cxcl1, Gsta5, Grin2c, and the C8orf46 homologue. The qPCR FFPE liver results were compared to the original liver microarray data and to qPCR results using RNA from fresh frozen liver. Archival liver paraffin blocks yielded 30 to 50 ?g of degraded RNA that ranged in size from 0.1 to 4 kB. qPCR results from FFPE and fresh frozen liver samples were positively correlated (p ? 0.05) by regression analysis and showed good agreement in direction and proportion of change with microarray data for 11 of 14 genes. All 14 transcripts could be amplified from FFPE kidney RNA except the glutamate receptor gene Grin2c; however, only Abcb1b was significantly upregulated from control. Abundant constitutive transcripts, S18 and ?-actin, could be amplified from lung FFPE samples, but the narrow RNA size range (25-500 bp length) prevented consistent detection of target transcripts. Overall, a discrete gene signature derived from prior transcript profiling and representing cell cycle progression, DNA damage response, and xenosensor and detoxication pathways was successfully applied to archival liver and kidney by qPCR and indicated that gene expression changes in response to subchronic AFB1 exposure occurred predominantly in the liver, the primary target for AFB1-induced tumors. We conclude that an evaluation of gene signatures in archival tissues can be an important toxicological tool for evaluating critical molecular events associated with chemical exposures. PMID:22545673

Merrick, B Alex; Auerbach, Scott S; Stockton, Patricia S; Foley, Julie F; Malarkey, David E; Sills, Robert C; Irwin, Richard D; Tice, Raymond R

2012-05-21

340

High frequency reference electrode  

DOEpatents

A high frequency reference electrode for electrochemical experiments comprises a mercury-calomel or silver-silver chloride reference electrode with a layer of platinum around it and a layer of a chemically and electrically resistant material such as TEFLON around the platinum covering all but a small ring or "halo" at the tip of the reference electrode, adjacent to the active portion of the reference electrode. The voltage output of the platinum layer, which serves as a redox electrode, and that of the reference electrode are coupled by a capacitor or a set of capacitors and the coupled output transmitted to a standard laboratory potentiostat. The platinum may be applied by thermal decomposition to the surface of the reference electrode. The electrode provides superior high-frequency response over conventional electrodes.

Kronberg, James W. (Aiken, SC)

1994-01-01

341

Is Gene Transcription Involved in Seed Dry After-Ripening?  

PubMed Central

Orthodox seeds are living organisms that survive anhydrobiosis and may display dormancy, an inability to germinate at harvest. Seed germination potential can be acquired during a prolonged period of dry storage called after-ripening. The aim of this work was to determine if gene transcription is an underlying regulatory mechanism for dormancy alleviation during after-ripening. To identify changes in gene transcription strictly associated with the acquisition of germination potential but not with storage, we used seed storage at low relative humidity that maintains dormancy as control. Transcriptome profiling was performed using DNA microarray to compare change in gene transcript abundance between dormant (D), after-ripened non-dormant (ND) and after-ripened dormant seeds (control, C). Quantitative real-time polymerase chain reaction (qPCR) was used to confirm gene expression. Comparison between D and ND showed the differential expression of 115 probesets at cut-off values of two-fold change (p<0.05). Comparisons between both D and C with ND in transcript abundance showed that only 13 transcripts, among 115, could be specific to dormancy alleviation. qPCR confirms the expression pattern of these transcripts but without significant variation between conditions. Here we show that sunflower seed dormancy alleviation in the dry state is not related to regulated changes in gene expression. PMID:24466101

Meimoun, Patrice; Mordret, Ernest; Langlade, Nicolas B.; Balzergue, Sandrine; Arribat, Sandrine; Bailly, Christophe; El-Maarouf-Bouteau, Hayat

2014-01-01

342

Optical voltage reference  

SciTech Connect

An optical voltage reference for providing an alternative to a battery source. The optical reference apparatus provides a temperature stable, high precision, isolated voltage reference through the use of optical isolation techniques to eliminate current and impedance coupling errors. Pulse rate frequency modulation is employed to eliminate errors in the optical transmission link while phase-lock feedback is employed to stabilize the frequency to voltage transfer function.

Rankin, R.; Kotter, D.

1992-12-31

343

Optical voltage reference  

DOEpatents

An optical voltage reference for providing an alternative to a battery source is described. The optical reference apparatus provides a temperature stable, high precision, isolated voltage reference through the use of optical isolation techniques to eliminate current and impedance coupling errors. Pulse rate frequency modulation is employed to eliminate errors in the optical transmission link while phase-lock feedback is employed to stabilize the frequency to voltage transfer function. 2 figures.

Rankin, R.; Kotter, D.

1994-04-26

344

Optical voltage reference  

DOEpatents

An optical voltage reference for providing an alternative to a battery source. The optical reference apparatus provides a temperature stable, high precision, isolated voltage reference through the use of optical isolation techniques to eliminate current and impedance coupling errors. Pulse rate frequency modulation is employed to eliminate errors in the optical transmission link while phase-lock feedback is employed to stabilize the frequency to voltage transfer function.

Rankin, Richard (Ammon, ID); Kotter, Dale (Bingham County, ID)

1994-01-01

345

Detection of the Helicobacter pylori dupA gene is strongly affected by the PCR design.  

PubMed

The Helicobacter pylori virulence gene dupA is usually detected by PCR, but the primer binding sites used are highly variable. Our newly designed qPCR against a conserved region of dupA was positive in 64.2% of 394 clinical isolates while the positivity rate of the commonly used PCRs ranged from 29.9% to 37.8%. PMID:25128081

Abadi, Amin Talebi Bezmin; Loffeld, Ruud J L F; Constancia, Ashandra C; Wagenaar, Jaap A; Kusters, Johannes G

2014-11-01

346

Microarray comparison of the gene expression profiles in the adult vs. embryonic day 14 rat liver  

PubMed Central

The aim of the present study was to identify the differentially-expressed genes of embryonic day 14 (ED 14) rat liver in comparison to adult rat liver, which may provide specific information for the investigation of the hepatogenesis mechanism. The gene expression profiles of ED 14 and adult rat livers were investigated using microarray analysis (the Illumina RatRef-12 Expression BeadChip). Quantitative polymerase chain reaction (qPCR) analyses were conducted to confirm the gene expression. There were 787 genes upregulated in the embryonic liver. Based on the gene ontology classification system, which was analyzed by the database for annotation, visualization and integrated discovery software, a number of the upregulated genes were categorized into the distinct and differentially-expressed functional groups, including metabolism pathway, cell cycle, transcription, signal transduction, purine metabolism, cell structure, transportation and apoptosis. qPCR analyses confirmed the gene expression. Eleven upregulated genes were found in the ED 14 rat liver, which may provide specific information for the understanding of the molecular mechanisms that control hepatogenesis. These overexpressed genes are potential markers for identifying hepatic progenitor cells. PMID:25054008

ZHENG, JIE; YU, SHUNA; JIANG, ZHENGCHEN; SHI, CAIXING; LI, JIN; DU, XIAODONG; WANG, HAILIANG; JIANG, JIYING

2014-01-01

347

RAS Reference Reagents  

Cancer.gov

Posted: 09/22/2014 Posted: 09/22/2014 RAS Reference Reagents Reference Reagents Group An important priority of the RAS Initiative is to distribute highly validated materials and methods to the world-wide community of RAS researchers. Two panels of

348

HAZARDOUS WASTE MANAGEMENT REFERENCE  

E-print Network

Cathode Ray Tubes and Consumer Electronic Devices 20 Lamps 21 #12;Hazardous Waste Management Reference Guide Page 3 of 36 CHAPTER FIVE ­ WASTE MINIMIZATION 22 SUBSTITUTION 22 RECYCLING AND REDISTRIBUTION 22HAZARDOUS WASTE MANAGEMENT REFERENCE GUIDE Prepared by Environment, Health and Safety Office

Faraon, Andrei

349

An Online Reference System.  

ERIC Educational Resources Information Center

Describes a computer aid developed to assist in academic library reference service using the DataPhase Circulation System, an automated system that features full cataloging records in database and permits local programing. Access points (subject, type of reference work, course) and database structure and user screens are highlighted. (EJS)

Chisman, Janet; Treat, William

1984-01-01

350

Online Voucher Quick Reference  

E-print Network

Online Voucher Quick Reference NUFinancials Payments OnlineVoucherQuickReference 12/10/2013 - rb > Online Voucher Entry Page · or NUFinancials Accounts Payable Vouchers Add/Update Online Voucher/letters or barcode# on form). 2. Enter the Invoice Date (from invoice, date of service, or today's date). 3. Look up

Shull, Kenneth R.

351

Martindale's "The Reference Desk"  

NSDL National Science Digital Library

This online reference provides links to reference materials on a variety of science topics, including astronomy, biosciences, chemistry, engineering, geosciences, and many others. The links are organized by topics (education, entertainment, music, science, and so forth). Within each topic, the resources are arranged by type (dictionaries, databases, journals, web sites, and so forth).

Martindale, Jim

352

Activities Using References.  

ERIC Educational Resources Information Center

Elementary students are introduced to a wide variety of reference materials. Separate sections contain activities for using an atlas, "The Book of Lists," dictionaries, encyclopedias, "Familiar Quotations," games, "The Guiness Book of World Records," magazines, multi-