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1

Toward Enhanced MIQE Compliance: Reference Residual Normalization of qPCR Gene Expression Data.  

PubMed

Normalization of fluorescence-based quantitative real-time PCR (qPCR) data varies across quantitative gene expression studies, despite its integral role in accurate data quantification and interpretation. Identification of suitable reference genes plays an essential role in accurate qPCR normalization, as it ensures that uncorrected gene expression data reflect normalized data. The reference residual normalization (RRN) method presented here is a modified approach to conventional 2(-??Ct)qPCR normalization that increases mathematical transparency and incorporates statistical assessment of reference gene stability. RRN improves mathematical transparency through the use of sample-specific reference residuals (RR i ) that are generated from the mean Ct of one or more reference gene(s) that are unaffected by treatment. To determine stability of putative reference genes, RRN uses ANOVA to assess the effect of treatment on expression and subsequent equivalence-threshold testing to establish the minimum permitted resolution. Step-by-step instructions and comprehensive examples that demonstrate the influence of reference gene stability on target gene normalization and interpretation are provided. Through mathematical transparency and statistical rigor, RRN promotes compliance with Minimum Information for Quantitative Experiments and, in so doing, provides increased confidence in qPCR data analysis and interpretation. PMID:24982597

Edmunds, Richard C; McIntyre, Jenifer K; Luckenbach, J Adam; Baldwin, David H; Incardona, John P

2014-07-01

2

Toward Enhanced MIQE Compliance: Reference Residual Normalization of qPCR Gene Expression Data  

PubMed Central

Normalization of fluorescence-based quantitative real-time PCR (qPCR) data varies across quantitative gene expression studies, despite its integral role in accurate data quantification and interpretation. Identification of suitable reference genes plays an essential role in accurate qPCR normalization, as it ensures that uncorrected gene expression data reflect normalized data. The reference residual normalization (RRN) method presented here is a modified approach to conventional 2???CtqPCR normalization that increases mathematical transparency and incorporates statistical assessment of reference gene stability. RRN improves mathematical transparency through the use of sample-specific reference residuals (RRi) that are generated from the mean Ct of one or more reference gene(s) that are unaffected by treatment. To determine stability of putative reference genes, RRN uses ANOVA to assess the effect of treatment on expression and subsequent equivalence-threshold testing to establish the minimum permitted resolution. Step-by-step instructions and comprehensive examples that demonstrate the influence of reference gene stability on target gene normalization and interpretation are provided. Through mathematical transparency and statistical rigor, RRN promotes compliance with Minimum Information for Quantitative Experiments and, in so doing, provides increased confidence in qPCR data analysis and interpretation.

McIntyre, Jenifer K.; Luckenbach, J. Adam; Baldwin, David H.; Incardona, John P.

2014-01-01

3

Validation of reference genes for qPCR studies on Caco-2 cell differentiation  

Microsoft Academic Search

Validation of reference gene expression stabilities is a prerequisite for reliable normalization of qPCR data. The present study assessed the variation of six reference genes (ACTB, GAPDH, B2M, HPRT1, SDHA, YWHAZ) in Caco-2 cells under the influence of different growth supports and cultivation periods. Genes were ranked according to their stability using the geNorm software. To verify the influence of

Claudia Piana; Michael Wirth; Stefan Gerbes; Helmut Viernstein; Franz Gabor; Stefan Toegel

2008-01-01

4

Elasmobranch qPCR reference genes: a case study of hypoxia preconditioned epaulette sharks  

Microsoft Academic Search

BACKGROUND: Elasmobranch fishes are an ancient group of vertebrates which have high potential as model species for research into evolutionary physiology and genomics. However, no comparative studies have established suitable reference genes for quantitative PCR (qPCR) in elasmobranchs for any physiological conditions. Oxygen availability has been a major force shaping the physiological evolution of vertebrates, especially fishes. Here we examined

Kalle T Rytkönen; Gillian MC Renshaw; Kevin J Ashton; Grant Williams-Pritchard; Erica H Leder; Mikko Nikinmaa

2010-01-01

5

Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR  

Microsoft Academic Search

BACKGROUND: Real-time quantitative PCR (qPCR) is a method for rapid and reliable quantification of mRNA transcription. Internal standards such as reference genes are used to normalise mRNA levels between different samples for an exact comparison of mRNA transcription level. Selection of high quality reference genes is of crucial importance for the interpretation of data generated by real-time qPCR. RESULTS: In

Ann-Britt Nygard; Claus B Jørgensen; Susanna Cirera; Merete Fredholm

2007-01-01

6

Evaluation of candidate reference genes for qPCR in maize.  

PubMed

Quantitative real-time PCR (qPCR) is a powerful tool to measure gene expression levels. Accurate and reproducible results are dependent on the correct choice of the reference genes for data normalization. To date, screenings evaluating candidate reference gene stability for expression studies in maize have not been reported. In the present work, we analyzed the expression patterns of 12 genes in a set of 20 maize samples, obtained from different tissues of plants grown at various experimental conditions. Using genorm(PLUS), NormFinder and BestKeeper algorithms, the expression stability of three "classical" reference genes, such as ACT, TUB and 18S rRNA, and the newly identified candidates, was assessed. With respect to the algorithms, our results showed similar performance among genorm(PLUS), NormFinder and BestKeeper in evaluating the suitability of reference genes. Our data therefore showed that the currently and widely used reference genes for data normalization in maize were not the most stable expressed transcripts. Five of the new putative reference genes (CUL, FPGS, LUG, MEP and UBCP) exhibited the highest expression stability according to all algorithms. In conclusion, with this study, we provide a list of validated reference genes and their relative primer sequences to conduct reliable qPCR experiments in maize. PMID:22459324

Manoli, Alessandro; Sturaro, Alba; Trevisan, Sara; Quaggiotti, Silvia; Nonis, Alberto

2012-05-15

7

Identification of valid reference genes for the normalization of RT qPCR gene expression data in human brain tissue  

Microsoft Academic Search

BACKGROUND: Studies of gene expression in post mortem human brain can contribute to understanding of the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Quantitative real-time PCR (RT qPCR) is often used to analyse gene expression. The validity of results obtained using RT qPCR is reliant on accurate data normalization. Reference

David TR Coulson; Simon Brockbank; Joseph G Quinn; Suzanne Murphy; Rivka Ravid; G Brent Irvine; Janet A Johnston

2008-01-01

8

Selection of reference genes for gene expression studies in zucchini (Cucurbita pepo) using qPCR.  

PubMed

The zucchini (Cucurbita pepo) is an important food crop, the transcriptomics of which are a fundamental tool to accelerate the development of new varieties by breeders. However, the suitability of reference genes for data normalization in zucchini has not yet been studied. The aim of this study was to assess the suitability of 13 genes for their potential use as reference genes in quantitative real-time PCR. Assays were performed on 34 cDNA samples representing plants under different stresses and at different developmental stages. The application of geNorm and NormFinder software revealed that the use of a combination of UFP, EF-1A, RPL36aA, PP2A, and CAC genes for the different experimental sets was the best strategy for reliable normalization. In contrast, 18S rRNA and TUA were less stable and unsuitable for use as internal controls. These results provide the possibility to allow more accurate use of qPCR in this horticultural crop. PMID:21476515

Obrero, Angeles; Die, Jose V; Román, Belén; Gómez, Pedro; Nadal, Salvador; González-Verdejo, Clara I

2011-05-25

9

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.

2011-01-01

10

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

11

Selection of Reference Genes for Quantitative Real Time PCR (qPCR) Assays in Tissue from Human Ascending Aorta  

PubMed Central

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n?=?30; dilated aorta and normal valve n?=?10; normal aorta and BAV n?=?4; dilated aorta and BAV n?=?8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1?, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples.

Rueda-Martinez, Carmen; Lamas, Oscar; Mataro, Maria Jose; Robledo-Carmona, Juan; Sanchez-Espin, Gemma; Jimenez-Navarro, Manuel; Such-Martinez, Miguel; Fernandez, Borja

2014-01-01

12

Selection of reference genes for quantitative real time PCR (qPCR) assays in tissue from human ascending aorta.  

PubMed

Dilatation of the ascending aorta (AAD) is a prevalent aortopathy that occurs frequently associated with bicuspid aortic valve (BAV), the most common human congenital cardiac malformation. The molecular mechanisms leading to AAD associated with BAV are still poorly understood. The search for differentially expressed genes in diseased tissue by quantitative real-time PCR (qPCR) is an invaluable tool to fill this gap. However, studies dedicated to identify reference genes necessary for normalization of mRNA expression in aortic tissue are scarce. In this report, we evaluate the qPCR expression of six candidate reference genes in tissue from the ascending aorta of 52 patients with a variety of clinical and demographic characteristics, normal and dilated aortas, and different morphologies of the aortic valve (normal aorta and normal valve n = 30; dilated aorta and normal valve n = 10; normal aorta and BAV n = 4; dilated aorta and BAV n = 8). The expression stability of the candidate reference genes was determined with three statistical algorithms, GeNorm, NormFinder and Bestkeeper. The expression analyses showed that the most stable genes for the three algorithms employed were CDKN1?, POLR2A and CASC3, independently of the structure of the aorta and the valve morphology. In conclusion, we propose the use of these three genes as reference genes for mRNA expression analysis in human ascending aorta. However, we suggest searching for specific reference genes when conducting qPCR experiments with new cohort of samples. PMID:24841551

Rueda-Martínez, Carmen; Lamas, Oscar; Mataró, María José; Robledo-Carmona, Juan; Sánchez-Espín, Gemma; Jiménez-Navarro, Manuel; Such-Martínez, Miguel; Fernández, Borja

2014-01-01

13

Identification and validation of reference genes for qPCR detection of serum microRNAs in colorectal adenocarcinoma patients.  

PubMed

Serum microRNAs (miRNAs) have become a highlighted research hotspot, especially for their great potential as a novel promising non-invasive biomarker in cancer diagnosis. The most frequently used approach for serum miRNAs detection is quantitative real time polymerase chain reaction (qPCR). In order to obtain reliable qPCR data of miRNAs expression, the use of reference genes as endogenous control is undoubtly necessary. However, no systematic evaluation and validation of reference genes for normalizing qPCR analysis of serum miRNAs has been reported in colorectal adenocarcinoma. We firstly profiled pooled serum of colorectal adenocarcinoma, colorectal adenoma and healthy controls and selected a list of 13 miRNAs as candidate reference genes. U6 snRNA (U6) and above-mentioned 13 miRNAs were included in further confirmation by qPCR. As a result, 5 miRNAs (miR-151a-3p, miR-4446-3p, miR-221-3p, miR-93-5p and miR-3184-3p) were not detected in all samples and 2 miRNAs (miR-197-3p and miR-26a-5p) were relatively low with median Cq more than 35, and were excluded from further stability analysis. Then variable stability of other 6 miRNAs (miR-103b, miR-484, miR-16-5p, miR-3615, miR-18a-3p and miR-191-5p) and U6 were evaluated using two algorithms: geNorm and NormFinder which both identified miR-191-5p as the most stably expressed reference gene and selected miR-191-5p and U6 as the most stable pair of reference genes. After validating in an independent large cohorts and selecting miR-92a-3p as target miRNA to evaluate the effect of reference gene, we propose that combination of miR-191-5p and U6 could be used as reference genes for serum microRNAs qPCR data in colorectal adenocarcinoma, colorectal adenoma and healthy controls. PMID:24349425

Zheng, Guixi; Wang, Haiyan; Zhang, Xin; Yang, Yongmei; Wang, Lili; Du, Lutao; Li, Wei; Li, Juan; Qu, Ailin; Liu, Yimin; Wang, Chuanxin

2013-01-01

14

Selection of suitable reference genes for qPCR normalization under abiotic stresses in Oenanthe javanica (BI.) DC.  

PubMed

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

Jiang, Qian; Wang, Feng; Li, Meng-Yao; Ma, Jing; Tan, Guo-Fei; Xiong, Ai-Sheng

2014-01-01

15

Reference genes for qPCR assays in toxic metal and salinity stress in two flatworm model organisms.  

PubMed

The flatworm species Schmidtea mediterranea and Macrostomum lignano have become new and innovative model organisms in stem cell, regeneration and tissue homeostasis research. Because of their unique stem cell system, (lab) technical advantages and their phylogenetic position within the Metazoa, they are also ideal candidate model organisms for toxicity assays. As stress and biomarker screenings are often performed at the transcriptional level, the aim of this study was to establish a set of reference genes for qPCR experiments for these two model organisms in different stress situations. We examined the transcriptional stability of nine potential reference genes (actb, tubb, ck2, cox4, cys, rpl13, gapdh, gm2ap, plscr1) to assess those that are most stable during altered stress conditions (exposure to carcinogenic metals and salinity stress). The gene expression stability was evaluated by means of geNorm and NormFinder algorithms. Sets of best reference genes in these analyses varied between different stress situations, although gm2ap and actb were stably transcribed during all tested combinations. In order to demonstrate the impact of bad normalisation, the stress-specific gene hsp90 was normalised to different sets of reference genes. In contrast to the normalisation according to GeNorm and NormFinder, normalisation of hsp90 in Macrostomum lignano during cadmium stress did not show a significant difference when normalised to only gapdh. On the other hand an increase of variability was noticed when normalised to all nine tested reference genes together. Testing appropriate reference genes is therefore strongly advisable in every new experimental condition. PMID:22080432

Plusquin, Michelle; DeGheselle, Olivier; Cuypers, Ann; Geerdens, Ellen; Van Roten, Andromeda; Artois, Tom; Smeets, Karen

2012-03-01

16

Selection of reliable reference genes for qPCR studies on chondroprotective action  

Microsoft Academic Search

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

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

2007-01-01

17

Evaluation of qPCR reference genes in two genotypes of Populus for use in photoperiod and low-temperature studies  

PubMed Central

Background Quantitative PCR (qPCR) is a widely used technique for gene expression analysis. A common normalization method for accurate qPCR data analysis involves stable reference genes to determine relative gene expression. Despite extensive research in the forest tree species Populus, there is not a resource for reference genes that meet the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) standards for qPCR techniques and analysis. Since Populus is a woody perennial species, studies of seasonal changes in gene expression are important towards advancing knowledge of this important developmental and physiological trait. The objective of this study was to evaluate reference gene expression stability in various tissues and growth conditions in two important Populus genotypes (P. trichocarpa “Nisqually 1” and P. tremula x P. alba 717 1-B4) following MIQE guidelines. Results We evaluated gene expression stability in shoot tips, young leaves, mature leaves and bark tissues from P. trichocarpa and P. tremula. x P. alba grown under long-day (LD), short-day (SD) or SD plus low-temperatures conditions. Gene expression data were analyzed for stable reference genes among 18S rRNA, ACT2, CDC2, CYC063, TIP4-like, UBQ7, PT1 and ANT using two software packages, geNormPLUS and BestKeeper. GeNormPLUS ranked TIP4-like and PT1 among the most stable genes in most genotype/tissue combinations while BestKeeper ranked CDC2 and ACT2 among the most stable genes. Conclusions This is the first comprehensive evaluation of reference genes in two important Populus genotypes and the only study in Populus that meets MIQE standards. Both analysis programs identified stable reference genes in both genotypes and all tissues grown under different photoperiods. This set of reference genes was found to be suitable for either genotype considered here and may potentially be suitable for other Populus species and genotypes. These results provide a valuable resource for the Populus research community.

2012-01-01

18

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.

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

2014-01-01

19

Validation of reference genes for normalization of qPCR gene expression data from Coffea spp. hypocotyls inoculated with Colletotrichum kahawae  

PubMed Central

Background Coffee production in Africa represents a significant share of the total export revenues and influences the lives of millions of people, yet severe socio-economic repercussions are annually felt in result of the overall losses caused by the coffee berry disease (CBD). This quarantine disease is caused by the fungus Colletotrichum kahawae Waller and Bridge, which remains one of the most devastating threats to Coffea arabica production in Africa at high altitude, and its dispersal to Latin America and Asia represents a serious concern. Understanding the molecular genetic basis of coffee resistance to this disease is of high priority to support breeding strategies. Selection and validation of suitable reference genes presenting stable expression in the system studied is the first step to engage studies of gene expression profiling. Results In this study, a set of ten genes (S24, 14-3-3, RPL7, GAPDH, UBQ9, VATP16, SAND, UQCC, IDE and ?-Tub9) was evaluated to identify reference genes during the first hours of interaction (12, 48 and 72 hpi) between resistant and susceptible coffee genotypes and C. kahawae. Three analyses were done for the selection of these genes considering the entire dataset and the two genotypes (resistant and susceptible), separately. The three statistical methods applied GeNorm, NormFinder, and BestKeeper, allowed identifying IDE as one of the most stable genes for all datasets analysed, and in contrast GADPH and UBQ9 as the least stable ones. In addition, the expression of two defense-related transcripts, encoding for a receptor like kinase and a pathogenesis related protein 10, were used to validate the reference genes selected. Conclusion Taken together, our results provide guidelines for reference gene(s) selection towards a more accurate and widespread use of qPCR to study the interaction between Coffea spp. and C. kahawae.

2013-01-01

20

Murine 3T3-L1 Adipocyte Cell Differentiation Model: Validated Reference Genes for qPCR Gene Expression Analysis  

PubMed Central

Background Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). Methodology/Principal Findings Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. Conclusion Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

Arsenijevic, Tatjana; Gregoire, Francoise; Delforge, Valerie; Delporte, Christine; Perret, Jason

2012-01-01

21

Validation of reference genes aiming accurate normalization of qPCR data in soybean upon nematode parasitism and insect attack  

PubMed Central

Background Soybean pathogens and pests reduce grain production worldwide. Biotic interaction cause extensive changes in plant gene expression profile and the data produced by functional genomics studies need validation, usually done by quantitative PCR. Nevertheless, this technique relies on accurate normalization which, in turn, depends upon the proper selection of stable reference genes for each experimental condition. To date, only a few studies were performed to validate reference genes in soybean subjected to biotic stress. Here, we report reference genes validation in soybean during root-knot nematode (Meloidogyne incognita) parasitism and velvetbean caterpillar (Anticarsia gemmatalis) attack. Findings The expression stability of nine classical reference genes (GmCYP2, GmELF1A, GmELF1B, GmACT11, GmTUB, GmTUA5, GmG6PD, GmUBC2 and GmUBC4) was evaluated using twenty-four experimental samples including different organs, developmental stages, roots infected with M. incognita and leaves attacked by A. gemmatalis. Two different algorithms (geNorm and NormFinder) were used to determine expression stability. GmCYP2 and GmUBC4 are the most stable in different organs. Considering the developmental stages, GmELF1A and GmELF1B genes are the most stable. For spatial and temporal gene expression studies, normalization may be performed using GmUBC4, GmUBC2, GmCYP2 and GmACT11 as reference genes. Our data indicate that both GmELF1A and GmTUA5 are the most stable reference genes for data normalization obtained from soybean roots infected with M. incognita, and GmCYP2 and GmELF1A are the most stable in soybean leaves infested with A. gemmatalis. Conclusions Future expression studies using nematode infection and caterpilar infestation in soybean plant may utilize the reference gene sets reported here.

2013-01-01

22

Identification of a common reference gene pair for qPCR in human mesenchymal stromal cells from different tissue sources treated with VEGF  

PubMed Central

Background Human mesenchymal stromal cells from the bone marrow (BMSCs) are widely used as experimental regenerative treatment of ischemic heart disease, and the first clinical trials using adipose-derived stromal cells (ASCs) are currently being conducted. Regenerative mechanisms of BMSCs and ASCs are manifold and in vitro pretreatment of the cells with growth factors has been applied to potentially enhance these properties. When characterizing the transcriptional activity of these cellular mechanisms in vitro it is important to consider the effect of the growth factor treatment on reference genes (RGs) for the normalization of qPCR data. Results BMSCs and ASCs were stimulated with vascular endothelial growth factor A-165 (VEGF) for one week, and compared with un-stimulated cells from the same donor. The stability of nine RGs through VEGF treatment as well as the donor variation was assessed using the GenEx software with the subprograms geNorm and Normfinder. The procedure of stepwise elimination was validated by poor performance of eliminated RGs in a normalization experiment using vWF as target gene. Normfinder found the TATA box binding protein (TBP) to be the most stable single RG for both BMSCs and ASCs. The optimal number of RGs for ASCs was two, and the lowest variance for vWF normalization was found using TBP and YWHAZ. For BMSCs, the optimal number of RGs was four, while the two-RG combination producing the most similar results was TBP and YWHAZ. Conclusions A common reference gene, TBP, was found to be the most stable standalone gene, while TBP and YWHAZ were found to be the best two-RG combination for qPCR analyses for both BMSCs and ASCs through the VEGF stimulation. The presented stepwise elimination procedure was validated, while we found the final normalization experiment to be essential.

2014-01-01

23

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

24

Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue  

Microsoft Academic Search

Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous\\u000a reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference\\u000a genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase,\\u000a TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate\\u000a dehydrogenase complex, subunit

Antje Koppelkamm; Benedikt Vennemann; Tony Fracasso; Sabine Lutz-Bonengel; Ulrike Schmidt; Marielle Heinrich

2010-01-01

25

Validation of adequate endogenous reference genes for the normalisation of qPCR gene expression data in human post mortem tissue.  

PubMed

Gene expression analyses based on messenger RNA (mRNA) profiling require accurate data normalisation. When using endogenous reference genes, these have to be validated carefully. Therefore, we examined the transcript stability of 10 potential reference genes using quantitative real-time polymerase chain reaction: beta actin, 18S rRNA, glyceraldehyde-3-phosphate dehydrogenase, TATA box-binding protein, hypoxanthine phosphoribosyl-transferase I, beta-2-microglobulin, hydroxymethylbilane synthase, succinate dehydrogenase complex, subunit A, cyclophilin A and ubiquitin C. The aim of the current study was to assess which reference genes show stable mRNA levels in human post mortem cardiac muscle, skeletal muscle and brain tissue. Considering cardiac muscle tissue, CYCA and TBP were identified as the most stable while in skeletal muscle tissue, SDHA and TBP, and in brain tissue, SDHA and HMBS turned out to be the most stable. Furthermore, we recommend a minimum of four carefully validated endogenous control genes for reliable data normalisation in human post mortem tissue. Parameters influencing the stability of transcript amounts were found to be mainly the post mortem interval in cardiac muscle and skeletal muscle tissue and the donor's cause of death in skeletal muscle and brain samples. Further parameters like gender, age at death and body mass index were found to influence mRNA quantities in skeletal muscle only. The set of stable control genes identified in this study may be used in further studies if the composition of the samples is similar to the one used here. PMID:20300940

Koppelkamm, Antje; Vennemann, Benedikt; Fracasso, Tony; Lutz-Bonengel, Sabine; Schmidt, Ulrike; Heinrich, Marielle

2010-09-01

26

Reference gene validation for qPCR on normoxia- and hypoxia-cultured human dermal fibroblasts exposed to UVA: Is ?-actin a reliable normalizer for photoaging studies?  

Microsoft Academic Search

Data normalization of gene expression on human dermal fibroblasts (HDF) exposed to UVA has commonly been done using either GAPDH or ?-actin as reference genes without any validation of their expression stability. Since this aspect, important for accurate normalization, has been overlooked, we aimed to establish a suitable set of reference genes for studies on UVA-treated HDF cultured under both

F. Brugè; E. Venditti; L. Tiano; G. P. Littarru; E. Damiani

2011-01-01

27

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL  

PubMed Central

Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It remains to be clarified whether an autocrine or paracrine mechanism by overexpression of receptor tyrosine kinase ligands is responsible for the tumorigenesis of wt-GIST.

2010-01-01

28

NORMA-Gene: A simple and robust method for qPCR normalization based on target gene data  

PubMed Central

Background Normalization of target gene expression, measured by real-time quantitative PCR (qPCR), is a requirement for reducing experimental bias and thereby improving data quality. The currently used normalization approach is based on using one or more reference genes. Yet, this approach extends the experimental work load and suffers from assumptions that may be difficult to meet and to validate. Results We developed a data driven normalization algorithm (NORMA-Gene). An analysis of the performance of NORMA-Gene compared to reference gene normalization on artificially generated data-sets showed that the NORMA-Gene normalization yielded more precise results under a large range of parameters tested. Furthermore, when tested on three very different real qPCR data-sets NORMA-Gene was shown to be best at reducing variance due to experimental bias in all three data-sets compared to normalization based on the use of reference gene(s). Conclusions Here we present the NORMA-Gene algorithm that is applicable to all biological and biomedical qPCR studies, especially those that are based on a limited number of assayed genes. The method is based on a data-driven normalization and is useful for as little as five target genes comprising the data-set. NORMA-Gene does not require the identification and validation of reference genes allowing researchers to focus their efforts on studying target genes of biological relevance.

2011-01-01

29

NORMA-Gene: A simple and robust method for qPCR normalization based on target gene data  

Microsoft Academic Search

Background  Normalization of target gene expression, measured by real-time quantitative PCR (qPCR), is a requirement for reducing experimental\\u000a bias and thereby improving data quality. The currently used normalization approach is based on using one or more reference\\u000a genes. Yet, this approach extends the experimental work load and suffers from assumptions that may be difficult to meet and\\u000a to validate.\\u000a \\u000a \\u000a \\u000a \\u000a Results  We developed

Lars-Henrik Heckmann; Peter B Sørensen; Paul Henning Krogh; Jesper G Sørensen

2011-01-01

30

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.

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

31

Gene Expression Analysis by qPCR in Clinical Kidney Transplantation.  

PubMed

Patients with a kidney transplant may encounter chronic dysfunction of their graft. Once damage in the graft has established, therapeutic intervention is less efficient. Clinical parameters and morphologic evaluation of biopsies are used for determining diagnosis and prognosis of the patient. Quantitative polymerase chain reaction (qPCR) may be integrated in clinical practice to facilitate routine diagnostics, risk assessment with respect to graft outcome, and determination of the response to therapy by the patient. The success of qPCR assays is highly dependent on the adequacy of the methodological procedures performed. Here, we describe tips and tricks for processing patient material, RNA analysis, and qPCR primer design and gene expression analyses. PMID:24740229

Eikmans, Michael; Anholts, Jacqueline D H; Claas, Frans H J

2014-01-01

32

A Pipeline to Determine RT-QPCR Control Genes for Evolutionary Studies: Application to Primate Gene Expression across Multiple Tissues  

Microsoft Academic Search

Because many species-specific phenotypic differences are assumed to be caused by differential regulation of gene expression, many recent investigations have focused on measuring transcript abundance. Despite the availability of high-throughput platforms, quantitative real-time polymerase chain reaction (RT-QPCR) is often the method of choice because of its low cost and wider dynamic range. However, the accuracy of this technique heavily relies

Olivier Fedrigo; Lisa R. Warner; Adam D. Pfefferle; Courtney C. Babbitt; Peter Cruz-Gordillo; Gregory A. Wray; Jeff Demuth

2010-01-01

33

Reference genes for canine skin when using quantitative real-time PCR  

Microsoft Academic Search

Quantitative real-time PCR (qPCR) facilitates the quantification of mRNA expression. Accurate qPCR analysis of gene expression requires the normalisation of data using a reference or housekeeping gene which is expressed at a similar level in all tissues tested. GAPDH is the most well known and most widely used reference gene but many papers have demonstrated that it is not stably

Shona H. Wood; Dylan N. Clements; Neil A. McEwan; Tim Nuttall; Stuart D. Carter

2008-01-01

34

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

35

The use of reference gene selection programs to study the silvering transformation in a freshwater eel Anguilla australis: a cautionary tale  

Microsoft Academic Search

BACKGROUND: Quantitative real-time PCR (qPCR) has been the method of choice for the quantification of mRNA. Due to the various artifactual factors that may affect the accuracy of qPCR, internal reference genes are most often used to normalize qPCR data. Recently, many studies have employed computer programs such as GeNorm, BestKeeper and NormFinder in selecting reference genes, but very few

Alvin N Setiawan; P Mark Lokman

2010-01-01

36

Reference gene selection for quantitative PCR studies in sheep neutrophils.  

PubMed

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

37

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.

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

2013-01-01

38

A GeNorm algorithm-based selection of reference genes for quantitative real-time PCR in skin biopsies of healthy dogs and dogs with atopic dermatitis  

Microsoft Academic Search

Quantitative real time PCR (Q-PCR) is the method of choice to study mRNA expression levels. Since Q-PCR is very sensitive, normalization of the data with stably expressed reference genes if of utmost importance. The stability of reference genes depends on the tissue and the species of interest. Therefore, evaluation of the stability of reference genes must be performed for each

Yvette M. Schlotter; Eveline Z. Veenhof; Bas Brinkhof; Victor P. M. G. Rutten; Bart Spee; Ton Willemse; Louis C. Penning

2009-01-01

39

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data  

Microsoft Academic Search

BACKGROUND: Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping

Sinara Artico; Sarah M Nardeli; Osmundo Brilhante; Maria Fátima Grossi-de-Sa; Marcio Alves-Ferreira

2010-01-01

40

Selection of stable reference genes for quantitative real-time PCR in porcine gastrointestinal tissues  

Microsoft Academic Search

The selection of stable reference genes is an essential aspect of controlling for the many technical variables that influence the final outcome in quantitative real-time PCR (qPCR). In this study, we evaluated the suitability of 8 commonly used reference genes based on their efficiency and dynamic range in porcine gastrointestinal tissues. The genes included ACTB, B2M, GAPDH, HMBS, HPRT, PPIA,

M. T. Ryan; C. B. Collins; J. V. O'Doherty; T. Sweeney

2010-01-01

41

A multiplex qPCR gene dosage assay for rapid genotyping and large-scale population screening for deletional ?-thalassemia.  

PubMed

The predominant determinants of ?-thalassemia are deletions in the human ?-globin gene cluster. A rapid DNA-based assay is needed for mass screening in thalassemia-prevention programs. Herein, we established a novel quadruplex TaqMan qPCR gene dosage assay with two separate combination reactions. The assay directly determined the copy number of human ?-globin genes based on relative quantitation of three target genes (HBA2, HBA1, and HBZ or HBPA1) versus a control gene (CREBBP). The assay showed good accuracy, with mean intra-assay and interassay variations of 3.31% ± 1.02% and 5.49% ± 0.32%, respectively. The assay was evaluated using 678 pretyped clinical DNA samples containing six ?-thalassemia deletions in 13 genotypes and 186 normal samples previously screened by multiplex ligation-dependent probe amplification or gap PCR. As determined by the 2(-??Cq) method, deleted gene dosage ratios were 0.46 to 0.60 in heterozygotes, 0.0 in homozygotes, and 0.97 to 1.07 in nondeleted samples. We found 99.3% concordance between the quantitative PCR and multiplex ligation-dependent probe amplification or gap-PCR results. Furthermore, routine screening for ?-thalassemia deletions was performed on 3000 random samples in a blind analysis. Results for all 279 positives, which had different deletions, were fully coincident with results from standard methods. We also identified two novel deletions confirmed by multiplex ligation-dependent probe amplification. Assays using the novel method are simple and suitable for rapid genotyping and mass screening. PMID:23810501

Zhou, Wanjun; Wang, Ge; Zhao, Xuefeng; Xiong, Fu; Zhou, Shaoxiong; Peng, Jianming; Cheng, Youming; Xu, Shun; Xu, Xiangmin

2013-09-01

42

A sequence-based approach to identify reference genes for gene expression analysis  

PubMed Central

Background An important consideration when analyzing both microarray and quantitative PCR expression data is the selection of appropriate genes as endogenous controls or reference genes. This step is especially critical when identifying genes differentially expressed between datasets. Moreover, reference genes suitable in one context (e.g. lung cancer) may not be suitable in another (e.g. breast cancer). Currently, the main approach to identify reference genes involves the mining of expression microarray data for highly expressed and relatively constant transcripts across a sample set. A caveat here is the requirement for transcript normalization prior to analysis, and measurements obtained are relative, not absolute. Alternatively, as sequencing-based technologies provide digital quantitative output, absolute quantification ensues, and reference gene identification becomes more accurate. Methods Serial analysis of gene expression (SAGE) profiles of non-malignant and malignant lung samples were compared using a permutation test to identify the most stably expressed genes across all samples. Subsequently, the specificity of the reference genes was evaluated across multiple tissue types, their constancy of expression was assessed using quantitative RT-PCR (qPCR), and their impact on differential expression analysis of microarray data was evaluated. Results We show that (i) conventional references genes such as ACTB and GAPDH are highly variable between cancerous and non-cancerous samples, (ii) reference genes identified for lung cancer do not perform well for other cancer types (breast and brain), (iii) reference genes identified through SAGE show low variability using qPCR in a different cohort of samples, and (iv) normalization of a lung cancer gene expression microarray dataset with or without our reference genes, yields different results for differential gene expression and subsequent analyses. Specifically, key established pathways in lung cancer exhibit higher statistical significance using a dataset normalized with our reference genes relative to normalization without using our reference genes. Conclusions Our analyses found NDUFA1, RPL19, RAB5C, and RPS18 to occupy the top ranking positions among 15 suitable reference genes optimal for normalization of lung tissue expression data. Significantly, the approach used in this study can be applied to data generated using new generation sequencing platforms for the identification of reference genes optimal within diverse contexts.

2010-01-01

43

Identification of condition-specific reference genes from microarray data for locusts exposed to hypobaric hypoxia.  

PubMed

Real-time quantitative polymerase chain reaction (qPCR) is a routine and robust approach for measuring gene expression. The stability of reference genes in qPCR is crucial for the accurate quantification of gene expression. To provide reliable reference genes for studying the transcriptional responses of locust muscles to hypobaric hypoxia, we first examined the gene expression stability of the frequently used housekeeping genes 18S, GAPDH, and ?-actin. However, the expression of these three housekeeping genes was influenced by hypobaric hypoxia. Consequently, we identified five novel candidate reference genes from the locust microarray data. The gene expression stability of the five candidates, together with the three classical housekeeping genes, were evaluated using two distinct algorithms implemented in geNorm and NormFinder. GeNorm identified Ach (acetyl-CoA hydrolase) and Pgp (phosphoglycolate phosphatase-like) as the most stable genes and NormFinder further distinguished Ach as the most stable one. The validity of Ach as a reference gene was confirmed through comparison with 18S. This study exemplifies the necessity of validating reference genes before their application and the feasibility of identifying condition-specific reference genes from large-scale gene expression data. PMID:23650605

Zhao, De-Jian; Guo, Kun; Kang, Le

2012-01-01

44

175. Titering Gene Delivery Vectors by QPCR: Application to rSV40Derived Vectors  

Microsoft Academic Search

Several methods have been used for quantification of SV40 gene transfer vectors (rSV40). Each technique measures a different parameter of recombinant virus structure or function, and each has its own advantages and disadvantages. Titering by A260\\/A280 ratios, as is commonly used for Adenovirus, is unreliable because full-length rSV40 genome is indistinguishable from rSV40 fragments and non-viral DNA. Quantifying non-replicating viruses

Christine B. Mitchell; David S. Strayer

2005-01-01

45

Normalization of gene expression using SYBR green qPCR: a case for paraoxonase 1 and 2 in Alzheimer's disease brains.  

PubMed

Validating the expression stability of reference genes is crucial for reliable normalization of real-time quantitative PCR (qPCR) data, but relatively few studies have investigated this issue in brain human tissues. The present study thus aimed at identifying in human post-mortem brain tissues a set of suitable endogenous reference genes (ERG) for the expression analysis of potential candidate genes associated with Alzheimer's disease (AD). The mRNA levels of ten common ERGs (ACTB, GAPDH, GPS1, GUSB, M-RIP, PGK1, POL2RF, PPIA, UBE2D2, and YES1) were determined in the frontal cortex of autopsy-confirmed AD and non-demented control cases (n=20) using SYBR Green technology. Then, these levels were ranked according to their expression stability using three software applications: geNorm, NormFinder and BestKeeper. Whereas PPIA and UBE2D2 were among the ERGs with the most reliable expression, ACTB was the worst. Subsequently, using PPIA and UBE2D2 as ERGs for normalization, the mRNA levels of paraoxonase 1 (PON1) and paraoxonase 2 (PON2) were quantified in the frontal cortex of AD and control cases (n=80) and analyzed using the REST 2009 program. Our results indicate that both paraoxonases are expressed in the human frontal cortex and that PON2 but not PON1 mRNA levels are up-regulated in AD relative to non-demented controls. However, re-analysis of the results by ANCOVA indicated that the significance of the difference between AD and control groups depended upon the ERG used for normalization. The use of a computational method allowing the inclusion of possible confounding factors is thus recommended for the analysis of data. PMID:21672555

Leduc, Valérie; Legault, Véronique; Dea, Doris; Poirier, Judes

2011-08-30

46

Selection of suitable reference genes for quantitative gene expression studies in milk somatic cells of lactating cows (Bos indicus).  

PubMed

We assessed the suitability of 9 internal control genes (ICG) in milk somatic cells of lactating cows to find suitable reference genes for use in quantitative PCR (qPCR). Eighteen multiparous lactating Sahiwal cows were used, 6 in each of 3 lactation stages: early (25 ± 5 d in milk), mid (160 ± 15 d in milk), and late (275 ± 25 d in milk) lactation. Nine candidate reference genes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH), protein phosphatase 1 regulatory subunit 11 (PPP1R11), ?-actin (ACTB), ?-2 microglobulin (B2M), 40S ribosomal protein S15a (RPS15A), ubiquitously expressed transcript (UXT), mitochondrial GTPase 1 (MTG1), 18S rRNA (RN18S1), and ubiquitin (UBC)] were evaluated. Three genes, ?-casein (CSN2), lactoferrin (LTF), and cathelicidin (CAMP) were chosen as target genes. Very high amplification was observed in 7 ICG and very low level amplification was observed in 2 ICG (UXT and MTG1). Thus, UXT and MTG1 were excluded from further analysis. The qPCR data were analyzed by 2 software packages, geNorm and NormFinder, to determine suitable reference genes, based on their stability and expression. Overall, PPP1R11, ACTB, UBC, and GAPDH were stably expressed among all candidate reference genes. Therefore, these genes could be used as ICG for normalization of qPCR data in milk somatic cells through lactation. PMID:22612931

Varshney, N; Mohanty, A K; Kumar, S; Kaushik, J K; Dang, A K; Mukesh, M; Mishra, B P; Kataria, R; Kimothi, S P; Mukhopadhyay, T K; Malakar, D; Prakash, B S; Grover, S; Batish, V K

2012-06-01

47

Validation of Reference Genes for Expression Studies during Craniofacial Development in Arctic Charr  

PubMed Central

Arctic charr (Salvelinus alpinus) is a highly polymorphic species and in Lake Thingvallavatn, Iceland, four phenotypic morphs have evolved. These differences in morphology, especially in craniofacial structures are already apparent during embryonic development, indicating that genes important in the formation of the craniofacial features are expressed differentially between the morphs. In order to generate tools to examine these expression differences in Arctic charr, the aim of the present study was to identify reference genes for quantitative real-time PCR (qPCR). The specific aim was to select reference genes which are able to detect very small expression differences among different morphs. We selected twelve candidate reference genes from the literature, identified corresponding charr sequences using data derived from transcriptome sequencing (RNA-seq) and examined their expression using qPCR. Many of the candidate reference genes were found to be stably expressed, yet their quality-rank as reference genes varied considerably depending on the type of analysis used. In addition to commonly used software for reference gene validation, we used classical statistics to evaluate expression profiles avoiding a bias for reference genes with similar expression patterns (co-regulation). Based on these analyses we chose three reference genes, ACTB, UB2L3 and IF5A1 for further evaluation. Their consistency was assessed in an expression study of three known craniofacially expressed genes, sparc (or osteonectin), matrix metalloprotease 2 (mmp2) and sox9 (sex-determining region Y box 9 protein) using qPCR in embryo heads derived from four charr groups at three developmental time points. The three reference genes were found to be very suitable for studying expression differences between the morphotypes, enabling robust detection of small relative expression changes during charr development. Further, the results showed that sparc and mmp2 are differentially expressed in embryos of different Arctic charr morphotypes.

Ahi, Ehsan Pashay; Gu?brandsson, Johannes; Kapralova, Kalina H.; Franzdottir, Sigri?ur R.; Snorrason, Sigur?ur S.; Maier, Valerie H.; Jonsson, Zophonias O.

2013-01-01

48

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

49

Selection of reliable reference genes for RT-qPCR analysis.  

PubMed

Reference genes have become the method of choice for normalization of qPCR data. It has been demonstrated in many studies that reference gene validation is essential to ensure accurate and reliable results. This chapter describes how a pilot study can be set up to identify the best set of reference genes to be used for normalization of qPCR data. The data from such a pilot study should be analyzed with dedicated algorithms such as geNorm to rank genes according to their stability--a measure for how well they are suited for normalization. geNorm also provides insights into the optimal number of reference genes and the overall quality of the selected set of reference genes. Importantly, these results are always in function of the sample type being studied. Guidelines are provided on the interpretation of the results from geNorm pilot studies as well as for the continued monitoring of reference gene quality in subsequent studies. For screening studies including a large, unbiased set of genes (e.g., complete miRNome) an alternative normalization method can be used: global mean normalization. This chapter also describes how the data from such studies can be used to identify reference genes for subsequent validation studies on smaller sets of selected genes. PMID:24740218

Hellemans, Jan; Vandesompele, Jo

2014-01-01

50

Defining reference genes for quantitative real-time PCR analysis of anther development in rice.  

PubMed

Quantitative real-time polymerase chain reaction (qPCR) is one of the most accurate and widely used methods for gene expression analysis. However, the choice of reference genes for normalization is critical for accurate quantification of gene expression. As development of genomics, mining large-scale datasets such as microarray and RNA-sequencing data becomes a new approach for exploitation of new reference genes. In this study, we analyzed an RNA-sequencing dataset of rice anther and 167 microarray datasets involving different tissues and developing stages of rice anthers and pollens. We selected 12 candidate genes and other 5 reference genes, including ACT1, eEF-1?, GAPDH, Exp2, and CCDC72 used in previous studies, and evaluated their expression in eight tissues and different developmental stages of anthers in rice variety 9311 and Yuetai. UPF3, eIF4A-3, GAPDH, and PPP6 were identified as the most suitable reference genes for qPCR analysis of anther development in rice. The new candidate reference genes showed more stable expression than the traditionally used reference genes. These results provide a set of reliable reference genes for studies in rice anther developmental process. PMID:24492537

Ji, Yanxiao; Tu, Ping; Wang, Kun; Gao, Feng; Yang, Weilong; Zhu, Yingguo; Li, Shaoqing

2014-04-01

51

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

52

Selection of reference genes in Hedysarum coronarium under various stresses and stages of development.  

PubMed

The cultivation of Hedysarum coronarium has generated interest recently for its high yield as a fodder crop, its high protein content, and the presence of condensed tannins in its leaf and stem tissues. Gene expression studies can lead to a better understanding of the biological processes of live organisms. Specifically, reverse transcription followed by quantitative polymerase chain reaction (PCR) represents the most powerful technology for comparing the expression profiles of target genes. The use of reference genes as internal controls to normalize messenger RNA (mRNA) levels is a requirement of quantitative PCR (qPCR). Few studies on reference genes have been performed in plants, and no studies have been performed in H. coronarium. Therefore, the aim of this study was to identify and evaluate reference genes to use in qPCR in H. coronarium. Sulla tissues under two conditions of abiotic stress and at various stages of development were studied to determine adequate reference genes. To optimize the identity and number of reference genes, geNorm and BestKeeper software programs were employed. Based on the results of both analyses, TUA1, TUA2, and UBQ were found to be the most suitable reference genes, and the combination of these three genes was suggested for the accurate normalization of gene expression in sulla tissues. PMID:21036135

Cordoba, E M; Die, J V; González-Verdejo, C I; Nadal, S; Román, B

2011-02-15

53

Selection of reference genes for mRNA quantification in Trypanosoma brucei  

Microsoft Academic Search

Internal normalization is an established procedure that is necessary for accurate and reliable quantification of differentially regulated mRNAs. The profound changes of gene expression in parasitic life cycles pose a particular challenge on selection of appropriate reference genes for normalization, most importantly when using quantitative real time PCR (qPCR). Here we use the ranking algorithm implemented in the geNorm application

Martin Brenndörfer; Michael Boshart

2010-01-01

54

Importance of suitable reference gene selection for quantitative RT-PCR during ATDC5 cells chondrocyte differentiation.  

PubMed

Real-time quantitative reverse transcription-polymerase chain reaction (qPCR) is an efficient and accurate method to detect and compare patterns of gene expression. The reliability of qPCR is highly dependent on the selection of appropriate reference genes used for normalization. By analyzing 16 potential candidates of reference genes (GAPDH, Actb, 18 s, PGK1, Hprt, Tbp, Rpl5, B2M, Gusb, Ppia, UBC, Sdha, Eef1a1, H2afz, Tkt and Ldha) through geNorm, we identified Ppia, Tbp, Hprt and Eef1a1 as the most stable reference genes while UBC, B2M, Gusb as the least stable ones during the chondrocyte differentiation of ATDC5 cells. Considering the low expression of Eef1a1 and Tbp would cause divergent results for they failed to provide accurate normalization for RNA extraction and reverse transcription efficiency, we recommended the use of Ppia and Hprt as the most suitable genes to normalize qPCR. In addition, although GAPDH, Actb and 18 s were usually adopted in most of studies using ATDC5 cells, they were found unstable and then were not ideal reference genes for qPCR assay in ATDC5 cells chondrocyte differentiation. Also, we further confirmed that the Ppia and Hprt worked well during chondrocyte differentiation of mouse mesenchymal cells. PMID:23705012

Zhai, Zhichen; Yao, Yongchang; Wang, Yingjun

2013-01-01

55

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

PubMed Central

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. coli. However, the choice of reliable reference genes has not been systematically validated. The objective of this study is to identify a set of reliable reference genes for transcription analysis in recombinant protein over-expression studies in E. coli. Results In this study, the meta-analysis of 240 sets of single-channel Affymetrix microarray data representing over-expressions of 63 distinct recombinant proteins in various E. coli strains identified twenty candidate reference genes that were stably expressed across all conditions. The expression of these twenty genes and two commonly used reference genes, rrsA encoding ribosomal RNA 16S and ihfB, was quantified by qPCR in E. coli cells over-expressing four genes of the 1-Deoxy-D-Xylulose 5-Phosphate pathway. From these results, two independent statistical algorithms identified three novel reference genes cysG, hcaT, and idnT but not rrsA and ihfB as highly invariant in two E. coli strains, across different growth temperatures and induction conditions. Transcriptomic data normalized by the geometric average of these three genes demonstrated that genes of the lycopene synthetic pathway maintained steady expression upon enzyme overexpression. In contrast, the use of rrsA or ihfB as reference genes led to the mis-interpretation that lycopene pathway genes were regulated during enzyme over-expression. Conclusion This study identified cysG/hcaT/idnT to be reliable novel reference genes for transcription analysis in recombinant protein producing E. coli.

2011-01-01

56

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.

Ahlgren, Nathan A.; Rocap, Gabrielle

2012-01-01

57

Reference gene for primary culture of prostate cancer cells.  

PubMed

Selection of reference genes to normalize mRNA levels between samples is critical for gene expression studies because their expression can vary depending on the tissues or cells used and the experimental conditions. We performed ten cell cultures from samples of prostate cancer. Cells were divided into three groups: control (with no transfection protocol), cells transfected with siRNA specific to knockdown the androgen receptor and cells transfected with inespecific siRNAs. After 24 h, mRNA was extracted and gene expression was analyzed by Real-time qPCR. Nine candidates to reference genes for gene expression studies in this model were analyzed (aminolevulinate, delta-, synthase 1 (ALAS1); beta-actin (ACTB); beta-2-microglobulin (B2M); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine phosphoribosyltransferase 1 (HPRT1); succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA); TATA box binding protein (TBP); ubiquitin C (UBC); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ)). Expression stability was calculated NormFinder algorithm to find the most stable genes. NormFinder calculated SDHA as the most stable gene and the gene with the lowest intergroup and intragroup variation, and indicated GAPDH and SDHA as the best combination of two genes for the purpose of normalization. Androgen receptor mRNA expression was evaluated after normalization by each candidate gene and showed statistical difference in the transfected group compared to control group only when normalized by combination of GAPDH and SDHA. Based on the algorithm analysis, the combination of SDHA and GAPDH should be used to normalize target genes mRNA levels in primary culture of prostate cancer cells submitted to transfection with siRNAs. PMID:23269617

Souza, Aline Francielle Damo; Brum, Ilma Simoni; Neto, Brasil Silva; Berger, Milton; Branchini, Gisele

2013-04-01

58

The choice of reference gene affects statistical efficiency in quantitative PCR data analysis  

PubMed Central

Quantitative polymerase chain reaction (qPCR), a highly sensitive method of measuring gene expression, is widely used in biomedical research. To produce reliable results, it is essential to use stably expressed reference genes (RGs) for data normalization so that sample-to-sample variation can be controlled. In this study, we examine the effect of different RGs on statistical efficiency by analyzing a qPCR data set that contains 12 target genes and 3 RGs. Our results show that choosing the most stably expressed RG for data normalization does not guarantee reduced variance or improved statistical efficiency. We also provide a formula for determining when data normalization will improve statistical efficiency and hence increase the power of statistical tests in data analysis.

Guo, Yi; Pennell, Michael L.; Pearl, Dennis K.; Knobloch, Thomas J.; Fernandez, Soledad; Weghorst, Christopher M.

2014-01-01

59

An examination of MS candidate genes identified as differentially regulated in multiple sclerosis plaque tissue, using absolute and comparative real-time Q-PCR analysis.  

PubMed

In our laboratory, we have developed methods in real-time detection and quantitative-polymerase chain reaction (Q-PCR) to analyse the relative levels of gene expression in post mortem brain tissues. We have then applied this method to examine differences in gene activity between normal white matter (NWM) and plaque tissue from multiple sclerosis (MS) patients. Genes were selected based on their association with pathology and through identification by previously conducted global gene expression analysis. Plaque tissue was obtained from secondary progressive (SP) patients displaying chronic active, as well as acute pathologies; while NWM from the same location was obtained from age- and sex-matched controls (normal patients). In this study, we used both SYBR Green I supplementation and commercially available mixes to assess both comparative and absolute levels of gene activity. The results of both methods compared favourably for four of the five genes examined (P < 0.05, Pearsons), while differences in gene expression between chronic active and acute pathologies were also identified. For example, a >50-fold increase in osteopontin (Spp1) and inositol 1-4-5 phosphate 3 kinase B (Itpkb) levels in acute plaques contrasted with the 5-fold or less increase in chronic active plaques (P < 0.05, unpaired t test). By contrast, there was no significant difference in the levels of the MS marker and calcium-dependent protease (Calpain, Capns1) in MS plaque tissue. In summary, Q-PCR analysis using SYBR Green I has allowed us to economically obtain what may be clinically significant information from small amounts of the CNS, providing an opportunity for further clinical investigations. PMID:15905117

Tajouri, L; Mellick, A S; Tourtellotte, A; Nagra, R M; Griffiths, L R

2005-07-01

60

Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR.  

PubMed

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant-pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1?, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions. PMID:23029521

Liu, Deshui; Shi, Lindan; Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2012-01-01

61

Validation of Reference Genes for Gene Expression Studies in Virus-Infected Nicotiana benthamiana Using Quantitative Real-Time PCR  

PubMed Central

Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR (qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16 commonly used housekeeping genes (GAPDH, 18S, EF1?, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm, NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall, and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A, F-BOX and L23 as reference genes was illustrated by expression-level analysis of AGO2 and RdR6 in virus-infected N. benthamiana leaves. This is the first study to systematically examine and evaluate the stability of different reference genes in N. benthamiana. Our results not only provide researchers studying these viruses a shortlist of potential housekeeping genes to use as normalisers for qPCR experiments, but should also guide the selection of appropriate reference genes for gene expression studies of N. benthamiana under other biotic and abiotic stress conditions.

Han, Chenggui; Yu, Jialin; Li, Dawei; Zhang, Yongliang

2012-01-01

62

Reference Genes for Quantitative Gene Expression Studies in Multiple Avian Species  

PubMed Central

Quantitative real-time PCR (qPCR) rapidly and reliably quantifies gene expression levels across different experimental conditions. Selection of suitable reference genes is essential for meaningful normalization and thus correct interpretation of data. In recent years, an increasing number of avian species other than the chicken has been investigated molecularly, highlighting the need for an experimentally validated pan-avian primer set for reference genes. Here we report testing a set for 14 candidate reference genes (18S, ABL, GAPDH, GUSB, HMBS, HPRT, PGK1, RPL13, RPL19, RPS7, SDHA, TFRC, VIM, YWHAZ) on different tissues of the mallard (Anas platyrhynchos), domestic chicken (Gallus gallus domesticus), common crane (Grus grus), white-tailed eagle (Haliaeetus albicilla), domestic turkey (Meleagris gallopavo f. domestica), cockatiel (Nymphicus hollandicus), Humboldt penguin (Sphenicus humboldti), ostrich (Struthio camelus) and zebra finch (Taeniopygia guttata), spanning a broad range of the phylogenetic tree of birds. Primer pairs for six to 11 genes were successfully established for each of the nine species. As a proof of principle, we analyzed expression levels of 10 candidate reference genes as well as FOXP2 and the immediate early genes, EGR1 and CFOS, known to be rapidly induced by singing in the avian basal ganglia. We extracted RNA from microbiopsies of the striatal song nucleus Area X of adult male zebra finches after they had sang or remained silent. Using three different statistical algorithms, we identified five genes (18S, PGK1, RPS7, TFRC, YWHAZ) that were stably expressed within each group and also between the singing and silent conditions, establishing them as suitable reference genes. In conclusion, the newly developed pan-avian primer set allows accurate normalization and quantification of gene expression levels in multiple avian species.

Meyer, Anne; Scharff, Constance; Gruber, Achim D.

2014-01-01

63

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

64

Selection of Suitable Endogenous Reference Genes for Relative Copy Number Detection in Sugarcane  

PubMed Central

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential “single copy” genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3—high copy number group, TST-1 and PRR-1—medium copy number group, P4H-1, APRT-2 and CYC-2—low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane.

Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

2014-01-01

65

Selection of suitable endogenous reference genes for relative copy number detection in sugarcane.  

PubMed

Transgene copy number has a great impact on the expression level and stability of exogenous gene in transgenic plants. Proper selection of endogenous reference genes is necessary for detection of genetic components in genetically modification (GM) crops by quantitative real-time PCR (qPCR) or by qualitative PCR approach, especially in sugarcane with polyploid and aneuploid genomic structure. qPCR technique has been widely accepted as an accurate, time-saving method on determination of copy numbers in transgenic plants and on detection of genetically modified plants to meet the regulatory and legislative requirement. In this study, to find a suitable endogenous reference gene and its real-time PCR assay for sugarcane (Saccharum spp. hybrids) DNA content quantification, we evaluated a set of potential "single copy" genes including P4H, APRT, ENOL, CYC, TST and PRR, through qualitative PCR and absolute quantitative PCR. Based on copy number comparisons among different sugarcane genotypes, including five S. officinarum, one S. spontaneum and two S. spp. hybrids, these endogenous genes fell into three groups: ENOL-3-high copy number group, TST-1 and PRR-1-medium copy number group, P4H-1, APRT-2 and CYC-2-low copy number group. Among these tested genes, P4H, APRT and CYC were the most stable, while ENOL and TST were the least stable across different sugarcane genotypes. Therefore, three primer pairs of P4H-3, APRT-2 and CYC-2 were then selected as the suitable reference gene primer pairs for sugarcane. The test of multi-target reference genes revealed that the APRT gene was a specific amplicon, suggesting this gene is the most suitable to be used as an endogenous reference target for sugarcane DNA content quantification. These results should be helpful for establishing accurate and reliable qualitative and quantitative PCR analysis of GM sugarcane. PMID:24857916

Xue, Bantong; Guo, Jinlong; Que, Youxiong; Fu, Zhiwei; Wu, Luguang; Xu, Liping

2014-01-01

66

Identification of superior reference genes for data normalisation of expression studies via quantitative PCR in hybrid roses (Rosa hybrida)  

PubMed Central

Background Gene expression studies are a prerequisite for understanding the biological function of genes. Because of its high sensitivity and easy use, quantitative PCR (qPCR) has become the gold standard for gene expression quantification. To normalise qPCR measurements between samples, the most prominent technique is the use of stably expressed endogenous control genes, the so called reference genes. However, recent studies show there is no universal reference gene for all biological questions. Roses are important ornamental plants for which there has been no evaluation of useful reference genes for gene expression studies. Results We used three different algorithms (BestKeeper, geNorm and NormFinder) to validate the expression stability of nine candidate reference genes in different rose tissues from three different genotypes of Rosa hybrida and in leaves treated with various stress factors. The candidate genes comprised the classical "housekeeping genes" (Actin, EF-1?, GAPDH, Tubulin and Ubiquitin), and genes showing stable expression in studies in Arabidopsis (PP2A, SAND, TIP and UBC). The programs identified no single gene that showed stable expression under all of the conditions tested, and the individual rankings of the genes differed between the algorithms. Nevertheless the new candidate genes, specifically, PP2A and UBC, were ranked higher as compared to the other traditional reference genes. In general, Tubulin showed the most variable expression and should be avoided as a reference gene. Conclusions Reference genes evaluated as suitable in experiments with Arabidopsis thaliana were stably expressed in roses under various experimental conditions. In most cases, these genes outperformed conventional reference genes, such as EF1-? and Tubulin. We identified PP2A, SAND and UBC as suitable reference genes, which in different combinations may be used for normalisation in expression analyses via qPCR for different rose tissues and stress treatments. However, the vast genetic variation found within the genus Rosa, including differences in ploidy levels, might also influence expression stability of reference genes, so that future research should also consider different genotypes and ploidy levels.

2011-01-01

67

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.

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

2013-01-01

68

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

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

2009-01-01

69

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.

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

2014-01-01

70

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.

2013-01-01

71

Evaluation of coffee reference genes for relative expression studies by quantitative real-time RT-PCR  

Microsoft Academic Search

Accuracy in quantitative real-time polymerase chain reaction (qPCR) requires the use of stable endogenous controls. Normalization\\u000a with multiple reference genes is the gold standard, but their identification is a laborious task, especially in species with\\u000a limited sequence information. Coffee (Coffea ssp.) is an important agricultural commodity and, due to its economic relevance, is the subject of increasing research in\\u000a genetics

Fernanda Cruz; Samara Kalaoun; Paula Nobile; Carlos Colombo; Juliana Almeida; Leila M. G. Barros; Eduardo Romano; Maria Fátima Grossi-de-Sá; Maité Vaslin; Marcio Alves-Ferreira

2009-01-01

72

Validation of reference genes for quantitative expression analysis by real-time rt-PCR in four lepidopteran insects.  

PubMed

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

73

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.

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

2012-01-01

74

Technical note: Validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli.  

PubMed

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a powerful tool to assess responses of MEC to pathogens. Although internal standards such as reference genes are required for qPCR to yield valid data, the validation of proper genes to quantify mRNA transcripts in MEC exposed to pro-inflammatory stimuli has never been reported. In this study, 10 commonly used reference genes belonging to different functional classes (ACTB, ATP5B, EIF2B2, GAPDH, PPIA, SDHA, SUZ12, UXT, YWHAZ, and 18s rRNA) were analyzed by qPCR to determine the most stable in bovine MEC unstimulated and stimulated with mastitis pathogens (Staphylococcus aureus or Escherichia coli), microbial agonists of the innate immune system (lipoteichoic acid and muramyl dipeptide, or lipopolysaccharide), or proinflammatory cytokines (IL-17A and tumor necrosis factor-?). An M value was used as a measure of gene stability as determined using the geNorm application. This study demonstrated that the expression of the 10 reference genes was stable under the different experimental conditions. These data will be useful for bovine mastitis research in selecting reference genes and validating reverse transcription-qPCR data. PMID:21524534

Bougarn, S; Cunha, P; Gilbert, F B; Meurens, F; Rainard, P

2011-05-01

75

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.

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

2014-01-01

76

Reference Gene Screening for Analyzing Gene Expression Across Goat Tissue  

PubMed Central

Real-time quantitative PCR (qRT-PCR) is one of the important methods for investigating the changes in mRNA expression levels in cells and tissues. Selection of the proper reference genes is very important when calibrating the results of real-time quantitative PCR. Studies on the selection of reference genes in goat tissues are limited, despite the economic importance of their meat and dairy products. We used real-time quantitative PCR to detect the expression levels of eight reference gene candidates (18S, TBP, HMBS, YWHAZ, ACTB, HPRT1, GAPDH and EEF1A2) in ten tissues types sourced from Boer goats. The optimal reference gene combination was selected according to the results determined by geNorm, NormFinder and Bestkeeper software packages. The analyses showed that tissue is an important variability factor in genes expression stability. When all tissues were considered, 18S, TBP and HMBS is the optimal reference combination for calibrating quantitative PCR analysis of gene expression from goat tissues. Dividing data set by tissues, ACTB was the most stable in stomach, small intestine and ovary, 18S in heart and spleen, HMBS in uterus and lung, TBP in liver, HPRT1 in kidney and GAPDH in muscle. Overall, this study provided valuable information about the goat reference genes that can be used in order to perform a proper normalisation when relative quantification by qRT-PCR studies is undertaken.

Zhang, Yu; Zhang, Xiao-Dong; Liu, Xing; Li, Yun-Sheng; Ding, Jian-Ping; Zhang, Xiao-Rong; Zhang, Yun-Hai

2013-01-01

77

Reference gene selection for quantitative real-time PCR normalization in Caragana intermedia under different abiotic stress conditions.  

PubMed

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

78

The use of laser microdissection in the identification of suitable reference genes for normalization of quantitative real-time PCR in human FFPE epithelial ovarian tissue samples.  

PubMed

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene. PMID:24776823

Cai, Jing; Li, Tao; Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

2014-01-01

79

The Use of Laser Microdissection in the Identification of Suitable Reference Genes for Normalization of Quantitative Real-Time PCR in Human FFPE Epithelial Ovarian Tissue Samples  

PubMed Central

Quantitative real-time PCR (qPCR) is a powerful and reproducible method of gene expression analysis in which expression levels are quantified by normalization against reference genes. Therefore, to investigate the potential biomarkers and therapeutic targets for epithelial ovarian cancer by qPCR, it is critical to identify stable reference genes. In this study, twelve housekeeping genes (ACTB, GAPDH, 18S rRNA, GUSB, PPIA, PBGD, PUM1, TBP, HRPT1, RPLP0, RPL13A, and B2M) were analyzed in 50 ovarian samples from normal, benign, borderline, and malignant tissues. For reliable results, laser microdissection (LMD), an effective technique used to prepare homogeneous starting material, was utilized to precisely excise target tissues or cells. One-way analysis of variance (ANOVA) and nonparametric (Kruskal-Wallis) tests were used to compare the expression differences. NormFinder and geNorm software were employed to further validate the suitability and stability of the candidate genes. Results showed that epithelial cells occupied a small percentage of the normal ovary indeed. The expression of ACTB, PPIA, RPL13A, RPLP0, and TBP were stable independent of the disease progression. In addition, NormFinder and geNorm identified the most stable combination (ACTB, PPIA, RPLP0, and TBP) and the relatively unstable reference gene GAPDH from the twelve commonly used housekeeping genes. Our results highlight the use of homogeneous ovarian tissues and multiple-reference normalization strategy, e.g. the combination of ACTB, PPIA, RPLP0, and TBP, for qPCR in epithelial ovarian tissues, whereas GAPDH, the most commonly used reference gene, is not recommended, especially as a single reference gene.

Huang, Bangxing; Cheng, Henghui; Ding, Hui; Dong, Weihong; Xiao, Man; Liu, Ling; Wang, Zehua

2014-01-01

80

Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data  

PubMed Central

Background Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes. Results By the use of two distinct algorithms, implemented by geNorm and NormFinder, we have assessed the gene expression of nine candidate reference genes in cotton: GhACT4, GhEF1?5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, Gh?TUB3 and GhUBQ14. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of GhPP2A1 and GhUBQ14 genes were the most stable across all samples and also when distinct plants organs are examined. GhACT4 and GhUBQ14 present more stable expression during flower development, GhACT4 and GhFBX6 in the floral verticils and GhMZA and GhPTB during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development. Conclusion We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of GhUBQ14 and GhPP2A1 housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; GhACT4 and GhUBQ14 for flower development, GhACT4 and GhFBX6 for the floral organs and GhMZA and GhPTB for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.

2010-01-01

81

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

82

Validation of reference genes from Eucalyptus spp. under different stress conditions  

PubMed Central

Background The genus Eucalyptus consists of approximately 600 species and subspecies and has a physiological plasticity that allows some species to propagate in different regions of the world. Eucalyptus is a major source of cellulose for paper manufacturing, and its cultivation is limited by weather conditions, particularly water stress and low temperatures. Gene expression studies using quantitative reverse transcription polymerase chain reaction (qPCR) require reference genes, which must have stable expression to facilitate the comparison of the results from analyses using different species, tissues, and treatments. Such studies have been limited in eucalyptus. Results Eucalyptus globulus Labill, Eucalyptus urograndis (hybrid from Eucalyptus urophylla S.T. Blake X Eucalyptus grandis Hill ex-Maiden) and E. uroglobulus (hybrid from E. urograndis X E. globulus) were subjected to different treatments, including water deficiency and stress recovery, low temperatures, presence or absence of light, and their respective controls. Except for treatment with light, which examined the seedling hypocotyl or apical portion of the stem, the expression analyses were conducted in the apical and basal parts of the stem. To select the best pair of genes, the bioinformatics tools GeNorm and NormFinder were compared. Comprehensive analyses that did not differentiate between species, treatments, or tissue types, showed that IDH (isocitrate dehydrogenase), SAND (SAND protein), ACT (actin), and A-Tub (?-tubulin) genes were the most stable. IDH was the most stable gene in all of the treatments. Conclusion Comparing these results with those of other studies on eucalyptus, we concluded that five genes are stable in different species and experimental conditions: IDH, SAND, ACT, A-Tub, and UBQ (ubiquitin). It is usually recommended a minimum of two reference genes is expression analysis; therefore, we propose that IDH and two others genes among the five identified genes in this study should be used as reference genes for a wide range of conditions in eucalyptus.

2012-01-01

83

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

84

Reference Genes for Real-Time PCR Quantification of MicroRNAs and Messenger RNAs in Rat Models of Hepatotoxicity  

PubMed Central

Hepatotoxicity is associated with major changes in liver gene expression induced by xenobiotic exposure. Understanding the underlying mechanisms is critical for its clinical diagnosis and treatment. MicroRNAs are key regulators of gene expression that control mRNA stability and translation, during normal development and pathology. The canonical technique to measure gene transcript levels is Real-Time qPCR, which has been successfully modified to determine the levels of microRNAs as well. However, in order to obtain accurate data in a multi-step method like RT-qPCR, the normalization with endogenous, stably expressed reference genes is mandatory. Since the expression stability of candidate reference genes varies greatly depending on experimental factors, the aim of our study was to identify a combination of genes for optimal normalization of microRNA and mRNA qPCR expression data in experimental models of acute hepatotoxicity. Rats were treated with four traditional hepatotoxins: acetaminophen, carbon tetrachloride, D-galactosamine and thioacetamide, and the liver expression levels of two groups of candidate reference genes, one for microRNA and the other for mRNA normalization, were determined by RT-qPCR in compliance with the MIQE guidelines. In the present study, we report that traditional reference genes such as U6 spliceosomal RNA, Beta Actin and Glyceraldehyde-3P-dehydrogenase altered their expression in response to classic hepatotoxins and therefore cannot be used as reference genes in hepatotoxicity studies. Stability rankings of candidate reference genes, considering only those that did not alter their expression, were determined using geNorm, NormFinder and BestKeeper software packages. The potential candidates whose measurements were stable were further tested in different combinations to find the optimal set of reference genes that accurately determine mRNA and miRNA levels. Finally, the combination of MicroRNA-16/5S Ribosomal RNA and Beta 2 Microglobulin/18S Ribosomal RNA were validated as optimal reference genes for microRNA and mRNA quantification, respectively, in rat models of acute hepatotoxicity.

Lardizabal, Maria N.; Nocito, Ana L.; Daniele, Stella M.; Ornella, Leonardo A.; Palatnik, Javier F.; Veggi, Luis M.

2012-01-01

85

Putative reference genes for gene expression studies in propionate and ?-hydroxybutyrate treated bovine adipose tissue explants.  

PubMed

Accurate gene expression normalization using a stable reference gene (RG) improves the reliability of quantitative real-time PCR (qPCR) results. Therefore, a validation of RGs should be done before their use. Only few studies on RGs have been done in cattle, and none in bovine adipose tissue (AT) explants, therefore, we characterize the effects of an in vitro treatment with propionate and ?-hydroxybutyric acid (BHB) on the mRNA content of these RGs comparing the output data from the geNorm™ and the Normfinder(©) program. geNorm™ and Normfinder(©) estimated the most stable RGs in the following sequence for subcutaneous and for retroperitoneal AT explants treated with propionate: low density lipoprotein receptor-related protein 10 (LRP10) > hippocalcin-like 1 (HPCAL1) > glyceraldehyde-phosphate-dehydrogenase (GAPDH) > ribosomal protein S9 (RPS9) > RNA polymerase II (Pol II) > beta2 actin (ACTB) > 18S ribosomal RNA (18S rRNA). BHB treated AT explants yielded a different stability ranking for RGs using geNorm™: HPCAL1, GAPDH > Pol II > LRP10 > ACTB > RPS9 > 18S rRNA. Normfinder(©) estimated a different stability ranking for the RGs as shown in the following sequence for subcutaneous and retroperitoneal AT explants treated with BHB: HPCAL1 > Pol II > GAPDH > ACTB > LRP10 > RPS9 > 18S rRNA. Subsequent pairwise analysis of variation of RGs using geNorm™ suggested that LRP10, HPCAL1 and GAPDH should be used for accurate normalization of subcutaneous and retroperitoneal AT explants treated with propionate, while HPCAL1, GAPDH and Pol II should be used for BHB treatment. PMID:20579188

Hosseini, A; Sauerwein, H; Mielenz, M

2010-10-01

86

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.

2011-01-01

87

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.

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

88

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.

2014-01-01

89

Reference gene selection for normalization of PCR analysis in chicken embryo fibroblast infected with H5N1 AIV.  

PubMed

Chicken embryo fibroblasts (CEFs) are among the most commonly used cells for the study of interactions between chicken hosts and H5N1 avian influenza virus (AIV). In this study, the expression of eleven housekeeping genes typically used for the normalization of quantitative real-time PCR (QPCR) analysis in mammals were compared in CEFs infected with H5N1 AIV to determine the most reliable reference genes in this system. CEFs cultured from 10-day-old SPF chicken embryos were infected with 100 TCID(50) of H5N1 AIV and harvested at 3, 12, 24 and 30 hours post-infection. The expression levels of the eleven reference genes in infected and uninfected CEFs were determined by real-time PCR. Based on expression stability and expression levels, our data suggest that the ribosomal protein L4 (RPL4) and tyrosine 3-monooxygenase tryptophan 5-monooxygenase activation protein zeta polypeptide (YWHAZ) are the best reference genes to use in the study of host cell response to H5N1 AIV infection. However, for the study of replication levels of H5N1 AIV in CEFs, the ?-actin gene (ACTB) and the ribosomal protein L4 (RPL4) gene are the best references. PMID:21221921

Yue, Hua; Lei, Xiao-wen; Yang, Fa-long; Li, Ming-yi; Tang, Cheng

2010-12-01

90

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

91

Reference genes for normalising gene expression data in collagenase-induced rat intracerebral haemorrhage  

Microsoft Academic Search

BACKGROUND: The mechanisms of brain injury following intracerebral haemorrhage (ICH) are incompletely understood. Gene expression studies using quantitative real-time RT-PCR following ICH have increased our understanding of these mechanisms, however the inconsistent results observed may be related to inappropriate reference gene selection. Reference genes should be stably expressed across different experimental conditions, however, transcript levels of common reference genes have

Naomi L Cook; Timothy J Kleinig; Corinna van den Heuvel; Robert Vink

2010-01-01

92

Reference Gene Validation Software for Improved Normalization  

Microsoft Academic Search

Real-time PCR is the method of choice for expression analysis of a limited number of genes. The measured gene expression variation between subjects is the sum of the true biological variation and several confounding factors resulting in non- specific variation. The purpose of normalization is to remove the non-biological variation as much as possible. Several normalization strategies have been proposed,

J. Vandesompele; M. Kubista; M. W. Pfaffl

93

Gene expression studies in prostate cancer tissue: which reference gene should be selected for normalization?  

Microsoft Academic Search

Using quantitative reverse transcription–polymerase chain reaction (RT-PCR), reference genes are utilized as endogenous controls for relative quantification of target genes in gene profiling studies. The suitability of housekeeping genes for that purpose in prostate cancer tissue has not been sufficiently investigated so far. The objective of this study was to select from a panel of 16 potential candidate reference genes

Falk Ohl; Monika Jung; Chuanliang Xu; Carsten Stephan; Anja Rabien; Mick Burkhardt; Andreas Nitsche; Glen Kristiansen; Stefan A. Loening; Aleksandar Radoni?; Klaus Jung

2005-01-01

94

Expression of target and reference genes in Daphnia magna exposed to ibuprofen  

Microsoft Academic Search

BACKGROUND: Transcriptomic techniques are now being applied in ecotoxicology and toxicology to measure the impact of stressors and develop understanding of mechanisms of toxicity. Microarray technology in particular offers the potential to measure thousands of gene responses simultaneously. However, it is important that microarrays responses should be validated, at least initially, using real-time quantitative polymerase chain reaction (QPCR). The accurate

Lars-Henrik Heckmann; Richard Connon; Thomas H Hutchinson; Steve J Maund; Richard M Sibly; Amanda Callaghan

2006-01-01

95

Careful selection of reference genes is required for reliable performance of RT-qPCR in human normal and cancer cell lines.  

PubMed

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, André; Hacker, Neville F; Heinzelmann-Schwarz, Viola

2013-01-01

96

Evaluating reference genes to normalize gene expression in human epileptogenic brain tissues  

Microsoft Academic Search

Several reference genes have been used to quantify gene expression in human epilepsy surgery tissue. However, their reliability has not been validated in detail, although this is crucial in interpreting epilepsy-related changes of gene expression. We evaluated 12 potential reference genes in neocortical tissues resected from patients with temporal lobe epilepsy (TLE) with either few or many seizures (n=6 each)

Stephan Wierschke; Sylvain Gigout; Peter Horn; Thomas-Nicolas Lehmann; Christoph Dehnicke; Anja U. Bräuer; Rudolf A. Deisz

2010-01-01

97

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood  

Microsoft Academic Search

BACKGROUND: Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. METHODS: Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples

Boryana S Stamova; Michelle Apperson; Wynn L Walker; Yingfang Tian; Huichun Xu; Peter Adamczy; Xinhua Zhan; Da-Zhi Liu; Bradley P Ander; Isaac H Liao; Jeffrey P Gregg; Renee J Turner; Glen Jickling; Lisa Lit; Frank R Sharp

2009-01-01

98

Assessment of reference gene stability influenced by extremely divergent disease symptoms in Solanum lycopersicum L.  

PubMed

Tomato (Solanum lycopersicum L.) is one of the most important vegetables of great worldwide economic value. The scientific importance of the vegetable results from the fact that the genome of S. lycopersicum has been sequenced. This allows researchers to study fundamental mechanisms playing an essential role during tomato development and response to environmental factors contributing significantly to cell metabolism alterations. Parallel with the development of contemporary genetics and the constant increase in sequencing data, progress has to be aligned with improvement of experimental methods used for studying genes functions and gene expression levels, of which the quantitative polymerase chain reaction (qPCR) is still the most reliable. As well as with other nucleic acid-based methods used for comparison of the abundance of specific RNAs, the RT-qPCR data have to be normalised to the levels of RNAs represented stably in a cell. To achieve the goal, the so-called housekeeping genes (i.e., RNAs encoding, for instance, proteins playing an important role in the cell metabolism or structure maintenance), are used for normalisation of the target gene expression data. However, a number of studies have indicated the transcriptional instability of commonly used reference genes analysed in different situations or conditions; for instance, the origin of cells, tissue types, or environmental or other experimental conditions. The expression of ten common housekeeping genes of S. lycopersicum, namely EF1?, TUB, CAC, EXP, RPL8, GAPDH, TBP, ACT, SAND and 18S rRNA were examined during viral infections of tomato. Changes in the expression levels of the genes were estimated by comparison of the non-inoculated tomato plants with those infected with commonly known tomato viral pathogens, Tomato torrado virus, Cucumber mosaic virus, Tobacco mosaic virus and Pepino mosaic virus, inducing a diverse range of disease symptoms on the common host, ranging from mild leaves chlorosis to very severe stem necrosis. It is emphasised that despite the wide range of diverse disease symptoms it is concluded that ACT, CAC and EF1? could be used as the most suitable reference genes in studies of host-virus interactions in tomato. PMID:23994079

Wieczorek, Przemys?aw; Wrzesi?ska, Barbara; Obr?palska-St?plowska, Aleksandra

2013-12-01

99

Reference gene alternatives to Gapdh in rodent and human heart failure gene expression studies  

Microsoft Academic Search

BACKGROUND: Quantitative real-time RT-PCR (RT-qPCR) is a highly sensitive method for mRNA quantification, but requires invariant expression of the chosen reference gene(s). In pathological myocardium, there is limited information on suitable reference genes other than the commonly used Gapdh mRNA and 18S ribosomal RNA. Our aim was to evaluate and identify suitable reference genes in human failing myocardium, in rat

Trond Brattelid; Lisbeth H Winer; Finn Olav Levy; Knut Liestøl; Ole M Sejersted; Kristin B Andersson

2010-01-01

100

Pneumocystis sp. in bats evaluated by qPCR.  

PubMed

Molecular techniques have revealed a high prevalence of Pneumocystis colonization in wild mammals. Accurate quantification of Pneumocystis sp. is essential for the correct interpretation of many research experiments investigating this organism. The objectives of this study were to detect the presence of Pneumocystis sp. in bats by qPCR, and to distinguish colonization from infection. Probes and primers for real time PCR (qPCR) were designed based on the gene of major surface glycoprotein (MSG) of Pneumocystis sp., in order to analyze 195 lung tissue samples from bats captured (2007-2009). All samples were also analyzed by nested PCR, using oligonucleotide primers designed for the gene encoding the mitochondrial small subunit rRNA (mtSSU rRNA) to confirm the results. The qPCR assay was standardized using a standard curve made with the DNA extracted from bronchoalveolar lavage positive for Pneumocystis jirovecii. The average Ct was found to be between 13 and 14 (calibration curve) for the detection of infection with Pneumocystis sp. and above these values for colonization. It was considered as negative samples the ones that had Ct values equal to 50. Out of the total 195 samples, 47 (24.1%) bat lung DNA samples were positive for Pneumocystis sp. by qPCR. The most common bat species found were: Tadarida brasiliensis (23.4%), Histiotus velatus (17.0%), Desmodus rotundus (14.9%) and Molossus molossus (8.5%). The average cycle threshold of the positive samples (bats) was 25.8 and standard deviation was 1.7. The DNA samples with Ct values greater than 14 suggest that these animals might be colonized by Pneumocystis sp. Results obtained in this study demonstrated the usefulness of the qPCR procedure for identification of Pneumocystis sp. and for distinction between its colonizing or infectious status in bats. PMID:23380373

Cavallini Sanches, E M; Ferreiro, L; Andrade, C P; Pacheco, S M; Almeida, L L; Spanamberg, A; Wissmann, G

2013-03-01

101

Selection of reliable reference genes for gene expression studies in peach using real-time PCR  

Microsoft Academic Search

BACKGROUND: RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for

Zhaoguo Tong; Zhihong Gao; Fei Wang; Jun Zhou; Zhen Zhang

2009-01-01

102

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)  

PubMed Central

MicroRNAs (miRNAs) are single-stranded, 18–24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets. Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8. Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

Gordanpour, Aida; Nam, Robert K.; Sugar, Linda; Bacopulos, Stephanie; Seth, Arun

2012-01-01

103

Expression of reference genes and T helper 17 associated cytokine genes in the equine intestinal tract.  

PubMed

There is accumulating evidence for the involvement of pro-inflammatory cytokines associated with a T helper 17 response in intestinal disorders such as inflammatory bowel disease (IBD) in humans. The involvement of interleukin (IL)-17 or IL-23 in equine IBD has not been studied and most gene expression studies in the equine intestine have been limited to the use of a single non-validated reference gene. In this study, expression of the reference gene candidates ?2 microglobulin (?2M), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), histone H2A type 1, hypoxanthine-guanine phosphoribosyltransferase (HPRT), 60S ribosomal protein L32 (RPL32), succinate dehydrogenase complex subunit A (SDHA) and transferrin receptor 1 protein coding (TFRC)in the equine intestine was evaluated by quantitative PCR. Three to four reference genes were adequate for normalisation of gene expression in the healthy duodenum, mid-jejunum, colon and rectum, although each segment required a unique combination of reference genes. No combination of the evaluated genes was optimal for the caecum and ileum. Another combination of reference genes (GAPDH, HPRT, RPL32 and SDHA) was optimal for normalisation of rectal samples from healthy and IBD-affected horses, indicating that reference genes should be re-evaluated if material from diseased specimens is analysed. Basal expression of IL-12p40, IL-17A and IL-23p19 was detected in each segment, which will enable gene expression studies of these cytokines by relative quantification. PMID:23810185

Hjertner, Bernt; Olofsson, Karin M; Lindberg, Ronny; Fuxler, Lisbeth; Fossum, Caroline

2013-09-01

104

Selection and validation of reference genes for quantitative gene expression studies in Erythroxylum coca.  

PubMed

Real-time quantitative PCR is a powerful technique for the investigation of comparative gene expression, but its accuracy and reliability depend on the reference genes used as internal standards. Only genes that show a high level of expression stability are suitable for use as reference genes, and these must be identified on a case-by-case basis. Erythroxylum coca produces and accumulates high amounts of the pharmacologically active tropane alkaloid cocaine (especially in the leaves), and is an emerging model for the investigation of tropane alkaloid biosynthesis. The identification of stable internal reference genes for this species is important for its development as a model species, and would enable comparative analysis of candidate biosynthetic genes in the different tissues of the coca plant. In this study, we evaluated the expression stability of nine candidate reference genes in E. coca ( Ec6409, Ec10131, Ec11142, Actin, APT2, EF1?, TPB1, Pex4, Pp2aa3). The expression of these genes was measured in seven tissues (flowers, stems, roots and four developmental leaf stages) and the stability of expression was assessed using three algorithms (geNorm, NormFinder and BestKeeper). From our results we conclude that Ec10131 and TPB1 are the most appropriate internal reference genes in leaves (where the majority of cocaine is produced), while Ec10131 and Ec6409 are the most suitable internal reference genes across all of the tissues tested. PMID:24627771

Docimo, Teresa; Schmidt, Gregor W; Luck, Katrin; Delaney, Sven K; D'Auria, John C

2013-01-01

105

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

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

106

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.

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

107

Selection of reliable reference genes for gene expression studies in peach using real-time PCR  

PubMed Central

Background RT-qPCR is a preferred method for rapid and reliable quantification of gene expression studies. Appropriate application of RT-qPCR in such studies requires the use of reference gene(s) as an internal control to normalize mRNA levels between different samples for an exact comparison of gene expression level. However, recent studies have shown that no single reference gene is universal for all experiments. Thus, the identification of high quality reference gene(s) is of paramount importance for the interpretation of data generated by RT-qPCR. Only a few studies on reference genes have been done in plants and none in peach (Prunus persica L. Batsch). Therefore, the present study was conducted to identify suitable reference gene(s) for normalization of gene expression in peach. Results In this work, eleven reference genes were investigated in different peach samples using RT-qPCR with SYBR green. These genes are: actin 2/7 (ACT), cyclophilin (CYP2), RNA polymerase II (RP II), phospholipase A2 (PLA2), ribosomal protein L13 (RPL13), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S ribosomal RNA (18S rRNA), tubblin beta (TUB), tubblin alpha (TUA), translation elongation factor 2 (TEF2) and ubiquitin 10 (UBQ10). All eleven reference genes displayed a wide range of Cq values in all samples, indicating that they expressed variably. The stability of these genes except for RPL13 was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper, which produced highly comparable results. Conclusion Our study demonstrates that expression stability varied greatly between genes studied in peach. Based on the results from geNorm, NormFinder and BestKeeper analyses, for all the sample pools analyzed, TEF2, UBQ10 and RP II were found to be the most suitable reference genes with a very high statistical reliability, and TEF2 and RP II for the other sample series, while 18S rRNA, RPL13 and PLA2 were unsuitable as internal controls. GAPDH and ACT also performed poorly and were less stable in our analysis. To achieve accurate comparison of levels of gene expression, two or more reference genes must be used for data normalization. The combinations of TEF2/UBQ10/RP II and TEF2/RP II were suggested for use in all samples and subsets, respectively.

2009-01-01

108

Validation of Reference Genes for the Relative Quantification of Gene Expression in Human Epicardial Adipose Tissue  

Microsoft Academic Search

BackgroundRelative quantification is a commonly used method for assessing gene expression, however its accuracy and reliability is dependent upon the choice of an optimal endogenous control gene, and such choice cannot be made a priori. There is limited information available on suitable reference genes to be used for studies involving human epicardial adipose tissue. The objective of the current study

Kanta Chechi; Yves Gelinas; Patrick Mathieu; Yves Deshaies; Denis Richard

2012-01-01

109

Reference genes for the normalization of gene expression in eucalyptus species.  

PubMed

Gene expression analysis is increasingly important in biological research, with reverse transcription-quantitative PCR (RT-qPCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Considering the increased sensitivity, reproducibility and large dynamic range of this method, the requirements for proper internal reference gene(s) for relative expression normalization have become much more stringent. Given the increasing interest in the functional genomics of Eucalyptus, we sought to identify and experimentally verify suitable reference genes for the normalization of gene expression associated with the flower, leaf and xylem of six species of the genus. We selected 50 genes that exhibited the least variation in microarrays of E. grandis leaves and xylem, and E. globulus xylem. We further performed the experimental analysis using RT-qPCR for six Eucalyptus species and three different organs/tissues. Employing algorithms geNorm and NormFinder, we assessed the gene expression stability of eight candidate new reference genes. Classic housekeeping genes were also included in the analysis. The stability profiles of candidate genes were in very good agreement. PCR results proved that the expression of novel Eucons04, Eucons08 and Eucons21 genes was the most stable in all Eucalyptus organs/tissues and species studied. We showed that the combination of these genes as references when measuring the expression of a test gene results in more reliable patterns of expression than traditional housekeeping genes. Hence, novel Eucons04, Eucons08 and Eucons21 genes are the best suitable references for the normalization of expression studies in the Eucalyptus genus. PMID:22197885

de Oliveira, Luisa Abruzzi; Breton, Michèle Claire; Bastolla, Fernanda Macedo; Camargo, Sandro da Silva; Margis, Rogério; Frazzon, Jeverson; Pasquali, Giancarlo

2012-02-01

110

Expression stability of commonly used reference genes in canine articular connective tissues  

Microsoft Academic Search

BACKGROUND: The quantification of gene expression in tissue samples requires the use of reference genes to normalise transcript numbers between different samples. Reference gene stability may vary between different tissues, and between the same tissue in different disease states. We evaluated the stability of 9 reference genes commonly used in human gene expression studies. Real-time reverse transcription PCR and a

Duncan Ayers; Dylan N Clements; Fiona Salway; Philip JR Day

2007-01-01

111

Identification and validation of suitable endogenous reference genes for gene expression studies in human peripheral blood  

PubMed Central

Background Gene expression studies require appropriate normalization methods. One such method uses stably expressed reference genes. Since suitable reference genes appear to be unique for each tissue, we have identified an optimal set of the most stably expressed genes in human blood that can be used for normalization. Methods Whole-genome Affymetrix Human 2.0 Plus arrays were examined from 526 samples of males and females ages 2 to 78, including control subjects and patients with Tourette syndrome, stroke, migraine, muscular dystrophy, and autism. The top 100 most stably expressed genes with a broad range of expression levels were identified. To validate the best candidate genes, we performed quantitative RT-PCR on a subset of 10 genes (TRAP1, DECR1, FPGS, FARP1, MAPRE2, PEX16, GINS2, CRY2, CSNK1G2 and A4GALT), 4 commonly employed reference genes (GAPDH, ACTB, B2M and HMBS) and PPIB, previously reported to be stably expressed in blood. Expression stability and ranking analysis were performed using GeNorm and NormFinder algorithms. Results Reference genes were ranked based on their expression stability and the minimum number of genes needed for nomalization as calculated using GeNorm showed that the fewest, most stably expressed genes needed for acurate normalization in RNA expression studies of human whole blood is a combination of TRAP1, FPGS, DECR1 and PPIB. We confirmed the ranking of the best candidate control genes by using an alternative algorithm (NormFinder). Conclusion The reference genes identified in this study are stably expressed in whole blood of humans of both genders with multiple disease conditions and ages 2 to 78. Importantly, they also have different functions within cells and thus should be expressed independently of each other. These genes should be useful as normalization genes for microarray and RT-PCR whole blood studies of human physiology, metabolism and disease.

Stamova, Boryana S; Apperson, Michelle; Walker, Wynn L; Tian, Yingfang; Xu, Huichun; Adamczy, Peter; Zhan, Xinhua; Liu, Da-Zhi; Ander, Bradley P; Liao, Isaac H; Gregg, Jeffrey P; Turner, Renee J; Jickling, Glen; Lit, Lisa; Sharp, Frank R

2009-01-01

112

In search of suitable reference genes for gene expression studies of human renal cell carcinoma by real-time PCR  

Microsoft Academic Search

BACKGROUND: Housekeeping genes are commonly used as endogenous reference genes for the relative quantification of target genes in gene expression studies. No conclusive systematic study comparing the suitability of different candidate reference genes in clear cell renal cell carcinoma has been published to date. To remedy this situation, 10 housekeeping genes for normalizing purposes of RT-PCR measurements already recommended in

Monika Jung; Azizbek Ramankulov; Jan Roigas; Manfred Johannsen; Martin Ringsdorf; Glen Kristiansen; Klaus Jung

2007-01-01

113

Reference genes for gene expression studies on non-small cell lung cancer  

Microsoft Academic Search

Study Objective : The aim of this study was to test a panel of 6 reference genes in order to iden - tify and validate the most suitable reference genes for expression studies in paired healthy and non-small cell lung cancer tissues. Method : Quantitative real-time PCR followed by the NormFinder- and geNorm-based analysis was employed. The study involved 21

Peter Gresner; Jolanta Gromadzinska; Wojciech Wasowicz

2009-01-01

114

Reference gene selection for head and neck squamous cell carcinoma gene expression studies  

Microsoft Academic Search

BACKGROUND: It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD and TBP) for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC).

Benjamin Lallemant; Alexandre Evrard; Christophe Combescure; Heliette Chapuis; Guillaume Chambon; Caroline Raynal; Christophe Reynaud; Omar Sabra; Dominique Joubert; Frédéric Hollande; Jean-Gabriel Lallemant; Serge Lumbroso; Jean-Paul Brouillet

2009-01-01

115

Evaluation of RNA extraction methods and identification of putative reference genes for real-time quantitative polymerase chain reaction expression studies on olive (Olea europaea L.) fruits.  

PubMed

Genome wide transcriptomic surveys together with targeted molecular studies are uncovering an ever increasing number of differentially expressed genes in relation to agriculturally relevant processes in olive (Olea europaea L). These data need to be supported by quantitative approaches enabling the precise estimation of transcript abundance. qPCR being the most widely adopted technique for mRNA quantification, preliminary work needs to be done to set up robust methods for extraction of fully functional RNA and for the identification of the best reference genes to obtain reliable quantification of transcripts. In this work, we have assessed different methods for their suitability for RNA extraction from olive fruits and leaves and we have evaluated thirteen potential candidate reference genes on 21 RNA samples belonging to fruit developmental/ripening series and to leaves subjected to wounding. By using two different algorithms, GAPDH2 and PP2A1 were identified as the best reference genes for olive fruit development and ripening, and their effectiveness for normalization of expression of two ripening marker genes was demonstrated. PMID:22703380

Nonis, Alberto; Vezzaro, Alice; Ruperti, Benedetto

2012-07-11

116

Validation of internal reference genes for real-time quantitative polymerase chain reaction studies in the tick, Ixodes scapularis (Acari: Ixodidae).  

PubMed

Obtaining reliable gene expression data using real-time quantitative polymerase chain reaction (qPCR) is highly dependent on the choice of normalization method. We tested the expression stability of multiple candidate genes in the salivary glands (SG) and synganglia (SYN) of female Ixodes scapularis (Say) ticks in multiple blood-feeding phases. We found that the amount of total RNA in both the SG and SYN increases dramatically during tick feeding, with 34x and 5.8x increases from 62 and 7.1 ng of unfed tick, respectively. We tested candidate genes that were predicted from I. scapularis genome data to encode glyceraldehyde 3-phosphate dehydrogenase (gapdh), ribosomal protein L13A (l13a), TATA box-binding protein (tbp), ribosomal protein S4 (rps4), glucose 6-phosphate dehydrogenase (gpdh), and beta-glucuronidase (gusb). The geNorm and NormFinder algorithms were used to analyze data from different feeding phases (i.e., daily samples from unfed to fully engorged females over a 7-d period in three replicate experiments). We found that the rps4 and l13a genes showed highly stable expression patterns over the feeding duration in both the SG and SYN. Furthermore, the highly expressed rps4 gene makes it useful as a normalization factor when we perform studies using minute amounts of dissected tissue for qPCR. We conclude that rps4 and l13a, whether individually or as a pair, serve as suitable internal reference genes for qRT-PCR studies in the SG and SYN of I. scapularis. PMID:23427655

Koci, Juraj; Simo, Ladislav; Park, Yoonseong

2013-01-01

117

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

Microsoft Academic Search

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

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

2009-01-01

118

Equivalence test in quantitative reverse transcription polymerase chain reaction: confirmation of reference genes suitable for normalization  

Microsoft Academic Search

In quantitative reverse transcription–polymerase chain reaction (qRT–PCR), normalization using reference genes is a common useful approach, but the validation of suitable reference genes remains a crucial problem. Use of unconfirmed reference genes may lead to misinterpretation of the expression of target genes. The aim of this study was to adapt an adequate statistical approach to identify and validate reference genes

Florian Haller; Bettina Kulle; Stefanie Schwager; Bastian Gunawan; Anja von Heydebreck; Holger Sültmann; László Füzesi

2004-01-01

119

Validation of candidate reference genes in Bifidobacterium adolescentis for gene expression normalization.  

PubMed

Normalization is an essential prerequisite for producing accurate real-time PCR expression analyses. The objective of this study is the selection of a set of optimal reference genes in Bifidobacterium adolescentis gene expression studies under bile exposure. B adolescentis is a particularly abundant species in the human adults gut microbiota, exerting relevant probiotic activities. In the gastrointestinal tract, bile represents a hard challenge for bacterial survival, because of its toxic effect. The natural exposure to bile in the colonic environment induces cells adaptation and tolerance mechanisms in bifidobacteria, which determines changes in gene expression profile, influencing the expression levels of housekeeping genes. In this context, the stability of 9 putative reference genes (cysS, purB, recA, rpoB-L, GADPH-R, 16S rRNA, glnA1, gyrA2, sdhA) was examined in B. adolescentis exposed to bile extract, using two different software (BestKeeper and NormFinder). Both algorithms identified gyrA2 and sdhA as the most stable genes under our experimental conditions, while 16S rRNA is the least reliable HKGs. To our best knowledge, this is the first attempt to validate reference genes in Bifidobacterium spp. and the results offer an appropriate set of reference genes suitable for qRT-PCR studies on B. adolescentis strains under bile stress. PMID:24657392

Stenico, Verena; Baffoni, Loredana; Gaggìa, Francesca; Biavati, Bruno

2014-06-01

120

Evaluation of Reference Genes for Gene Expression Analysis Using Quantitative RT-PCR in Azospirillum brasilense  

PubMed Central

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.

McMillan, Mary; Pereg, Lily

2014-01-01

121

Genomic selection of reference genes for real-time PCR in human myocardium  

Microsoft Academic Search

BACKGROUND: Reliability of real-time PCR (RT-qPCR) data is dependent on the use of appropriate reference gene(s) for normalization. To date, no validated reference genes have been reported for normalizing gene expression in human myocardium. This study aimed to identify validated reference genes for use in gene expression studies of failed and non-failed human myocardium. METHODS: Bioinformatic analysis of published human

Anna P Pilbrow; Leigh J Ellmers; Michael A Black; Christine S Moravec; Wendy E Sweet; Richard W Troughton; A Mark Richards; Chris M Frampton; Vicky A Cameron

2008-01-01

122

Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain  

Microsoft Academic Search

BACKGROUND: Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as \\

Nigel C Noriega; Steven G Kohama; Henryk F Urbanski

2010-01-01

123

Selection of Reference Gene Expression in a Schizophrenia Brain Cohort  

PubMed Central

Objective To conduct postmortem human brain research into the neuropathological basis of schizophrenia, it is critical to establish cohorts that are well-characterised and well-matched. Our objective was to determine if specimen characteristics, including: diagnosis, age, postmortem interval (PMI), brain acidity (pH), and/or the agonal state of the subject at death related to RNA quality, and to determine the most appropriate reference gene mRNAs. Methods We selected a matched cohort of 74 cases (37 schizophrenia / schizoaffective disorder cases and 37 controls cases). Middle frontal gyrus tissue was pulverised, tissue pH was measured, RNA isolated for cDNA from each case, and RNA integrity number (RIN) measurements were assessed. Using RT-PCR, we measured nine housekeeper genes and calculated a geomean in each diagnostic group. Results We found that the RINs were very good (mean 7.3) and all nine housekeeper control genes were significantly correlated with RIN. Seven of nine housekeeper genes were also correlated with pH, and two clinical variables, agonal state and duration of illness did have an effect on some control mRNAs. No major impact of PMI or freezer time on housekeeper mRNAs was detected. Our results show that people with schizophrenia had significantly less PPIA, and SDHA and tended to have less GUSB and B2M mRNA suggesting that these control genes may not be good candidates for normalisation. Conclusions In our cohort, less than 10% variability in RIN values was detected and the diagnostic groups were well matched overall. Our cohort was adequately powered (0.80–0.90) to detect mRNA differences (25%) due to disease. Our study suggests that multiple factors should be considered in mRNA expression studies of human brain tissues. When schizophrenia cases are adequately matched to control cases subtle differences in gene expression can be reliably detected.

Weickert, Cynthia Shannon; Sheedy, Donna; Rothmond, Debora A.; Dedova, Irina; Fung, Samantha; Garrick, Therese; Wong, Jenny; Harding, Antony J.; Sivagnanansundaram, Sinthuja; Hunt, Clare; Duncan, Carlotta; Sundqvist, Nina; Tsai, Shan-Yuan; Anand, Jasna; Draganic, Daren; Harper, Clive

2010-01-01

124

Validation of reference genes for RT-qPCR studies of gene expression in banana fruit under different experimental conditions  

Microsoft Academic Search

Reverse transcription quantitative real-time PCR (RT-qPCR) is a sensitive technique for quantifying gene expression, but its\\u000a success depends on the stability of the reference gene(s) used for data normalization. Only a few studies on validation of\\u000a reference genes have been conducted in fruit trees and none in banana yet. In the present work, 20 candidate reference genes\\u000a were selected, and

Lei ChenHai-ying; Hai-ying Zhong; Jian-fei Kuang; Jian-guo Li; Wang-jin Lu; Jian-ye Chen

125

Reference gene selection for head and neck squamous cell carcinoma gene expression studies  

PubMed Central

Background It is no longer adequate to choose reference genes blindly. We present the first study that defines the suitability of 12 reference genes commonly used in cancer studies (ACT, ALAS, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPL27, RPS29, SHAD and TBP) for the normalization of quantitative expression data in the field of head and neck squamous cell carcinoma (HNSCC). Results Raw expression levels were measured by RT-qPCR in HNSCC and normal matched mucosa of 46 patients. We analyzed the expression stability using geNorm and NormFinder and compared the expression levels between subgroups. In HNSCC and/or normal mucosa, the four best normalization genes were ALAS, GAPDH, RPS18 and SHAD and the most stable combination of two genes was GAPDH-SHAD. We recommend using KALPHA-TBP for the study of T1-T2 tumors, RPL27-SHAD for T3-T4 tumors, KALPHA-SHAD for N0 tumors, and ALAS-TBP for N+ tumors. ACT, B2M, GAPDH, HMBS, HPRT, KALPHA, RPS18, RPS29, SHAD and TBP were slightly misregulated (<1.7-fold) between tumor and normal mucosa but can be used for normalization, depending on the resolution required for the assay. Conclusion In the field of HNSCC, this study will guide researchers in selecting the most appropriate reference genes from among 12 potentially suitable reference genes, depending on the specific setting of their experiments.

Lallemant, Benjamin; Evrard, Alexandre; Combescure, Christophe; Chapuis, Heliette; Chambon, Guillaume; Raynal, Caroline; Reynaud, Christophe; Sabra, Omar; Joubert, Dominique; Hollande, Frederic; Lallemant, Jean-Gabriel; Lumbroso, Serge; Brouillet, Jean-Paul

2009-01-01

126

Evaluation and selection of reliable reference genes for gene expression under abiotic stress in cotton (Gossypium hirsutum L.).  

PubMed

Reference genes are critical for normalization of the gene expression level of target genes. The widely used housekeeping genes may change their expression levels at different tissue under different treatment or stress conditions. Therefore, systematical evaluation on the housekeeping genes is required for gene expression analysis. Up to date, no work was performed to evaluate the housekeeping genes in cotton under stress treatment. In this study, we chose 10 housekeeping genes to systematically assess their expression levels at two different tissues (leaves and roots) under two different abiotic stresses (salt and drought) with three different concentrations. Our results show that there is no best reference gene for all tissues at all stress conditions. The reliable reference gene should be selected based on a specific condition. For example, under salt stress, UBQ7, GAPDH and EF1A8 are better reference genes in leaves; TUA10, UBQ7, CYP1, GAPDH and EF1A8 were better in roots. Under drought stress, UBQ7, EF1A8, TUA10, and GAPDH showed less variety of expression level in leaves and roots. Thus, it is better to identify reliable reference genes first before performing any gene expression analysis. However, using a combination of housekeeping genes as reference gene may provide a new strategy for normalization of gene expression. In this study, we found that combination of four housekeeping genes worked well as reference genes under all the stress conditions. PMID:23933278

Wang, Min; Wang, Qinglian; Zhang, Baohong

2013-11-01

127

Selection of appropriate reference genes for gene expression studies by quantitative real-time polymerase chain reaction in cucumber  

Microsoft Academic Search

Quantitative real-time polymerase chain reaction (QRT-PCR) has become one of the most widely used methods for gene expression analysis. However, the expression profile of a target gene may be misinterpreted due to unstable expression of the reference genes under different experimental conditions. Thus, a systematic evaluation of these reference genes is necessary before experiments are performed. In this study, 10

Hongjian Wan; Zhenguo Zhao; Chuntao Qian; Yihu Sui; Ahmed Abbas Malik; Jinfeng Chen

2010-01-01

128

Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells  

PubMed Central

Introduction The objective of this study was to find highly reliable internal-control genes (ICGs) for normalization of qPCR data from porcine adult mesenchymal stem cells induced to differentiate toward adipogenic and osteogenic lineages. Methods Stem cells were acquired from subcutaneous back fat and bone marrow of three castrated Yorkshire crossbred male pigs. Adipose and bone marrow-derived stem cells (ADSCs and BMSCs) were cultured in vitro with specific osteogenic or adipogenic differentiation medium for 4 weeks. Total RNA was extract for microarray (13,000 oligonucleotides) and qPCR analyses. Microarray data were used to uncover the most stably expressed genes (that is, potential ICGs). Co-regulation among potential ICGs was evaluated with Ingenuity Pathway Analysis. qPCR was performed on the non-coregulated ICGs candidates and on specific osteogenic (COL1A1) and adipogenic (DBI) genes. geNorm was used to uncover the most reliable ICGs by using qPCR data and the optimal number of ICGs to be used to calculate the normalization factor. Results Microarray data analysis revealed 27 potential ICGs. Among those, 10 genes without known co-regulation were selected to perform qPCR. geNorm performed on qPCR data uncovered high stability in expression ratio among the selected ICGs. However, especially reliable normalization was obtained by geometric mean of NSUN5, TIMM17B, and VPS4A. The effect of normalization, assessed on specific osteogenic (COL1A1) and adipogenic (DBI) genes, was apparent for the adipogenic and less apparent for the osteogenic differentiation. Conclusions The combination of microarray data and pairwise gene analysis allowed identification of novel and highly reliable ICGs for qPCR data normalization of adult porcine stem cells induced to differentiate to adipogenic and osteogenic lineages.

2010-01-01

129

Validation of reference genes in Solenopsis invicta in different developmental stages, castes and tissues.  

PubMed

To accurately assess gene expression levels, it is essential to normalize real-time quantitative PCR (RT-qPCR) data with suitable internal reference genes. For the red imported fire ant, Solenopsis invicta, reliable reference genes to assess the transcript expression levels of the target genes have not been previously investigated. In this study, we examined the expression levels of five candidate reference genes (rpl18, ef1-beta, act, GAPDH, and tbp) in different developmental stages, castes and tissues of S. invicta. To evaluate the suitability of these genes as endogenous controls, three software-based approaches (geNorm, BestKeeper and NormFinder) and one web-based comprehensive tool (RefFinder) were used to analyze and rank the tested genes. Furthermore, the optimal number of reference gene(s) was determined by the pairwise variation value. Our data showed that two of the five candidate genes, rpl18 and ef1-beta, were the most suitable reference genes because they have the most stable expression among different developmental stages, castes and tissues in S. invicta. Although widely used as reference gene in other species, in S. invicta the act gene has high variation in expression and was consequently excluded as a reliable reference gene. The two validated reference genes, rpl18 and ef1-beta, can be widely used for quantification of target gene expression with RT-qPCR technology in S. invicta. PMID:23469057

Cheng, Daifeng; Zhang, Zhiling; He, Xiaofang; Liang, Guangwen

2013-01-01

130

Selection of reference genes for expression studies with fish myogenic cell cultures  

Microsoft Academic Search

BACKGROUND: Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes

Neil I Bower; Ian A Johnston

2009-01-01

131

METHOD TO CLASSIFY ENVIRONMENTAL SAMPLES BASED ON MOLD ANALYSES BY QPCR  

EPA Science Inventory

A total of 82 quantitative PCR (QPCR) assays were used to identify and quantify different indoor molds in dust samples from the homes of six infants suffering from pulmonary hemorrhage and 26 reference homes in Cleveland, Ohio. No significant difference was seen in the total cell...

132

Identification of suitable reference genes for gene expression normalization in qRT-PCR analysis in watermelon.  

PubMed

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

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

2014-01-01

133

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.

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

2014-01-01

134

Optimizing stem-loop qPCR assays through multiplexed cDNA synthesis of U6 and miRNAs  

PubMed Central

We recently reported that hairpin (or stem-loop) priming is better-suited than polyA tailing to generate cDNA for plant microRNA qPCR. One major limitation of this method is the need to perform individual cDNA synthesis reactions for the reference gene and test miRNAs. Here, we report a novel fusion primer that allows multiplexed hairpin cDNA synthesis of the most-commonly used reference gene, nucleolar small RNA U6, together with test miRNAs. We also propose the use of miR1515 as a house keeping control for tropical legumes. We show that multiplexed cDNA synthesis does not result in loss of sensitivity and reduces the amount of RNA required for miRNA gene expression assays.

Turner, Marie; Adhikari, Sajag; Subramanian, Senthil

2013-01-01

135

Selection of reliable reference genes during THP1 monocyte differentiation into macrophages  

Microsoft Academic Search

BACKGROUND: Reliable reference genes are a vital prerequisite for any functional study employing quantitative real-time RT-PCR (RT-qPCR) for analyzing gene expression. Yet a proper selection and assessment of the chosen reference genes is only rarely included into a study. To date, no reference genes have been validated for differentiation of THP-1 monocytes. Here we report on the selection of validated

Marten B Maeß; Stefanie Sendelbach; Stefan Lorkowski

2010-01-01

136

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

137

Selection and validation of endogenous reference genes using a high throughput approach  

PubMed Central

Background Endogenous reference genes are commonly used to normalize expression levels of other genes with the assumption that the expression of the former is constant in different tissues and in different physiopathological conditions. Whether this assumption is correct it is, however, still matter of debate. In this study, we searched for stably expressed genes in 384 cDNA array hybridization experiments encompassing different tissues and cell lines. Results Several genes were identified whose expression was highly stable across all samples studied. The usefulness of 8 genes among them was tested by normalizing the relative gene expression against test genes whose expression pattern was known. The range of accuracy of individual endogenous reference genes was wide whereas consistent information could be obtained when information pooled from different endogenous reference genes was used. Conclusions This study suggests that even when the most stably expressed genes in array experiments are used as endogenous reference, significant variation in test gene expression estimates may occur and the best normalization is achieved when data from several endogenous reference genes are pooled together to minimize minimal but significant variation among samples. We are presently optimizing strategies for the preparation of endogenous reference gene mixtures that could yield information comparable to that of data pooled from individual endogenous reference gene normalizations.

Jin, Ping; Zhao, Yingdong; Ngalame, Yvonne; Panelli, Monica C; Nagorsen, Dirk; Monsurro, Vladia; Smith, Kina; Hu, Nan; Su, Hua; Taylor, Phil R; Marincola, Francesco M; Wang, Ena

2004-01-01

138

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions  

Microsoft Academic Search

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best

Valéria Mafra; Karen S. Kubo; Marcio Alves-Ferreira; Marcelo Ribeiro-Alves; Rodrigo M. Stuart; Leonardo P. Boava; Carolina M. Rodrigues; Marcos A. Machado

2012-01-01

139

Evaluation of new reference genes in papaya for accurate transcript normalization under different experimental conditions.  

PubMed

Real-time reverse transcription PCR (RT-qPCR) is a preferred method for rapid and accurate quantification of gene expression studies. Appropriate application of RT-qPCR requires accurate normalization though the use of reference genes. As no single reference gene is universally suitable for all experiments, thus reference gene(s) validation under different experimental conditions is crucial for RT-qPCR analysis. To date, only a few studies on reference genes have been done in other plants but none in papaya. In the present work, we selected 21 candidate reference genes, and evaluated their expression stability in 246 papaya fruit samples using three algorithms, geNorm, NormFinder and RefFinder. The samples consisted of 13 sets collected under different experimental conditions, including various tissues, different storage temperatures, different cultivars, developmental stages, postharvest ripening, modified atmosphere packaging, 1-methylcyclopropene (1-MCP) treatment, hot water treatment, biotic stress and hormone treatment. Our results demonstrated that expression stability varied greatly between reference genes and that different suitable reference gene(s) or combination of reference genes for normalization should be validated according to the experimental conditions. In general, the internal reference genes EIF (Eukaryotic initiation factor 4A), TBP1 (TATA binding protein 1) and TBP2 (TATA binding protein 2) genes had a good performance under most experimental conditions, whereas the most widely present used reference genes, ACTIN (Actin 2), 18S rRNA (18S ribosomal RNA) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were not suitable in many experimental conditions. In addition, two commonly used programs, geNorm and Normfinder, were proved sufficient for the validation. This work provides the first systematic analysis for the selection of superior reference genes for accurate transcript normalization in papaya under different experimental conditions. PMID:22952972

Zhu, Xiaoyang; Li, Xueping; Chen, Weixin; Chen, Jianye; Lu, Wangjin; Chen, Lei; Fu, Danwen

2012-01-01

140

Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses.  

PubMed

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies. PMID:24573225

Borowski, Joyce Moura; Galli, Vanessa; da Silva Messias, Rafael; Perin, Ellen Cristina; Buss, Julieti Hugh; Dos Anjos E Silva, Sérgio Delmar; Rombaldi, Cesar Valmor

2014-06-01

141

Ribosomal Protein L13a as a Reference Gene for Human Bone Marrow-Derived Mesenchymal Stromal Cells During Expansion, Adipo-, Chondro-, and Osteogenesis  

PubMed Central

In the field of human mesenchymal stromal cell (MSC) research, quantitative real-time reverse transcription–polymerase chain reaction (qPCR) is the method of choice to study changes in gene expression patterns upon differentiation, application of stimuli, or of factors such as inhibitors or siRNAs. To reliably detect small changes, the use of a reference gene (RG) that is stably expressed under all conditions is essential. The large number of different RGs used in the field and the lack of validation of their suitability make the comparison between studies impossible. Therefore, this work aims to establish one single RG for mesodermal differentiation studies that use MSCs. Seven commonly used RGs (glyceraldehyde-3-phosphate dehydrogenase [GAPDH], ribosomal protein L13a [RPL13a], beta-actin [ACTB], tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta-polypeptide [YWHAZ], eukaryotic translational elongation factor 1 alpha [EF1?], ?2-microglobulin [B2M], and 18S ribosomal RNA [18S]) were investigated concerning their mRNA expression stability during expansion of bone marrow-derived MSCs up to four passages as well as during their adipo-, chondro-, and osteogenenic differentiation on days 9, 16, and 22 after induction. RPL13a was validated for qPCR studies of MSCs (bone marrow- and placenta-derived) and, additionally, for primary human bone cells (HBCs) and the osteosarcoma cell line MG-63. GAPDH and ACTB, the two most frequently used RGs, showed the highest expression variance. The superior performance of RPL13a should make it the RG of choice for all MSC studies addressing mesodermal differentiation.

Studer, Deborah; Lischer, Stefanie; Jochum, Wolfram; Ehrbar, Martin; Zenobi-Wong, Marcy

2012-01-01

142

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.

2012-01-01

143

Identification of valid reference genes for gene expression studies of human stomach cancer by reverse transcription-qPCR  

Microsoft Academic Search

BACKGROUND: Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for the analysis of gene expression. Target gene expression levels are usually normalized to a consistently expressed reference gene also known as internal standard, in the same sample. However, much effort has not been expended thus far in the search for reference genes suitable for the study

Hyun-Wook Rho; Byoung-Chan Lee; Eun-Seok Choi; Il-Ju Choi; Yeon-Su Lee; Sung-Ho Goh

2010-01-01

144

Quantification of E. coli O157 and STEC in feces of farm animals using direct multiplex real time PCR (qPCR) and a modified most probable number assay comprised of immunomagnetic bead separation and qPCR detection.  

PubMed

To better understand Escherichia coli O157:H7 on-farm transmission dynamics requires sensitive methods for quantification of a broad range of concentrations of target organisms. For this purpose, a multiplex real time PCR (qPCR) assay was developed for quantification of O157 E. coli from 1g fecal samples of cattle and other animal species, targeting the Shiga toxin genes (stx1 and stx2) and the O157 somatic antigen gene, per. The multiplex qPCR assay provided specific detection across a broad range of bacterial concentrations with a lower limit of detection (LOD) of 10(1) genome copies which is equivalent to 10(1) bacteria. However, the LOD, when direct qPCR was applied to quantification of the targets in the feces of dairy cattle, was 10(3) genome copies per gram of feces. Enumeration below the threshold for direct qPCR was performed using a modified most probable number (mMPN) method whereby E. coli O157 in enriched samples was isolated using immunomagnetic bead separation (IMS) and detected using qPCR, thus reducing the time and logistic constraints of biochemical/serological/gel analysis. Application of the mMPN (IMS/qPCR) assay to samples that were negative when tested using direct qPCR alone permitted quantification of low levels of E. coli O157 below levels detectable with direct qPCR. The direct qPCR and mMPN (IMS/qPCR) assays were applied to fecal samples from dairy, beef, swine and poultry feces. This approach can be employed to gain a better understanding of the patterns of infection in animals for analysis of on-farm transmission dynamics, for evaluating the effects of on-farm control strategies and for risk assessment in public health. PMID:24530484

Guy, Rebecca A; Tremblay, Donald; Beausoleil, Louise; Harel, Josée; Champagne, Marie-Josée

2014-04-01

145

Reference Genes for Accurate Transcript Normalization in Citrus Genotypes under Different Experimental Conditions  

PubMed Central

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress.

Mafra, Valeria; Kubo, Karen S.; Alves-Ferreira, Marcio; Ribeiro-Alves, Marcelo; Stuart, Rodrigo M.; Boava, Leonardo P.; Rodrigues, Carolina M.; Machado, Marcos A.

2012-01-01

146

Reference genes for accurate transcript normalization in citrus genotypes under different experimental conditions.  

PubMed

Real-time reverse transcription PCR (RT-qPCR) has emerged as an accurate and widely used technique for expression profiling of selected genes. However, obtaining reliable measurements depends on the selection of appropriate reference genes for gene expression normalization. The aim of this work was to assess the expression stability of 15 candidate genes to determine which set of reference genes is best suited for transcript normalization in citrus in different tissues and organs and leaves challenged with five pathogens (Alternaria alternata, Phytophthora parasitica, Xylella fastidiosa and Candidatus Liberibacter asiaticus). We tested traditional genes used for transcript normalization in citrus and orthologs of Arabidopsis thaliana genes described as superior reference genes based on transcriptome data. geNorm and NormFinder algorithms were used to find the best reference genes to normalize all samples and conditions tested. Additionally, each biotic stress was individually analyzed by geNorm. In general, FBOX (encoding a member of the F-box family) and GAPC2 (GAPDH) was the most stable candidate gene set assessed under the different conditions and subsets tested, while CYP (cyclophilin), TUB (tubulin) and CtP (cathepsin) were the least stably expressed genes found. Validation of the best suitable reference genes for normalizing the expression level of the WRKY70 transcription factor in leaves infected with Candidatus Liberibacter asiaticus showed that arbitrary use of reference genes without previous testing could lead to misinterpretation of data. Our results revealed FBOX, SAND (a SAND family protein), GAPC2 and UPL7 (ubiquitin protein ligase 7) to be superior reference genes, and we recommend their use in studies of gene expression in citrus species and relatives. This work constitutes the first systematic analysis for the selection of superior reference genes for transcript normalization in different citrus organs and under biotic stress. PMID:22347455

Mafra, Valéria; Kubo, Karen S; Alves-Ferreira, Marcio; Ribeiro-Alves, Marcelo; Stuart, Rodrigo M; Boava, Leonardo P; Rodrigues, Carolina M; Machado, Marcos A

2012-01-01

147

Evaluation of Candidate Reference Genes for Gene Expression Normalization in Brassica juncea Using Real Time Quantitative RT-PCR  

PubMed Central

The real time quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important to gain insight into function of genes. Given the increased sensitivity, ease and reproducibility of qRT-PCR, the requirement of suitable reference genes for normalization has become important and stringent. It is now known that the expression of internal control genes in living organism vary considerably during developmental stages and under different experimental conditions. For economically important Brassica crops, only a couple of reference genes are reported till date. In this study, expression stability of 12 candidate reference genes including ACT2, ELFA, GAPDH, TUA, UBQ9 (traditional housekeeping genes), ACP, CAC, SNF, TIPS-41, TMD, TSB and ZNF (new candidate reference genes), in a diverse set of 49 tissue samples representing different developmental stages, stress and hormone treated conditions and cultivars of Brassica juncea has been validated. For the normalization of vegetative stages the ELFA, ACT2, CAC and TIPS-41 combination would be appropriate whereas TIPS-41 along with CAC would be suitable for normalization of reproductive stages. A combination of GAPDH, TUA, TIPS-41 and CAC were identified as the most suitable reference genes for total developmental stages. In various stress and hormone treated samples, UBQ9 and TIPS-41 had the most stable expression. Across five cultivars of B. juncea, the expression of CAC and TIPS-41 did not vary significantly and were identified as the most stably expressed reference genes. This study provides comprehensive information that the new reference genes selected herein performed better than the traditional housekeeping genes. The selection of most suitable reference genes depends on the experimental conditions, and is tissue and cultivar-specific. Further, to attain accuracy in the results more than one reference genes are necessary for normalization.

Chandna, Ruby; Augustine, Rehna; Bisht, Naveen C.

2012-01-01

148

Identification and Validation of Reference Genes for Transcript Normalization in Strawberry (Fragaria x ananassa) Defense Responses  

PubMed Central

Strawberry (Fragaria spp) is an emerging model for the development of basic genomics and recombinant DNA studies among rosaceous crops. Functional genomic and molecular studies involve relative quantification of gene expression under experimental conditions of interest. Accuracy and reliability are dependent upon the choice of an optimal reference control transcript. There is no information available on validated endogenous reference genes for use in studies testing strawberry-pathogen interactions. Thirteen potential pre-selected strawberry reference genes were tested against different tissues, strawberry cultivars, biotic stresses, ripening and senescent conditions, and SA/JA treatments. Evaluation of reference candidate’s suitability was analyzed by five different methodologies, and information was merged to identify best reference transcripts. A combination of all five methods was used for selective classification of reference genes. The resulting superior reference genes, FaRIB413, FaACTIN, FaEF1? and FaGAPDH2 are strongly recommended as control genes for relative quantification of gene expression in strawberry. This report constitutes the first systematic study to identify and validate optimal reference genes for accurate normalization of gene expression in strawberry plant defense response studies.

Amil-Ruiz, Francisco; Garrido-Gala, Jose; Blanco-Portales, Rosario; Folta, Kevin M.; Munoz-Blanco, Juan; Caballero, Jose L.

2013-01-01

149

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

150

Selection of Reliable Reference Genes for Gene Expression Studies Using Real-Time PCR in Tung Tree during Seed Development  

PubMed Central

Quantitative real-time PCR (RT-qPCR) has become an accurate and widely used technique to analyze expression levels of selected genes. It is very necessary to select appropriate reference genes for gene expression normalization. In the present study, we assessed the expression stability of 11 reference genes including eight traditional housekeeping genes and three novel genes in different tissues/organs and developing seeds from four cultivars of tung tree. All 11 reference genes showed a wide range of Ct values in all samples, indicating that they differently expressed. Three softwares – geNorm, NormFinder and BestKeeper – were used to determine the stability of these references except for ALB (2S albumin), which presented a little divergence. The results from the three softwares showed that ACT7 (Actin7a), UBQ (Ubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase) and EF1? (elongation factor 1-?) were the most stable reference genes across all of the tested tung samples and tung developing seeds, while ALB (2S albumin) was unsuitable as internal controls. ACT7, EF1? (elongation factor1-beta), GAPDH and TEF1 (transcription elongation factor 1) were the top four choices for different tissues/organs whereas LCR69 did not favor normalization of RT-qPCR in these tissues/organs. Meanwhile, the expression profiles of FAD2 and FADX were realized using stable reference genes. The relative quantification of the FAD2 and FADX genes varied according to the internal controls and the number of internal controls. The results further proved the importance of the choice of reference genes in the tung tree. These stable reference genes will be employed in normalization and quantification of transcript levels in future expression studies of tung genes.

Han, Xiaojiao; Lu, Mengzhu; Chen, Yicun; Zhan, Zhiyong; Cui, Qinqin; Wang, Yangdong

2012-01-01

151

Selection of valid reference genes for expression studies of hepatic cell lines under IFN-? treatment.  

PubMed

The proper selection of reference genes to normalize the quantitative real-time PCR (RT-qPCR) results under particular experimental conditions is crucial for validation of the gene quantification data. Herein, using SYBR green RT-qPCR, five reference genes (GAPDH, ACTB, HMBS, HPRT-1 and TBP) were evaluated to determine the most stable reference genes in hepatic cell lines (Huh-7 and HepG(2)) under IFN-? treatment conditions. Analyses by geNorm program ranked GAPDH and HPRT-1 in Huh-7 and that of ACTB and HMBS in HepG(2) cells as the most stable reference genes under IFN-? treatment. While, same reference gene pairs were ranked by NormFinder program in Huh-7 cells, GAPDH was assessed as the most stable gene in HepG(2) group by this program, implying the importance of the employed algorithm in comparative interpretation of the data. Finally, cumulative analyses by one-way ANOVA, geNorm and NormFinder programs indicated that use of two reference genes (HMBS and GAPDH) in Huh-7 and three (HMBS, ACTB and GAPDH) in HepG(2) cells would greatly improve the normalization of the RT-qPCR data under IFN-?. Data presented in this paper will aid the selection of the most stable reference genes in RT-qPCR studies on evaluation of hepatic viral proteins and IFN pathway. PMID:22982679

Hashemi, Atieh; Roohvand, Farzin; Ghahremani, Mohammad-Hossein

2012-10-01

152

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.

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

2014-01-01

153

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

154

Reference gene selection for qRT-PCR in Caragana korshinskii Kom. under different stress conditions.  

PubMed

Caragana korshinskii Kom., which is widely distributed in the northwest China and Mongolia, is an important forage bush belonging to the legume family with high economic and ecological value. Strong tolerance ability to various stresses makes C. korshinskii Kom. a valuable species for plant stress research. In this study, suitable reference genes for quantitative real-time reverse transcription PCR (qRT-PCR) were screened from 11 candidate reference genes, including ACT, GAPDH, EF1?, UBQ, TUA, CAP, TUB, TUB3, SKIP1, SKIP5-1 and SKIP5-2. A total of 129 samples under drought, heat, cold, salt, ABA and high pH treatment were profiled, and software such as geNORM, NormFinder and BestKeeper were used for reference gene evaluation and selection. Different suitable reference genes were selected under different stresses. Across all 129 samples, GAPDH, EF1? and SKIP5-1 were found to be the most stable reference genes, and EF1?+SKIP5-1 is the most stable reference gene combination. Conversely, TUA, TUB and SKIP1 were not suitable for using as reference genes owing to their great expression variation under some stress conditions. The relative expression levels of CkWRKY1 were detected using the stable and unstable reference genes and their applicability was confirmed. These results provide some stable reference genes and reference gene combinations for qRT-PCR under different stresses in C. korshinskii Kom. for future research work, and indicate that CkWRKY1 plays essential roles in response to stresses in C. korshinskii. PMID:24452712

Yang, Qi; Yin, Jiajia; Li, Gao; Qi, Liwang; Yang, Feiyun; Wang, Ruigang; Li, Guojing

2014-04-01

155

Selection of Reference Genes for Quantitative Gene Expression Studies in Platycladus orientalis (Cupressaceae) Using Real-Time PCR  

Microsoft Academic Search

Platycladus orientalis is a tree species that is highly resistant, widely adaptable, and long-lived, with lifespans of even thousands of years. To explore the mechanisms underlying these characteristics, gene expressions have been investigated at the transcriptome level by RNA-seq combined with a digital gene expression (DGE) technique. So, it is crucial to have a reliable set of reference genes to

Ermei Chang; Shengqing Shi; Jianfeng Liu; Tielong Cheng; Liang Xue; Xiuyan Yang; Wenjuan Yang; Qian Lan; Zeping Jiang

2012-01-01

156

Comprehensive Selection of Reference Genes for Gene Expression Normalization in Sugarcane by Real Time Quantitative RT-PCR  

PubMed Central

The increasingly used real time quantitative reverse transcription-PCR (qRT-PCR) method for gene expression analysis requires one or several reference gene(s) acting as normalization factor(s). In order to facilitate gene expression studies in sugarcane (Saccharum officinarum), a non-model plant with limited genome information, the stability of 13 candidate reference genes was evaluated. The geNorm, NormFinder and deltaCt methods were used for selecting stably expressed internal controls across different tissues and under various experimental treatments. These results revealed that, among these 13 candidate reference genes, GAPDH, eEF-1a and eIF-4? were the most stable and suitable for use as normalization factors across all various experimental samples. In addition, APRT could be a candidate for examining the relationship between gene copy number and transcript levels in sugarcane tissue samples. According to the results evaluated by geNorm, combining CUL and eEF-1? in hormone treatment experiments; CAC and CUL in abiotic stress tests; GAPDH, eEF-1a and CUL in all treatment samples plus CAC, CUL, APRT and TIPS-41 in cultivar tissues as groups for normalization would lead to more accurate and reliable expression quantification in sugarcane. This is the first systematic validation of reference genes for quantification of transcript expression profiles in sugarcane. This study should provide useful information for selecting reference genes for more accurate quantification of gene expression in sugarcane and other plant species.

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

2014-01-01

157

Technical note: Validation of candidate reference genes for normalization of quantitative PCR in bovine mammary epithelial cells responding to inflammatory stimuli  

Microsoft Academic Search

Mammary epithelial cells (MEC) participate in the first line of defense of the mammary gland to invading pathogens. In vitro culture of MEC is widely used as a model to study the capacity of these cells to sense and respond to mastitis-causing bacteria. Analysis of gene expression by quantitative PCR (qPCR) following exposure to bacteria or bacterial constituents is a

S. Bougarn; P. Cunha; F. B. Gilbert; F. Meurens; P. Rainard

2011-01-01

158

Stability of expression of reference genes in porcine peripheral blood mononuclear and dendritic cells  

Microsoft Academic Search

Real-time quantitative PCR (RT-qPCR) is a critical tool used to evaluate changes in gene expression. The precision of this tool is reliant upon the selection of reference genes whose expression remains unaltered in culture conditions and following stimulation. Stably expressed reference genes are used to normalize data so observed changes in expression are not due to artifacts but rather reflect

Marina R. Facci; Gael Auray; Francois Meurens; Rachelle Buchanan; Jill van Kessel; Volker Gerdts

2011-01-01

159

Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon  

Microsoft Academic Search

BACKGROUND: Salmonid fishes are among the most widely studied model fish species but reports on systematic evaluation of reference genes in qRT-PCR studies is lacking. RESULTS: The stability of six potential reference genes was examined in eight tissues of Atlantic salmon (Salmo salar), to determine the most suitable genes to be used in quantitative real-time RT-PCR analyses. The relative transcription

Pål A Olsvik; Kai K Lie; Ann-Elise O Jordal; Tom O Nilsen; Ivar Hordvik

2005-01-01

160

Validation of reference genes for gene expression studies in peanut by quantitative real-time RT-PCR.  

PubMed

Quantitative real-time reverse transcription PCR (qRT-PCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. Only a few studies on the reference genes have been done with peanut to date. In the present study, 14 potential reference genes in peanut were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms. Expression stability was assessed by qRT-PCR across 32 biological samples, including various tissue types, seed developmental stages, salt and cold treatments. The results showed that the best-ranked references genes differed across the samples. UKN1, UKN2, TUA5 and ACT11 were the most stable across all the tested samples. A combination of ACT11, TUA5, UKN2, PEPKR1 and TIP41 would be appropriate as a reference panel for normalizing gene expression data across the various tissues tested, whereas the combination of TUA5 and UKN1 was the most suitable for seed developmental stages. TUA5 and EF1b exhibited the most stable expression under cold treatment. For salt-treated leaves, TUA5 and UKN2 were the most stably expressed and HDC and UKN1 for salt-treated roots. The relative gene expression level of peanut Cys(2)/His(2)-type zinc finger protein gene AhZFP1 was analyzed in order to validate the reference genes selected for this study. These results provide guidelines for the selection of reference genes under different experimental conditions and also a foundation for more accurate and widespread use of qRT-PCR in peanut gene analysis. PMID:22203160

Chi, Xiaoyuan; Hu, Ruibo; Yang, Qingli; Zhang, Xiaowen; Pan, Lijuan; Chen, Na; Chen, Mingna; Yang, Zhen; Wang, Tong; He, Yanan; Yu, Shanlin

2012-02-01

161

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

162

Identification of Internal Reference Genes for Gene Expression Normalization between the Two Sexes in Dioecious White Campion  

PubMed Central

Quantitative real time (qRT)-PCR is a precise and efficient method for studying gene expression changes between two states of interest, and is frequently used for validating interesting gene expression patterns in candidate genes initially identified in genome-wide expression analyses, such as RNA-seq experiments. For an adequate normalisation of qRT-PCR data, it is essential to have reference genes available whose expression intensities are constant among the different states of interest. In this study we present and validate a catalogue of traditional and newly identified reference genes that were selected from RNA-seq data from multiple individuals from the dioecious plant Silene latifolia with the aim of studying gene expression differences between the two sexes in both reproductive and vegetative tissues. The catalogue contains more than 15 reference genes with both stable expression intensities and a range of expression intensities in flower buds and leaf tissues. These reference genes were used to normalize expression differences between reproductive and vegetative tissues in eight candidate genes with sex-biased expression. Our results suggest a trend towards a reduced sex-bias in sex-linked gene expression in vegetative tissues. In this study, we report on the systematic identification and validation of internal reference genes for adequate normalization of qRT-PCR-based analyses of gene expression differences between the two sexes in S. latifolia. We also show how RNA-seq data can be used efficiently to identify suitable reference genes in a wide diversity of species.

Zemp, Niklaus; Minder, Aria; Widmer, Alex

2014-01-01

163

Identification of reference genes for quantitative real-time PCR in the bovine mammary gland during the lactation cycle.  

PubMed

Achieving greater understanding of the genomic influence on milk synthesis in dairy cows represents a daunting challenge. Bovine-specific microarrays have allowed for high-throughput gene expression analysis of the mammary transcriptome. However, real-time PCR (qPCR) still represents the method of choice for accurate expression profiling of small numbers of genes and verification of key microarray relationships. This method is extremely sensitive but requires data normalization to account for analytical errors. Ideally, expression of genes used as internal controls should not be affected by specific treatments or physiological state. Mammary biopsies were collected from five cows each at -15, 1, 15, 30, 60, 120, and 240 days relative to parturition for gene expression profiling. We evaluated expression of nine genes (RPS9, ACTB, GAPD, GTP, ITGB4BP, MRPL39, RPS23, RPS15, and UXT) that could serve as internal controls in mammary tissue using qPCR. Due to gradual increases in mammary RNA concentration (mug/mg tissue) over lactation, all genes investigated experienced a dilution effect. We used pairwise comparison of expression ratios to analyze the reliability of these genes as internal controls. UXT, RPS9, and RPS15 had the most stable expression ratios across cow and time. We also assessed co-regulation among genes through network analysis. Network analysis suggested co-regulation among most of the genes examined, with MYC playing a central role. Pairwise comparison was suitable for finding appropriate internal controls in mammary gland tissue. Results showed that the geometrical average of UXT, RPS9, and RPS15 expression could be used as internal control for longitudinal mammary gene expression profiling. PMID:17284669

Bionaz, Massimo; Loor, Juan J

2007-05-11

164

Evaluation of putative reference genes for gene expression normalization in soybean by quantitative real-time RT-PCR  

PubMed Central

Background Real-time quantitative reverse transcription PCR (RT-qPCR) data needs to be normalized for its proper interpretation. Housekeeping genes are routinely employed for this purpose, but their expression level cannot be assumed to remain constant under all possible experimental conditions. Thus, a systematic validation of reference genes is required to ensure proper normalization. For soybean, only a small number of validated reference genes are available to date. Results A systematic comparison of 14 potential reference genes for soybean is presented. These included seven commonly used (ACT2, ACT11, TUB4, TUA5, CYP, UBQ10, EF1b) and seven new candidates (SKIP16, MTP, PEPKR1, HDC, TIP41, UKN1, UKN2). Expression stability was examined by RT-qPCR across 116 biological samples, representing tissues at various developmental stages, varied photoperiodic treatments, and a range of soybean cultivars. Expression of all 14 genes was variable to some extent, but that of SKIP16, UKN1 and UKN2 was overall the most stable. A combination of ACT11, UKN1 and UKN2 would be appropriate as a reference panel for normalizing gene expression data among different tissues, whereas the combination SKIP16, UKN1 and MTP was most suitable for developmental stages. ACT11, TUA5 and TIP41 were the most stably expressed when the photoperiod was altered, and TIP41, UKN1 and UKN2 when the light quality was changed. For six different cultivars in long day (LD) and short day (SD), their expression stability did not vary significantly with ACT11, UKN2 and TUB4 being the most stable genes. The relative gene expression level of GmFTL3, an ortholog of Arabidopsis FT (FLOWERING LOCUS T) was detected to validate the reference genes selected in this study. Conclusion None of the candidate reference genes was uniformly expressed across all experimental conditions, and the most suitable reference genes are conditional-, tissue-specific-, developmental-, and cultivar-dependent. Most of the new reference genes performed better than the conventional housekeeping genes. These results should guide the selection of reference genes for gene expression studies in soybean.

Hu, Ruibo; Fan, Chengming; Li, Hongyu; Zhang, Qingzhu; Fu, Yong-Fu

2009-01-01

165

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

166

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.

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

167

Identification of suitable reference genes in bone marrow stromal cells from osteoarthritic donors.  

PubMed

Bone marrow stromal cells (BMSCs) are key cellular components for musculoskeletal tissue engineering strategies. Furthermore, recent data suggest that BMSCs are involved in the development of Osteoarthritis (OA) being a frequently occurring degenerative joint disease. Reliable reference genes for the molecular evaluation of BMSCs derived from donors exhibiting OA as a primary co-morbidity have not been reported on yet. Hence, the aim of the study was to identify reference genes suitable for comparative gene expression analyses using OA-BMSCs. Passage 1 bone marrow derived BMSCs were isolated from n=13 patients with advanced stage idiopathic hip osteoarthritis and n=15 age-matched healthy donors. The expression of 31 putative reference genes was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) using a commercially available TaqMan(®) assay. Calculating the coefficient of variation (CV), mRNA expression stability was determined and afterwards validated using geNorm and NormFinder algorithms. Importin 8 (IPO8), TATA box binding protein (TBP), and cancer susceptibility candidate 3 (CASC3) were identified as the most stable reference genes. Notably, commonly used reference genes, e.g. beta-actin (ACTB) and beta-2-microglobulin (B2M) were among the most unstable genes. For normalization of gene expression data of OA-BMSCs the combined use of IPO8, TBP, and CASC3 gene is recommended. PMID:24080205

Schildberg, Theresa; Rauh, Juliane; Bretschneider, Henriette; Stiehler, Maik

2013-11-01

168

Selection of Reference Genes for Quantitative Real-Time PCR in Bamboo (Phyllostachys edulis)  

PubMed Central

Background The Moso bamboo (Phyllostachys edulis) is one of the most important forestry resources and plays essential ecological roles in southern China. A draft nuclear genome sequence is expected to be publicly available in the near future; an explosion of gene expression data related to the unique traits of Moso bamboo will undoubtedly follow. Reverse transcription quantitative real-time PCR ((RT-)qPCR) is a widely used method for gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. Result In this study, 14 candidate reference genes were chosen, and their expression levels were assessed by (RT-)qPCR in a set of six tissue samples (root, stem, mature stem, leaf, flower, and leaf sheath) and at two developmental stages (before and after flowering) in bamboo specimens obtained in three locations. The stability and suitability of the candidate reference genes were validated using the geNorm, NormFinder and BestKeeper programs. The results showed that TIP41 and NTB were suitable reference genes across all the tissues and at the different developmental stages examined in this study. While the expression of the NTB, TIP41 and UBQ were the mostly stable in different plant tissues samples, the expression of the TIP41, NTB and CAC were ranked the most stable in bamboo plants at various developmental stages. AP2-like gene was further assessed by using the reference genes TIP41 and NTB in comparison to ACT. Significant difference of the expression profile of AP2-like demonstrated the importance of choosing adequate reference genes in bamboo. Conclusion TIP41 and NTB were found to be homogeneously expressed and were adequate for normalization purposes, showing equivalent transcript levels in different samples. They are therefore the recommended reference genes for measuring gene expression in P. edulis.

Fan, Chunjie; Ma, Jinmin; Guo, Qirong; Li, Xiaotie; Wang, Hui; Lu, Mengzhu

2013-01-01

169

Microarray analysis of relative gene expression stability for selection of internal reference genes in the rhesus macaque brain  

PubMed Central

Background Normalization of gene expression data refers to the comparison of expression values using reference standards that are consistent across all conditions of an experiment. In PCR studies, genes designated as "housekeeping genes" have been used as internal reference genes under the assumption that their expression is stable and independent of experimental conditions. However, verification of this assumption is rarely performed. Here we assess the use of gene microarray analysis to facilitate selection of internal reference sequences with higher expression stability across experimental conditions than can be expected using traditional selection methods. We recently demonstrated that relative gene expression from qRT-PCR data normalized using GAPDH, ALG9 and RPL13A expression values mirrored relative expression using quantile normalization in Robust Multichip Analysis (RMA) on the Affymetrix® GeneChip® rhesus Macaque Genome Array. Having shown that qRT-PCR and Affymetrix® GeneChip® data from the same hormone replacement therapy (HRT) study yielded concordant results, we used quantile-normalized gene microarray data to identify the most stably expressed among probe sets for prospective internal reference genes across three brain regions from the HRT study and an additional study of normally menstruating rhesus macaques (cycle study). Gene selection was limited to 575 previously published human "housekeeping" genes. Twelve animals were used per study, and three brain regions were analyzed from each animal. Gene expression stabilities were determined using geNorm, NormFinder and BestKeeper software packages. Results Sequences co-annotated for ribosomal protein S27a (RPS27A), and ubiquitin were among the most stably expressed under all conditions and selection criteria used for both studies. Higher annotation quality on the human GeneChip® facilitated more targeted analysis than could be accomplished using the rhesus GeneChip®. In the cycle study, multiple probe sets annotated for actin, gamma 1 (ACTG1) showed high signal intensity and were among the most stably expressed. Conclusions Using gene microarray analysis, we identified genes showing high expression stability under various sex-steroid environments in different regions of the rhesus macaque brain. Use of quantile-normalized microarray gene expression values represents an improvement over traditional methods of selecting internal reference genes for PCR analysis.

2010-01-01

170

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

171

Analysis of fungal gene expression by Real Time quantitative PCR.  

PubMed

The Real-Time quantitative PCR (qPCR) method has become central for the quantification of gene expression as well as other applications. The major advantages of qPCR are the utilization of small amount of template, high sensitivity and the ability to detect products during the reaction. After selecting qPCR among other options (northern blot, semi-quantitative PCR), one should consider several factors. The first and critical step in qPCR of fungi is the selection of an appropriate growth medium and RNA extraction method, which will avoid accumulation of inhibitors. In this chapter, we focus on detection of the accumulating product with the Syber Green dye, but other detection technologies, such as hybridization probes, might be considered as well. Accurate qPCR analysis with Syber Green depends mainly on optimal PCR reaction, and therefore it is important to design primers that will avoid formation of interfering structures. It is possible to use absolute quantification of the template in the sample, or to conduct a relative analysis, as described in this protocol. In the relative analysis method, expression of the gene of interest is compared with expression of a reference gene. According to our experience as well as according to the literature, it is recommended to use at least three reference genes in order to obtain reliable results. PMID:20238263

Ish-Shalom, Shahar; Lichter, Amnon

2010-01-01

172

Evaluation of reference genes for human chondrocytes cultured in several different thermal environments.  

PubMed

Abstract Purpose: The purpose of this study was to identify reference genes showing stable expression in chondrocytes cultured under several different thermal environments and in different culture systems. Materials and methods: Human articular chondrocytes were cultured by monolayer or pellet culture system at 32?°C, 37?°C, and 41?°C for 3 days. Thereafter, the total RNA was extracted, and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) was performed. The qRT-PCR data was analysed using three different algorithms (geNorm, NormFinder, and BestKeeper) to identify reference genes exhibiting stable expression from among the seven candidate reference genes (B2M, ACTB, GAPDH, HSPCB, RPL13a, YWHAZ, and 18S). Results: The candidate reference genes, except for HSPCB and YWHAZ, showed systematic variations in expression. In the monolayer culture, RPL13a was the most stable gene identified using NormFinder and BestKeeper; on using geNorm, ACTB and GAPDH showed the highest expression stability. In the pellet culture, ACTB was the most stable gene identified using NormFinder and BestKeeper, whereas GAPDH and RPL13a were the most stable reference genes as determined using geNorm. In the combined group, B2M and GAPDH were the most stable genes identified using geNorm, whereas RPL13a and YWHAZ were the most stable as per NormFinder and BestKeeper, respectively. The best combination of two candidate reference genes among all the groups determined using NormFinder was RPL13a and YWHAZ. Conclusion: The combination of RPL13a and YWHAZ might be suitable as reference genes for human chondrocytes cultured at 32?°C, 37?°C, and 41?°C in monolayer, pellet, or combined cultures. PMID:24773042

Ito, Akira; Aoyama, Tomoki; Tajino, Junichi; Nagai, Momoko; Yamaguchi, Shoki; Iijima, Hirotaka; Zhang, Xiangkai; Akiyama, Haruhiko; Kuroki, Hiroshi

2014-05-01

173

Establishing references for gene expression analyses by RT-qPCR in Theobroma cacao tissues.  

PubMed

Lack of continuous progress in Theobroma cacao (Malvaceae) breeding, especially associated with seed quality traits, requires more efficient selection methods based on genomic information. Reverse transcript quantitative PCR (RT-qPCR) has become the method of choice for gene expression analysis, but relative expression analysis requires various reference genes, which must be stable across various biological conditions. We sought suitable reference genes for various tissues of cacao, especially developing seeds. Ten potential reference genes were analyzed for stability at various stages of embryo development, leaves, stems, roots, flowers, and pod epicarp; seven of them were also evaluated in shoot tips treated either with hormones (salicylate; ethefon; methyl-jasmonate) or after inoculation with the fungus Moniliophthora perniciosa (Marasmiaceae sensu lato). For developing embryos, the three most stable genes were actin (ACT), polyubiquitin (PUB), and ribosomal protein L35 (Rpl35). In the analyses of various tissues, the most stable genes were malate dehydrogenase (MDH), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and acyl-carrier protein B (ACP B). GAPDH, MDH and tubulin (TUB) were the most appropriate for normalization when shoot apexes were treated with hormones, while ACT, TUB and Rpl35 were the most appropriate after inoculation with M. perniciosa. We conclude that for each plant system and biological or ontogenetical condition, there is a need to define suitable reference genes. This is the first report to define reference genes for expression studies in cacao. PMID:22095481

Pinheiro, T T; Litholdo, C G; Sereno, M L; Leal, G A; Albuquerque, P S B; Figueira, A

2011-01-01

174

Evaluation of suitable reference genes for gene expression studies in bronchoalveolar lavage cells from horses with inflammatory airway disease  

Microsoft Academic Search

BACKGROUND: The stability of reference genes has a tremendous effect on the results of relative quantification of genes expression by quantitative polymerase chain reaction. Equine Inflammatory Airway Disease (IAD) is a common condition often treated with corticosteroids. The diagnosis of IAD is based on clinical signs and bronchoalveolar lavage (BAL) fluid cytology. The aim of this study was to identify

Laura Beekman; Triin Tohver; Rkia Dardari; Renaud Léguillette

2011-01-01

175

GAPDH, 18S rRNA and YWHAZ are Suitable Endogenous Reference Genes for Relative Gene Expression Studies in Placental Tissues from Human Idiopathic Fetal Growth Restriction  

Microsoft Academic Search

Comparative gene expression studies in the placenta may provide insights into molecular mechanisms of important genomic alterations in pregnancy disorders. Endogenous reference genes often referred to as housekeeping genes, are routinely used to normalise gene expression levels. For this reason, it is important that these genes be empirically evaluated for stability between placental samples including samples from complicated pregnancies. To

P. Murthi; E. Fitzpatrick; A. J. Borg; S. Donath; S. P. Brennecke; B. Kalionis

2008-01-01

176

An efficient RNA isolation procedure and identification of reference genes for normalization of gene expression in blueberry.  

PubMed

Application of transcriptomics approaches can greatly enhance our understanding of blueberry physiology. The success of transcriptomics approaches is dependent on the extraction of high-quality RNA which is complicated by the abundance of polyphenolics and polysaccharides in blueberry. Additionally, transcriptomics requires the accurate quantification of transcript abundance. Quantitative real-time polymerase chain reaction (qRT-PCR) is a robust method to determine transcript abundance. Normalization of gene expression using stably expressed reference genes is essential in qRT-PCR. An evaluation of the stability of expression of reference genes has not yet been reported in blueberry. The objectives of this study were to develop an effective procedure for extracting RNA from different organs and to evaluate potential reference genes for qRT-PCR analyses in blueberry. RNA of high quality and yield was extracted from eight and six organs of rabbiteye and southern highbush blueberry, respectively, using a modified cetyltrimethyl ammonium bromide-based method. The expression stability of 12 reference genes was evaluated. UBIQUITIN-CONJUGATING ENZYME (UBC28), RNA HELICASE-LIKE (RH8), CLATHRIN ADAPTER COMPLEXES MEDIUM SUBUNIT FAMILY PROTEIN (CACSa), and POLYUBIQUITIN (UBQ3b) were the most stably expressed genes across multiple organs in both blueberry species. Further, the expression stability of the reference genes in the branch abscission zone following treatment with fruit abscission-inducing compounds was analyzed. CACSa, RH8, and UBC28 were the most stably expressed genes in the abscission zone under abscission-inducing conditions. We suggest a preliminary evaluation of UBC28, CACSa, RH8, and UBQ3b to identify the most suitable reference genes for the experimental conditions under consideration in blueberry. PMID:21761237

Vashisth, Tripti; Johnson, Lisa Klima; Malladi, Anish

2011-12-01

177

Selection of optimal reference genes for quantitative RT-PCR studies of boar spermatozoa cryopreservation.  

PubMed

Reference genes can be used to normalize mRNA levels across different samples for the exact comparison of the mRNA expression level. It is important to select reference genes with high quality for the accurate interpretation of qRT-PCR data. Although several studies have attempted to validate reference genes in pigs, no validation studies have been performed on spermatozoa samples frozen with different cryoprotectants. In this study, 11 commonly used reference genes (ACTB, B2M, GAPDH, HPRT1, RPL4, SDHA, YWHAZ, PPIA, PGK1, S18, and BLM) were investigated in boar spermatozoa frozen with six different cryoprotectants using qRT-PCR. The expression stability of these reference genes in different samples was evaluated using geNorm (qbase(plus) software), NormFinder, and BestKeeper. The geNorm results revealed that PGK1, ACTB, and RPL4 exhibit high expression stability in all of the samples, and the NormFinder results indicated that GAPDH is the most stable gene. Furthermore, the BestKeeper results indicated that the three most stable genes are PPIA, GAPDH, and RPL4 and that S18, B2M and BLM are the three least stable genes. There are a number of differences in the ranking order of the reference genes obtained using the different algorithms. In conclusion, GAPDH, RPL4, and PPIA were the three most stable genes in frozen boar spermatozoa, as determined based on the cycle threshold coefficient of variation (Ct CV%) and the comprehensive ranking order, and this finding is consistent with the BestKeeper results. PMID:24440873

Zeng, Changjun; He, Lian; Peng, Wenpei; Ding, Li; Tang, Keyi; Fang, Donghui; Zhang, Yan

2014-02-01

178

Detection of Streptococcus equi subspecies equi using a triplex qPCR assay  

PubMed Central

Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2 h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria.

Webb, Katy; Barker, Colin; Harrison, Tihana; Heather, Zoe; Steward, Karen F.; Robinson, Carl; Newton, J. Richard; Waller, Andrew S.

2013-01-01

179

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

180

Verification of reference genes for relative quantification of gene expression by real-time reverse transcription PCR in the pig  

Microsoft Academic Search

The aim of this study was to develop a set of reliable reference genes for quantification of mRNA expression in the pig. The\\u000a mRNA expression stability in pig tissues was studied for 4 genes:EEF1A1, GAPDH, HPRT1 andTOP2B. The level of expression was characterized byCt values for each gene and each tissue. By using the geNorm algorithm, the stability of the

Kate?ina Svobodová; Karel Bílek; Aleš Knoll

2008-01-01

181

Evaluation of endogenous reference genes for analysis of gene expression with real-time RT-PCR during planarian regeneration  

Microsoft Academic Search

It is important that endogenous reference genes for real-time RT-PCR be empirically evaluated for stability in different cell\\u000a types, developmental stages, and\\/or sample treatment. To select the most stable endogenous reference genes during planarian\\u000a regeneration, three housekeeping genes, 18S rRNA, ACTB and DjEF2, were identified and established expression levels by real-time RT-PCR. The data were analyzed by GeNorm and NormFinder

Yan-qing YuwenZi-mei; Zi-mei Dong; Qing-hua Wang; Xiao-juan Sun; Chang-ying Shi; Guang-wen Chen

182

Leukocyte count affects expression of reference genes in canine whole blood samples  

Microsoft Academic Search

BACKGROUND: The dog is frequently used as a model for hematologic human diseases. In this study the suitability of nine potential reference genes for quantitative RT-PCR studies in canine whole blood was investigated. FINDINGS: The expression of these genes was measured in whole blood samples of 263 individual dogs, representing 73 different breeds and a group of 40 mixed breed

Christine J Piek; Bas Brinkhof; Jan Rothuizen; Aldo Dekker; Louis C Penning

2011-01-01

183

Reference Gene Selection for Quantitative Real-time PCR Normalization in Quercus suber  

PubMed Central

The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1?, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks.

Marum, Liliana; Miguel, Andreia; Ricardo, Candido P.; Miguel, Celia

2012-01-01

184

Reference gene selection for quantitative real-time PCR normalization in Quercus suber.  

PubMed

The use of reverse transcription quantitative PCR technology to assess gene expression levels requires an accurate normalization of data in order to avoid misinterpretation of experimental results and erroneous analyses. Despite being the focus of several transcriptomics projects, oaks, and particularly cork oak (Quercus suber), have not been investigated regarding the identification of reference genes suitable for the normalization of real-time quantitative PCR data. In this study, ten candidate reference genes (Act, CACs, EF-1?, GAPDH, His3, PsaH, Sand, PP2A, ß-Tub and Ubq) were evaluated to determine the most stable internal reference for quantitative PCR normalization in cork oak. The transcript abundance of these genes was analysed in several tissues of cork oak, including leaves, reproduction cork, and periderm from branches at different developmental stages (1-, 2-, and 3-year old) or collected in different dates (active growth period versus dormancy). The three statistical methods (geNorm, NormFinder, and CV method) used in the evaluation of the most suitable combination of reference genes identified Act and CACs as the most stable candidates when all the samples were analysed together, while ß-Tub and PsaH showed the lowest expression stability. However, when different tissues, developmental stages, and collection dates were analysed separately, the reference genes exhibited some variation in their expression levels. In this study, and for the first time, we have identified and validated reference genes in cork oak that can be used for quantification of target gene expression in different tissues and experimental conditions and will be useful as a starting point for gene expression studies in other oaks. PMID:22529976

Marum, Liliana; Miguel, Andreia; Ricardo, Cândido P; Miguel, Célia

2012-01-01

185

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.

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

2013-01-01

186

Identification and validation of reference genes for gene expression analysis using quantitative PCR in Spodoptera litura (Lepidoptera: Noctuidae).  

PubMed

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

Lu, Yanhui; Yuan, Miao; Gao, Xiwu; Kang, Tinghao; Zhan, Sha; Wan, Hu; Li, Jianhong

2013-01-01

187

Evaluation and identification of reliable reference genes for toxicological study in Caenorhabditis elegans.  

PubMed

To identify reliable reference genes for toxicological studies, 16 commonly-used reference genes were selected as candidates to evaluate their expression stabilities under experimental conditions in Caenorhabditis elegans. Sixteen candidates were composed of 12 protein-coding genes and 4 non-coding RNAs, they were act-2, ama-1, arp-6, cdc-42, csq-1, eif-3.C, idhg-1, mdh, pmp-3, rbd-1, tba-1, Y45F10D.4, 18S rRNA, Ce234, U18, and U6. Larval stage 1 synchronized hermaphrodites were exposed to benzo-?-pyrene (B?P), chlorpyrifos, diazinon, gossypol, zinc oxide nanoparticles, and the vehicle control DMSO for 30 h, respectively. Expression stabilities of candidate genes were analyzed using 4 independent evaluating approaches (BestKeeper, the delta Ct approach, geNorm, and NormFinder) followed by a comprehensive method. Results showed that there were slight differences in ranking order between evaluation methods due to their different assumptions and computations. The results also showed that responses of candidate genes to different chemicals were distinct, 18S rRNA was the best for B?P and chlorpyrifos, tba-1 was the most stable gene for diazinon and gossypol treatments, while pmp-3 was more stable for zinc oxide exposure. Additionally, results demonstrated that combinations of multiple genes were more reliable than individual gene, suggesting selecting two or more candidates as reference genes may generate more reliable results for toxicological studies. PMID:24510408

Wu, Hongmei; Taki, Faten A; Zhang, Yanqiong; Dobbins, Dorothy L; Pan, Xiaoping

2014-05-01

188

Quantitative evaluation and selection of reference genes in mouse oocytes and embryos cultured in vivo and in vitro  

Microsoft Academic Search

BACKGROUND: Real-time PCR is an efficient tool to measure transcripts and provide valuable quantitative information on gene expression of preimplantation stage embryos. Finding valid reference genes for normalization is essential to interpret the real-time PCR results accurately, and understand the biological dynamics during early development. The use of reference genes also known as housekeeping genes is the most widely applied

Solomon Mamo; Arpad Baji Gal; Szilard Bodo; Andras Dinnyes

2007-01-01

189

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.

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

2005-01-01

190

Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria.  

PubMed

Cyanobacteria are a group of photosynthetic prokaryotes that have a diverse morphology, minimal nutritional requirements and metabolic plasticity that has made them attractive organisms to use in biotechnological applications. The use of these organisms as cell factories requires the knowledge of their physiology and metabolism at a systems level. For the quantification of gene transcripts real-time quantitative polymerase chain reaction (RT-qPCR) is the standard technique. However, to obtain reliable RT-qPCR results the use and validation of reference genes is mandatory. Towards this goal we have selected and analyzed twelve candidate reference genes from three morphologically distinct cyanobacteria grown under routinely used laboratory conditions. The six genes exhibiting less variation in each organism were evaluated in terms of their expression stability using geNorm, NormFinder and BestKeeper. In addition, the minimum number of reference genes required for normalization was determined. Based on the three algorithms, we provide a list of genes for cyanobacterial RT-qPCR data normalization. To our knowledge, this is the first work on the validation of reference genes for cyanobacteria constituting a valuable starting point for future works. PMID:22496882

Pinto, Filipe; Pacheco, Catarina C; Ferreira, Daniela; Moradas-Ferreira, Pedro; Tamagnini, Paula

2012-01-01

191

Impact of thawing on reference gene expression stability in renal cell carcinoma samples.  

PubMed

More and more samples are obtained from biobanks for biomedical research; however, some of these samples may undergo thawing before processing. We aim to evaluate the reference gene expression stability in thawed renal cell carcinoma samples. Sixteen matched malignant and nonmalignant renal tissue samples were obtained and each sample was divided into 4 aliquots before being snap frozen and stored at -80°C. By quantitative real-time polymerase chain reaction, a time-course study was conducted on the thawed tissue to evaluate the expression stability of a panel of the 10 most frequently used reference genes in renal cell carcinom samples: ACTB, ALAS1, B2M, GAPDH, HMBS, HPRT, PPIA, RPLP0,TBP, and TUBB. As shown by geNorm M values, PPIA was the most stable gene at the 0-, 15-, and 30-minute time points (M=0.82, 0.85, and 0.76, respectively), whereas GAPDH was ranked last at the 5-, 15-, and 30-minute time points (M=1.38, 1.44, and 1.39, respectively). A positive correlation was found by linear regression between the thawing time and 2 to the power of crossing point values of all candidate reference genes (P<0.05). The mean coefficient of variance of all reference genes increased significantly at time points 5, 15, and 30 minutes compared with 0 minutes (P<0.01). In conclusion, using the geNorm algorithm, PPIA was identified as the most stably expressed gene between malignant and nonmalignant renal tissue samples that were thawed for similar time periods. All the reference genes showed high variations along with the thawing time; it should be recommended to use a combination of several candidate reference genes when comparing samples thawed for different time periods. PMID:22847160

Ma, Yi; Dai, HuiLi; Kong, XianMing; Wang, LiMin

2012-09-01

192

Validation of putative reference genes for gene expression studies in human hepatocellular carcinoma using real-time quantitative RT-PCR  

Microsoft Academic Search

BACKGROUND: Reference genes, which are often referred to as housekeeping genes are frequently used to normalize mRNA levels between different samples in quantitative reverse transcription polymerase chain reaction (qRT-PCR). The selection of reference genes is critical for gene expression studies because the expression of these genes may vary among tissues or cells and may change under certain circumstances. Here, a

Vito R Cicinnati; Qingli Shen; Georgios C Sotiropoulos; Arnold Radtke; Guido Gerken; Susanne Beckebaum

2008-01-01

193

Validation of reference genes for quantitative RT-PCR studies of gene expression in perennial ryegrass (Lolium perenne L.)  

PubMed Central

Background Perennial ryegrass (Lolium perenne L.) is an important pasture and turf crop. Biotechniques such as gene expression studies are being employed to improve traits in this temperate grass. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is among the best methods available for determining changes in gene expression. Before analysis of target gene expression, it is essential to select an appropriate normalisation strategy to control for non-specific variation between samples. Reference genes that have stable expression at different biological and physiological states can be effectively used for normalisation; however, their expression stability must be validated before use. Results Existing Serial Analysis of Gene Expression data were queried to identify six moderately expressed genes that had relatively stable gene expression throughout the year. These six candidate reference genes (eukaryotic elongation factor 1 alpha, eEF1A; TAT-binding protein homolog 1, TBP-1; eukaryotic translation initiation factor 4 alpha, eIF4A; YT521-B-like protein family protein, YT521-B; histone 3, H3; ubiquitin-conjugating enzyme, E2) were validated for qRT-PCR normalisation in 442 diverse perennial ryegrass (Lolium perenne L.) samples sourced from field- and laboratory-grown plants under a wide range of experimental conditions. Eukaryotic EF1A is encoded by members of a multigene family exhibiting differential expression and necessitated the expression analysis of different eEF1A encoding genes; a highly expressed eEF1A (h), a moderately, but stably expressed eEF1A (s), and combined expression of multigene eEF1A (m). NormFinder identified eEF1A (s) and YT521-B as the best combination of two genes for normalisation of gene expression data in perennial ryegrass following different defoliation management in the field. Conclusions This study is unique in the magnitude of samples tested with the inclusion of numerous field-grown samples, helping pave the way to conduct gene expression studies in perennial biomass crops under field-conditions. From our study several stably expressed reference genes have been validated. This provides useful candidates for reference gene selection in perennial ryegrass under conditions other than those tested here.

2010-01-01

194

Reference gene selection for gene expression studies using RT-qPCR in virus-infected planthoppers  

PubMed Central

Background Planthoppers not only severely affect crops by causing mechanical damage when feeding but are also vectors of several plant virus species. The analysis of gene expression in persistently infected planthoppers might unveil the molecular basis of viral transmission. Quantitative real-time RT-PCR (RT-qPCR) is currently the most accurate and sensitive method used for quantitative gene expression analysis. In order to normalize the resulting quantitative data, reference genes with constant expression during the experimental procedures are needed. Results Partial sequences of the commonly used reference genes actin (ACT), ?1-tubulin (TUB), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), elongation factor 1 alpha (EF1A), ribosomal protein S18 (RPS18) and polyubiquitin C (UBI) from Delphacodes kuscheli, a planthopper capable of persistently transmitting the plant fijivirus Mal de Río Cuarto virus (MRCV), were isolated for the first time. Specific RT-qPCR primers were designed and the expression stability of these genes was assayed in MRCV-infective and naïve planthoppers using geNorm, Normfinder and BestKeeper tools. The overall analysis showed that UBI, followed by 18S and ACT, are the most suitable genes as internal controls for quantitative gene expression studies in MRCV-infective planthoppers, while TUB and EF1A are the most variable ones. Moreover, EF1A was upregulated by MRCV infection. Conclusions A RT-qPCR platform for gene expression analysis in the MRCV-infected planthopper vector Delphacodes kuscheli was developed. Our work is the first report on reference gene selection in virus-infected insects, and might serve as a precedent for future gene expression studies on MRCV and other virus-planthopper pathosystems.

2011-01-01

195

Evaluation of reference genes for studies of gene expression in the bovine liver, kidney, pituitary, and thyroid  

Microsoft Academic Search

Expression patterns of candidate genes with important functions in animal metabolism can help to identify potential molecular\\u000a markers for cattle production traits. Reverse transcription followed by polymerase chain reaction is a method for rapid and\\u000a accurate mRNA quantification. However, for exact comparison of mRNA quantity in various samples or tissues, it is important\\u000a to choose appropriate reference genes. In cattle,

Pawe? Lisowski; Mariusz Pierzcha?a; Joanna Go?cik; Chandra S. Pareek; Lech Zwierzchowski

2008-01-01

196

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

197

Selection of reference genes for gene expression studies in PBMC from Bama miniature pig under heat stress.  

PubMed

Heat stress decreases immune function and increases disease susceptibility in stressed animals, which are important factors for industry and public health. We investigated the molecular mechanisms underlying heat stress by profiling the expression of target genes involved in the cellular response in the blood of Bama miniature pigs (Sus scrofa domestica) over 21 days with the use of quantitative RT-PCR (qRT-PCR). Reliable standards were established for the normalization of qRT-PCR. Six potential reference genes were ranked by their stability using the geNorm and NormFinder programs. Ribosomal protein L4 (RPL4) and TATA-box-binding protein (TBP) ranked as the two most stably expressed genes, except on day 21 when beta-2-microglobulin (B2M) was the most stable. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ribosomal RNA 18S (18SRNA) were discarded as reference genes due to their unstable expression patterns. When B2M and TBP genes were selected as standards in combination, rather than GAPDH, a significant upregulation in Toll-like receptor 2 (TLR2) expression was observed after 21 consecutive days of heat stress. These findings suggest that selection of an ideal reference gene is a key step in comparison of transcription profiles in Bama miniature pigs. PMID:21820186

Xiang-Hong, Ju; Yan-Hong, Yong; Han-Jin, Xu; Li-Long, An; Ying-Mei, Xu; Pei-Rong, Jiao; Ming, Liao

2011-11-15

198

An alternative and sensitive method based on LCM and Q-PCR for HER2 testing in breast cancer.  

PubMed

Nowadays, HER2 testing in breast cancer represents a necessity for both prognostic and therapy. Despite widespread use of immunohistochemistry (IHC) for assessing HER2 status, there are some limitations to identify truly negative or positive HER2 cases. Fluorescence in situ hybridization (FISH) or chromogenic in situ hybridization (CISH) could solve the equivocal HER2 IHC cases but there is no consensus on which is the best method. Consequently, finding a sensitive method for HER2 testing is critical for the management of the disease. In addition, tumor heterogeneity is an important factor which could affect accuracy of molecular diagnostics. Laser capture micro-dissection (LCM) is used to isolate pure cell populations from heterogeneous tumor tissue. The combination between LCM and quantitative polymerase chain reaction (Q-PCR), the gold standard in molecular biology for quantifying gene amplification levels, could define an important tool to improve the molecular diagnostics of HER2 status.In our pilot study we used LCM and Q-PCR to evaluate HER2 gene amplification for invasive breast carcinoma samples. The samples were selected based on HER2 status assessed by IHC and CISH. Our results demonstrated high sensitivity of Q-PCR for assessing HER2 DNA amplification as well as a good concordance between Q-PCR and IHC/ CISH assay. PMID:24878813

Fetica, Bogdan; Balacescu, Ovidiu; Balacescu, Loredana; Rus, Meda; Berindan-Neagoe, Ioana

2014-01-01

199

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes  

Microsoft Academic Search

Real time quantitative reverse transcription–polymerase chain reaction (qRT–PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes,

Attila Balogh; György Paragh; Attila Juhász; Tamás Köbling; Dániel Tör?csik; Viktória Varga; Gabriella Emri; Irén Horkay; Beáta Scholtz; Éva Remenyik

2008-01-01

200

Selection of referent transcript for normalization of gene expression in cervical cytology samples.  

PubMed

Real-time polymerase chain reaction is widely used in gene expression studies but this requires an appropriate referent gene for data normalization. So far, no gold standard is available and the selection has to be empirically validated. The aim of this study was to identify the most stable referent gene in exfoliated cervical cells with different degrees of cervical pathology. Seventy-five samples were used, 18 were from normal cervices, 18 from cervical intraepithelial neoplasia (CIN) 1, 17 from CIN 2, 17 from CIN 3, and 5 from squamous cell carcinoma. Using NormFinder, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was found to be the most stably expressed referent gene with a stability value of 0.37 across all lesion grades. Followed by RPL4 (stability value of 0.77) and ?-actin (0.77), large ribosomal protein P0 (1.01), and PKG1 (1.02). The results of expression stability by geNorm showed that normal cervices were more varied, with stability values ranging from 2.85 to 3.46, with ?-actin performing slightly better than GAPDH (M: 2.85 vs. 2.98). For the CIN 1 to 3, GAPDH was determined to be the most stably expressed gene (M: 0.94 to 1.37). The next most stable gene expressed was PKG1 (M: 1.02 to 1.44). However, the sample size for squamous cell carcinoma was too small for justification. In conclusion, overall GAPDH is the most stable referent gene expressed across all cervical lesion grades and is most suitable for normalization in gene expression studies. PMID:24517915

Cheung, Jo Lai Ken; Cheung, Tak Hong; Yu, Mei Yung; Yeung, Apple Chung Man; Chan, Paul Kay Sheung

2014-02-01

201

Guideline to reference gene selection for quantitative real-time PCR  

Microsoft Academic Search

Today, quantitative real-time PCR is the method of choice for rapid and reliable quantification of mRNA transcription. However, for an exact comparison of mRNA transcription in different samples or tissues it is crucial to choose the appropriate reference gene. Recently glyceraldehyde 3-phosphate dehydrogenase and ?-actin have been used for that purpose. However, it has been reported that these genes as

Aleksandar Radoni?; Stefanie Thulke; Ian M. Mackay; Olfert Landt; Wolfgang Siegert; Andreas Nitschea

2004-01-01

202

Allele diversity for abiotic stress responsive candidate genes in chickpea reference set using gene based SNP markers  

PubMed Central

Chickpea is an important food legume crop for the semi-arid regions, however, its productivity is adversely affected by various biotic and abiotic stresses. Identification of candidate genes associated with abiotic stress response will help breeding efforts aiming to enhance its productivity. With this objective, 10 abiotic stress responsive candidate genes were selected on the basis of prior knowledge of this complex trait. These 10 genes were subjected to allele specific sequencing across a chickpea reference set comprising 300 genotypes including 211 genotypes of chickpea mini core collection. A total of 1.3 Mbp sequence data were generated. Multiple sequence alignment (MSA) revealed 79 SNPs and 41 indels in nine genes while the CAP2 gene was found to be conserved across all the genotypes. Among 10 candidate genes, the maximum number of SNPs (34) was observed in abscisic acid stress and ripening (ASR) gene including 22 transitions, 11 transversions and one tri-allelic SNP. Nucleotide diversity varied from 0.0004 to 0.0029 while polymorphism information content (PIC) values ranged from 0.01 (AKIN gene) to 0.43 (CAP2 promoter). Haplotype analysis revealed that alleles were represented by more than two haplotype blocks, except alleles of the CAP2 and sucrose synthase (SuSy) gene, where only one haplotype was identified. These genes can be used for association analysis and if validated, may be useful for enhancing abiotic stress, including drought tolerance, through molecular breeding.

Roorkiwal, Manish; Nayak, Spurthi N.; Thudi, Mahendar; Upadhyaya, Hari D.; Brunel, Dominique; Mournet, Pierre; This, Dominique; Sharma, Prakash C.; Varshney, Rajeev K.

2014-01-01

203

Allele diversity for abiotic stress responsive candidate genes in chickpea reference set using gene based SNP markers.  

PubMed

Chickpea is an important food legume crop for the semi-arid regions, however, its productivity is adversely affected by various biotic and abiotic stresses. Identification of candidate genes associated with abiotic stress response will help breeding efforts aiming to enhance its productivity. With this objective, 10 abiotic stress responsive candidate genes were selected on the basis of prior knowledge of this complex trait. These 10 genes were subjected to allele specific sequencing across a chickpea reference set comprising 300 genotypes including 211 genotypes of chickpea mini core collection. A total of 1.3 Mbp sequence data were generated. Multiple sequence alignment (MSA) revealed 79 SNPs and 41 indels in nine genes while the CAP2 gene was found to be conserved across all the genotypes. Among 10 candidate genes, the maximum number of SNPs (34) was observed in abscisic acid stress and ripening (ASR) gene including 22 transitions, 11 transversions and one tri-allelic SNP. Nucleotide diversity varied from 0.0004 to 0.0029 while polymorphism information content (PIC) values ranged from 0.01 (AKIN gene) to 0.43 (CAP2 promoter). Haplotype analysis revealed that alleles were represented by more than two haplotype blocks, except alleles of the CAP2 and sucrose synthase (SuSy) gene, where only one haplotype was identified. These genes can be used for association analysis and if validated, may be useful for enhancing abiotic stress, including drought tolerance, through molecular breeding. PMID:24926299

Roorkiwal, Manish; Nayak, Spurthi N; Thudi, Mahendar; Upadhyaya, Hari D; Brunel, Dominique; Mournet, Pierre; This, Dominique; Sharma, Prakash C; Varshney, Rajeev K

2014-01-01

204

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

205

A universal reference sample derived from clone vector for improved detection of differential gene expression  

PubMed Central

Background Using microarrays by co-hybridizing two samples labeled with different dyes enables differential gene expression measurements and comparisons across slides while controlling for within-slide variability. Typically one dye produces weaker signal intensities than the other often causing signals to be undetectable. In addition, undetectable spots represent a large problem for two-color microarray designs and most arrays contain at least 40% undetectable spots even when labeled with reference samples such as Stratagene's Universal Reference RNAs™. Results We introduce a novel universal reference sample that produces strong signal for all spots on the array, increasing the average fraction of detectable spots to 97%. Maximizing detectable spots on the reference image channel also decreases the variability of microarray data allowing for reliable detection of smaller differential gene expression changes. The reference sample is derived from sequence contained in the parental EST clone vector pT7T3D-Pac and is called vector RNA (vRNA). We show that vRNA can also be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This reference sample can be made inexpensively in large quantities as a renewable resource that is consistent across experiments. Conclusion Results of this study show that vRNA provides a useful universal reference that yields high signal for almost all spots on a microarray, reduces variation and allows for comparisons between experiments and laboratories. Further, it can be used for quality control of microarray printing and PCR product quality, detection of hybridization anomalies, and simplification of spot finding and segmentation tasks. This type of reference allows for detection of small changes in differential expression while reference designs in general allow for large-scale multivariate experimental designs. vRNA in combination with reference designs enable systems biology microarray experiments of small physiologically relevant changes.

Khan, Rishi L; Gonye, Gregory E; Gao, Guang; Schwaber, James S

2006-01-01

206

Direct qPCR quantification of unprocessed forensic casework samples.  

PubMed

The current short tandem repeat (STR) typing workflow for forensic casework samples involves sample collection, sample screening, DNA extraction, DNA qPCR quantification and STR amplification. Although very effective and powerful, this workflow still has room for improvements. For example, the screening assays in practice do not provide DNA related information and also do not work with touch DNA samples, which make up of the majority of the property crime samples. It is known that not all DNA samples have equal probative values. Considering the DNA backlog situation crime laboratories face today, an effective screening tool would be highly desirable. It would allow forensic scientists to prioritize the DNA samples so that the limited resources would be first spent on samples that would have better chances of producing informative STR profiles. qPCR assay does provide DNA quantity and gender information and would be an ideal screening tool. However, prior to quantification, sample extraction and purification are required. By the time a DNA sample is ready for qPCR quantification, time and resources have already been spent on samples that should have been given low priority or excluded from further processing if DNA quantity and gender information were known. To overcome this problem, a direct quantification technology is developed to allow qPCR quantification of casework samples without the need for DNA extraction and purification. The key to a direct qPCR assay is the PE-Swab, a novel sample collection device. A small sample punch can be generated from a PE-Swab and placed in a qPCR reaction for quantification. After optimizing the punch size and the quantification software baseline setting, accurate DNA quantification can be obtained from a sample without the need to carry out DNA extraction and purification. Proof of concept studies were done with low lever touch samples as well as blood samples. The PE-Swab also allows direct STR amplification of casework samples without the need for DNA extraction. Besides its potential as a screening tool, the direct qPCR assay can also be used to normalize the DNA input for a direct STR amplification reaction. The feasibility of the direct qPCR/direct STR amplification workflow was demonstrated with touch DNA samples and blood stain samples. PMID:24705062

Liu, Jason Yingjie

2014-07-01

207

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.

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

2014-01-01

208

The Gene Ontology's Reference Genome Project: a unified framework for functional annotation across species.  

PubMed

The Gene Ontology (GO) is a collaborative effort that provides structured vocabularies for annotating the molecular function, biological role, and cellular location of gene products in a highly systematic way and in a species-neutral manner with the aim of unifying the representation of gene function across different organisms. Each contributing member of the GO Consortium independently associates GO terms to gene products from the organism(s) they are annotating. Here we introduce the Reference Genome project, which brings together those independent efforts into a unified framework based on the evolutionary relationships between genes in these different organisms. The Reference Genome project has two primary goals: to increase the depth and breadth of annotations for genes in each of the organisms in the project, and to create data sets and tools that enable other genome annotation efforts to infer GO annotations for homologous genes in their organisms. In addition, the project has several important incidental benefits, such as increasing annotation consistency across genome databases, and providing important improvements to the GO's logical structure and biological content. PMID:19578431

2009-07-01

209

SPUD qPCR Assay Confirms PREXCEL-Q Software's Ability to Avoid qPCR Inhibition  

PubMed Central

Real-time quantitative polymerase chain reaction is subject to inhibition by substances that co-purify with nucleic acids during isolation and preparation of samples. Such materials alter the activity of reverse transcriptase (RT) and thermostable DNA polymerase enzymes on which the assay depends. When removal of inhibitory substances by column or reagent-based methods fails or is incomplete, the remaining option of appropriately, precisely and differentially diluting samples and standards to non-inhibitory concentrations is often avoided due to the logistic problem it poses. To address this, we invented the PREXCEL-Q software program to automate the process of calculating the non-inhibitory dilutions for all samples and standards after a preliminary test plate has been performed on an experimental sample mixture. The SPUD assay was used to check for inhibition in each PREXCEL-Q-designed qPCR reaction. When SPUD amplicons or SPUD amplicon-containing plasmids were spiked equally into each qPCR reaction, all reactions demonstrated complete absence of qPCR inhibition. Reactions spiked with ~15,500 SPUD amplicons yielded a Cq of 27.39 ± 0.28 (at ~80.8% efficiency), while reactions spiked with ~7,750 SPUD plasmids yielded a Cq of 23.82 ± 0.15 (at ~97.85% efficiency). This work demonstrates that PREXCEL-Q sample and standard dilution calculations ensure avoidance of qPCR inhibition.

Gallup, J.M.; Sow, F.B.; Van Geelen, A.; Ackermann, M.R.

2009-01-01

210

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.

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

2013-01-01

211

Evaluation of suitable reference genes for gene expression studies in porcine alveolar macrophages in response to LPS and LTA  

PubMed Central

Background To obtain reliable quantitative real-time PCR data, normalization relative to stable housekeeping genes (HKGs) is required. However, in practice, expression levels of 'typical' housekeeping genes have been found to vary between tissues and under different experimental conditions. To date, validation studies of reference genes in pigs are relatively rare and have never been performed in porcine alveolar macrophages (AMs). In this study, expression stability of putative housekeeping genes were identified in the porcine AMs in response to the stimulation with two pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Three different algorithms (geNorm, Normfinder and BestKeeper) were applied to assess the stability of HKGs. Results The mRNA expression stability of nine commonly used reference genes (B2M, BLM, GAPDH, HPRT1, PPIA, RPL4, SDHA, TBP and YWHAZ) was determined by qRT-PCR in AMs that were stimulated by LPS and LTA in vitro. mRNA expression levels of all genes were found to be affected by the type of stimulation and duration of the stimulation (P < 0.0001). geNorm software revealed that SDHA, B2M and RPL4 showed a high expression stability in the irrespective to the stimulation group, while SDHA, YWHAZ and RPL4 showed high stability in non-stimulated control group. In all cases, GAPDH showed the least stability in geNorm. NormFinder revealed that SDHA was the most stable gene in all the groups. Moreover, geNorm software suggested that the geometric mean of the three most stable genes would be the suitable combination for accurate normalization of gene expression study. Conclusions There was discrepancy in the ranking order of reference genes obtained by different analysing algorithms. In conclusion, the geometric mean of the SDHA, YWHAZ and RPL4 seemed to be the most appropriate combination of HKGs for accurate normalization of gene expression data in porcine AMs without knowing the type of bacterial pathogenic status of the animals.

2012-01-01

212

Systematic validation of candidate reference genes for qRT-PCR normalization under iron deficiency in Arabidopsis.  

PubMed

A reliable result obtained by qRT-PCR highly depends on accurate transcript normalization using stably expressed reference genes. However, the transcript levels of traditional reference genes are not always stable. Also, the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated in Arabidopsis under iron deficiency. Our results revealed that three novel reference genes (SAND, YLS8 and TIP41-like) were identified and validated as suitable reference genes for qRT-PCR normalization in both iron deprivation (the addition of Ferrozine to the medium) and starvation (withdrawal of iron from the medium) conditions. This conclusion was also confirmed by publicly available microarray data. In addition, when using SAND, YLS8 and TIP41-like as multiple reference genes, the expression patterns of FIT1 and IRT1, two iron deficiency marker genes, were approximately similar with that reported previously. However, a weaker inducible response was obtained from qRT-PCR by normalizating EF-1? alone. Together, we proposed that the combination of SAND, YLS8 and TIP41-like can be used for accurate normalization of gene expression in iron deficiency research. These results provide a valuable evidence for the importance of adequate reference genes in qRT-PCR normalization, insisting on the use of appropriate reference gene validation in all transcriptional analyses. PMID:23547009

Han, Bin; Yang, Zheng; Samma, Muhammad Kaleem; Wang, Ren; Shen, Wenbiao

2013-06-01

213

Rapid QPCR-based assay for fecal Bacteroides spp. as a tool for assessing fecal contamination in recreational waters.  

PubMed

Concentrations of fecal indicator bacteria (FIB; e.g. Escherichia coli, and Enterococcus sp.) can only be used in limited ways for determining the source of fecal contamination in recreational waters because they cannot distinguish human from non-human fecal contamination. Several Bacteroides spp. have been suggested as potential alternative indicators. We have developed a rapid, culture-independent method for quantifying fecal Bacteroides spp. using quantitative PCR (QPCR) targeting the 16S rRNA gene. The assay specifically targets and quantifies the most common human Bacteroides spp. The details of the method are presented, including analyses of a wide range of fecal samples from different organisms. Specificity and performance of the QPCR assay were also tested via a laboratory experiment where human sewage and gull guano were inoculated into a range of environmental water samples. Concentrations of fecal Bacteroides spp., total Enterococcus sp., Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus were measured using QPCR, and total Enterococcus sp. and E. coli were quantified by membrane filtration (MF). Samples spiked with gull guano were highly concentrated with total Enterococcus sp., E. coli, E. faecalis, and E. casseliflavus, demonstrating that these indicators are prominent in animal feces. On the other hand, fecal Bacteroides spp. concentrations were high in samples containing sewage and were relatively low in samples spiked with gull guano. Sensitivity and specificity results suggest that the rapid fecal Bacteroides spp. QPCR assay may be a useful tool to effectively predict the presence and concentration of human-specific fecal pollution. PMID:19631958

Converse, Reagan R; Blackwood, A Denene; Kirs, Marek; Griffith, John F; Noble, Rachel T

2009-11-01

214

Sampling Daphnia's expressed genes: preservation, expansion and invention of crustacean genes with reference to insect genomes  

PubMed Central

Background Functional and comparative studies of insect genomes have shed light on the complement of genes, which in part, account for shared morphologies, developmental programs and life-histories. Contrasting the gene inventories of insects to those of the nematodes provides insight into the genomic changes responsible for their diversification. However, nematodes have weak relationships to insects, as each belongs to separate animal phyla. A better outgroup to distinguish lineage specific novelties would include other members of Arthropoda. For example, crustaceans are close allies to the insects (together forming Pancrustacea) and their fascinating aquatic lifestyle provides an important comparison for understanding the genetic basis of adaptations to life on land versus life in water. Results This study reports on the first characterization of cDNA libraries and sequences for the model crustacean Daphnia pulex. We analyzed 1,546 ESTs of which 1,414 represent approximately 787 nuclear genes, by measuring their sequence similarities with insect and nematode proteomes. The provisional annotation of genes is supported by expression data from microarray studies described in companion papers. Loci expected to be shared between crustaceans and insects because of their mutual biological features are identified, including genes for reproduction, regulation and cellular processes. We identify genes that are likely derived within Pancrustacea or lost within the nematodes. Moreover, lineage specific gene family expansions are identified, which suggest certain biological demands associated with their ecological setting. In particular, up to seven distinct ferritin loci are found in Daphnia compared to three in most insects. Finally, a substantial fraction of the sampled gene transcripts shares no sequence similarity with those from other arthropods. Genes functioning during development and reproduction are comparatively well conserved between crustaceans and insects. By contrast, genes that were responsive to environmental conditions (metal stress) and not sex-biased included the greatest proportion of genes with no matches to insect proteomes. Conclusion This study along with associated microarray experiments are the initial steps in a coordinated effort by the Daphnia Genomics Consortium to build the necessary genomic platform needed to discover genes that account for the phenotypic diversity within the genus and to gain new insights into crustacean biology. This effort will soon include the first crustacean genome sequence.

Colbourne, John K; Eads, Brian D; Shaw, Joseph; Bohuski, Elizabeth; Bauer, Darren J; Andrews, Justen

2007-01-01

215

Validation of reference genes for quantitative expression analysis by real-time RT-PCR in Saccharomyces cerevisiae  

Microsoft Academic Search

BACKGROUND: Real-time RT-PCR is the recommended method for quantitative gene expression analysis. A compulsory step is the selection of good reference genes for normalization. A few genes often referred to as HouseKeeping Genes (HSK), such as ACT1, RDN18 or PDA1 are among the most commonly used, as their expression is assumed to remain unchanged over a wide range of conditions.

Marie-Ange Teste; Manon Duquenne; Jean M François; Jean-Luc Parrou

2009-01-01

216

Evaluation of suitable reference genes for normalization of real-time reverse transcription PCR analysis in colon cancer  

Microsoft Academic Search

BACKGROUND: Real-time reverse transcription PCR (qRT-PCR) is frequently used for gene expression quantification due to its methodological reproducibility and sensitivity. The gene expression is quantified by normalization to one or more reference genes which are presumed stably expressed throughout a given experiment. The aim of this study was to validate a standardized experimental setup to identifying reference genes for normalization

Lise Aa Sørby; Solveig N Andersen; Ida RK Bukholm; Morten B Jacobsen

2010-01-01

217

Validation of reference genes for estimating wound age in contused rat skeletal muscle by quantitative real-time PCR  

Microsoft Academic Search

Gene expression profiling by quantitative real-time PCR (RT-qPCR) is a valuable tool in forensic science for estimating the\\u000a age of a wound. To accurately assess gene expression levels over time in injured tissue, the genes used as internal reference\\u000a standards must be carefully validated for transcriptional stability. This study examined the transcriptional stability of\\u000a nine potential reference genes (?-actin, GAPDH,

Jun-hong Sun; Liang-huan Nan; Cai-rong Gao; Ying-yuan Wang

218

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.

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

2014-01-01

219

Identification of reference genes for reverse transcription quantitative real-time PCR normalization in pepper ( Capsicum annuum L.)  

Microsoft Academic Search

Reverse transcription quantitative real-time polymerase chain reaction (qRT-PCR) is a commonly used technology for gene expression and transcriptome analysis. Normalization is a process that is necessary to accurately analyze qRT-PCR data. Stability of reference gene expression is required for this process. Due to the large variation in expression levels of reference genes obtained from different experimental conditions, gene expression stabilities

Hongjian Wan; Wei Yuan; Meiying Ruan; Qingjing Ye; Rongqing Wang; Zhimiao Li; Guozhi Zhou; Zhuping Yao; Jing Zhao; Shujun Liu; Yuejian Yang

220

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

221

Quantification of Gene Expression after Painful Nerve Injury: Validation of Optimal Reference Genes  

Microsoft Academic Search

Stably expressed housekeeping genes (HKGs) are necessary for standardization of transcript measurement by quantitative real-time\\u000a polymerase chain reaction (qRT-PCR). Peripheral nerve injury disrupts expression of numerous genes in sensory neurons, but\\u000a the stability of conventional HKGs has not been tested in this context. We examined the stability of candidate HKGs during\\u000a nerve injury, including the commonly used 18S ribosomal RNA,

Madhavi Latha Yadav Bangaru; Frank Park; Andy Hudmon; J. Bruce McCallum; Quinn H. Hogan

222

Evaluation of suitable reference genes for gene expression studies in bovine muscular tissue  

Microsoft Academic Search

BACKGROUND: Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RTqPCR) is a technique used to measure mRNA species copy number as a way to determine key genes involved in different biological processes. However, the expression level of these key genes may vary among tissues or cells not only as a consequence of differential expression but also due to different factors,

Raquel Pérez; Isabel Tupac-Yupanqui; Susana Dunner

2008-01-01

223

Analysis of multiple transcriptomes of the African oil palm (Elaeis guineensis) to identify reference genes for RT-qPCR.  

PubMed

The African oil palm (Elaeis guineensis), which is grown in tropical and subtropical regions, is a highly productive oil-bearing crop. For gene expression-based analyses such as reverse transcription-quantitative real time PCR (RT-qPCR), reference genes are essential to provide a baseline with which to quantify relative gene expression. Normalization using reliable reference genes is critical in correctly interpreting expression data from RT-qPCR. In order to identify suitable reference genes in African oil palm, 17 transcriptomes of different tissues obtained from NCBI were systematically assessed for gene expression variation. In total, 53 putative candidate reference genes with coefficient of variation values <3.0 were identified: 18 in reproductive tissue and 35 in vegetative tissue. Analysis for enriched functions showed that approximately 90% of identified genes were clustered in cell component gene functions, and 12 out of 53 genes were traditional housekeeping genes. We selected and validated 16 reference genes chosen from leaf tissue transcriptomes by using RT-qPCR in sets of cold, drought and high salinity treated samples, and ranked expression stability using statistical algorithms geNorm, Normfinder and Bestkeeper. Genes encoding actin, adenine phosphoribosyltransferase and eukaryotic initiation factor 4A genes were the most stable genes over the cold, drought and high salinity stresses. Identification of stably expressed genes as reference gene candidates from multiple transcriptome datasets was found to be reliable and efficient, and some traditional housekeeping genes were more stably expressed than others. We provide a useful molecular genetic resource for future gene expression studies in African oil palm, facilitating molecular genetics approaches for crop improvement in this species. PMID:24862192

Xia, Wei; Mason, Annaliese S; Xiao, Yong; Liu, Zheng; Yang, Yaodong; Lei, Xintao; Wu, Xiaoming; Ma, Zilong; Peng, Ming

2014-08-20

224

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

225

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.

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

2014-01-01

226

Identification of appropriate reference genes for normalizing transcript expression by quantitative real-time PCR in Litsea cubeba.  

PubMed

Quantitative real-time PCR has emerged as a highly sensitive and widely used method for detection of gene expression profiles, via which accurate detection depends on reliable normalization. Since no single control is appropriate for all experimental treatments, it is generally advocated to select suitable internal controls prior to use for normalization. This study reported the evaluation of the expression stability of twelve potential reference genes in different tissue/organs and six fruit developmental stages of Litsea cubeba in order to screen the superior internal reference genes for data normalization. Two softwares-geNorm, and NormFinder-were used to identify stability of these candidate genes. The cycle threshold difference and coefficient of variance were also calculated to evaluate the expression stability of candidate genes. F-BOX, EF1?, UBC, and TUA were selected as the most stable reference genes across 11 sample pools. F-BOX, EF1?, and EIF4? exhibited the highest expression stability in different tissue/organs and different fruit developmental stages. Besides, a combination of two stable reference genes would be sufficient for gene expression normalization in different fruit developmental stages. In addition, the relative expression profiles of DXS and DXR were evaluated by EF1?, UBC, and SAMDC. The results further validated the reliability of stable reference genes and also highlighted the importance of selecting suitable internal controls for L. cubeba. These reference genes will be of great importance for transcript normalization in future gene expression studies on L. cubeba. PMID:24162560

Lin, Liyuan; Han, Xiaojiao; Chen, Yicun; Wu, Qingke; Wang, Yangdong

2013-12-01

227

Comparison of five endogenous reference genes for specific PCR detection and quantification of Brassica napus.  

PubMed

Five previously reported Brassica napus endogenous reference genes, including acetyl-CoA carboxylase gene (BnACCg8), phosphoenolpyruvate carboxylase (PEP), oleoyl hydrolase gene (FatA), high-mobility-group protein I/Y gene (HMG-I/Y) and cruciferin A gene (CruA), were analyzed for their PCR specificity between B. napus and other species and the quantification stability among different B. napus cultivars. PCR and sequencing results indicated that none of these systems was species-specific as required by the genetically modified organism labeling policy. When these genes were employed in real-time PCR, BnACCg8 and HMG-I/Y systems showed relatively greater heterogeneity among 10 different cultivars. The sequencing results showed that the single nucleotide polymorphism in the primer binding sites was the potential source of the instability in the HMG-I/Y system. The bias of BnACCg8 was thought to be associated with the inconsistent copy number of this gene. PMID:20143854

Wu, Gang; Zhang, Li; Wu, Yuhua; Cao, Yinglong; Lu, Changming

2010-03-10

228

Taking qPCR to a higher level: Analysis of CNV reveals the power of high throughput qPCR to enhance quantitative resolution  

Microsoft Academic Search

This paper assesses the quantitative resolution of qPCR using copy number variation (CNV) as a paradigm. An error model is developed for real-time qPCR data showing how the precision of CNV determination varies with the number of replicates. Using samples with varying numbers of X chromosomes, experimental data demonstrates that real-time qPCR can readily distinguish four copes from five copies,

Suzanne Weaver; Simant Dube; Alain Mir; Jian Qin; Gang Sun; Ramesh Ramakrishnan; Robert C. Jones; Kenneth J. Livak

2010-01-01

229

Quantification of gene expression after painful nerve injury: validation of optimal reference genes.  

PubMed

Stably expressed housekeeping genes (HKGs) are necessary for standardization of transcript measurement by quantitative real-time polymerase chain reaction (qRT-PCR). Peripheral nerve injury disrupts expression of numerous genes in sensory neurons, but the stability of conventional HKGs has not been tested in this context. We examined the stability of candidate HKGs during nerve injury, including the commonly used 18S ribosomal RNA, ?-tubulin I and ?-tubulin III, actin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and hypoxanthine phosphoribosyltransferase 1 (HPRT1), and mitogen-activated protein kinase 6 (MAPK6). Total RNA for cDNA synthesis was isolated from dorsal root ganglia of rats at 3, 7, and 21 days following either skin incision alone or spinal nerve ligation, after which the axotomized and adjacent ganglia were analyzed separately. Relative stability of HKGs was determined using statistical algorithms geNorm and NormFinder. Both analyses identified MAPK6 and GAPDH as the two most stable HKGs for normalizing gene expression for qRT-PCR analysis in the context of peripheral nerve injury. Our findings indicate that a prior analysis of HKG expression levels is important for accurate normalization of gene expression in models of nerve injury. PMID:21863315

Bangaru, Madhavi Latha Yadav; Park, Frank; Hudmon, Andy; McCallum, J Bruce; Hogan, Quinn H

2012-03-01

230

Identification of reference genes for qRT-PCR in human lung squamous-cell carcinoma by RNA-Seq.  

PubMed

Although the accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) is highly dependent on the reliable reference genes, many commonly used reference genes are not stably expressed and as such are not suitable for quantification and normalization of qRT-PCR data. The aim of this study was to identify novel reliable reference genes in lung squamous-cell carcinoma. We used RNA sequencing (RNA-Seq) to survey the whole genome expression in 5 lung normal samples and 44 lung squamous-cell carcinoma samples. We evaluated the expression profiles of 15 commonly used reference genes and identified five additional candidate reference genes. To validate the RNA-Seq dataset, we used qRT-PCR to verify the expression levels of these 20 genes in a separate set of 100 pairs of normal lung tissue and lung squamous-cell carcinoma samples, and then analyzed these results using geNorm and NormFinder. With respect to 14 of the 15 common reference genes (B2M, GAPDH, GUSB, HMBS, HPRT1, IPO8, PGK1, POLR2A, PPIA, RPLP0, TBP, TFRC, UBC, and YWHAZ), the expression levels were either too low to be easily detected, or exhibited a high degree of variability either between lung normal and squamous-cell carcinoma samples, or even among samples of the same tissue type. In contrast, 1 of the 15 common reference genes (ACTB) and the 5 additional candidate reference genes (EEF1A1, FAU, RPS9, RPS11, and RPS14) were stably and constitutively expressed at high levels in all the samples tested. ACTB, EEF1A1, FAU, RPS9, RPS11, and RPS14 are ideal reference genes for qRT-PCR analysis of lung squamous-cell carcinoma, while 14 commonly used qRT-PCR reference genes are less appropriate in this context. PMID:24457517

Zhan, Cheng; Zhang, Yongxing; Ma, Jun; Wang, Lin; Jiang, Wei; Shi, Yu; Wang, Qun

2014-04-01

231

Identification and validation of reference genes for expression studies in human keratinocyte cell lines treated with and without interferon-? - a method for qRT-PCR reference gene determination.  

PubMed

Based on the exquisite sensitivity, reproducibility and wide dynamic range of quantitative reverse-transcription real-time polymerase chain reaction (qRT-PCR), it is currently the gold standard for gene expression studies. Target gene expression is calculated relative to a stably expressed reference gene. An ideal reference should be uniformly expressed during all experimental conditions within the given experimental system. However, no commonly applicable 'best' reference gene has been identified. Thus, endogenous controls must be determined for every experimental system. As no appropriate reference genes have been reported for immunological studies in keratinocytes, we aimed at identifying and validating a set of endogenous controls for these settings. An extensive validation of sixteen possible endogenous controls in a panel of 8 normal and transformed keratinocyte cell lines in experimental conditions with and without interferon-? was performed. RNA and cDNA quality was stringently controlled. Candidate reference genes were assessed by TaqMan(®) qRT-PCR. Two different statistical algorithms were used to determine the most stably and reproducibly expressed housekeeping genes. mRNA abundance was compared and reference genes with widely different ranges of expression than possible target genes were excluded. Subsequent geNorm and NormFinder analyses identified GAPDH, PGK1, IPO8 and PPIA as the most stably expressed genes in the keratinocyte panel under the given experimental conditions. We conclude that the geometric means of expression values of these four genes represents a robust normalization factor for qRT-PCR analyses in interferon-?-dependent gene expression studies in keratinocytes. The methodology and results herein may help other researchers by facilitating their choice of reference genes. PMID:22775998

Riemer, Angelika B; Keskin, Derin B; Reinherz, Ellis L

2012-08-01

232

A new set of reference genes for RT-qPCR assays in the yeast Dekkera bruxellensis.  

PubMed

The yeast Dekkera bruxellensis has been recently regarded as an important microorganism for bioethanol production owing to its ability to convert glucose, sucrose, and cellobiose to ethanol. The aim of this work was to validate a new set of reference genes for gene expression analysis by quantitative real-time PCR in D. bruxellensis and compare the influence of the method of choice for quantification of mRNA levels with the reliability of our data. Three candidate reference genes, DbEFA1, DbEFB1, and DbYNA1, were used in a quantitative analysis of 4 genes of interest, DbYNR1, DbTPS1, DbADH7, and DbUBA4, based on an approach for calculating the normalization factors by means of the geNorm applet. Each reference gene was also individually used for a 2(-??C(q)) (comparative C(q) method) calculation of the relative expression of genes of interest. Our results showed that the 3 reference genes provided enough stability and were complementary to the normalization factors method in different culture conditions. This work was able to confirm the usefulness of a previously reported reference gene, EFA1/TEF1, and increased the set of possible reference genes in D. bruxellensis to 4. Moreover, this can improve the reliability of the analysis of the regulation of gene expression in the industrial yeast D. bruxellensis. PMID:23210993

de Barros Pita, Will; Leite, Fernanda Cristina Bezerra; de Souza Liberal, Anna Theresa; Pereira, Luciana Filgueira; Carazzolle, Marcelo Falsarella; Pereira, Gonçalo Amarante; de Morais, Marcos Antonio

2012-12-01

233

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.

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

234

Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli).  

PubMed

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) is a technique widely used for quantification of mRNA transcription. Data normalization is an indispensable process for RT-qPCR and reference genes are most commonly used to normalize RT-qPCR and to reduce possible errors generated in the quantification of genes among several proposed methods. To date, RT-qPCR has been used in terms of gene expression studies in black rockfish (Sebastes schlegeli) but the majority of published RT-qPCR studies still lack proper validation of the reference genes. In the present study, mRNA transcription profiles of eight putative reference genes (18S rRNA, ACTB, GAPDH, TUBA, RPL17, EF1A, HPRT, and B2M) were examined using RT-qPCR in different tissues and larvae developmental stages of black rockfish. Three common statistical algorithms (geNorm, NormFinder, and BestKeeper) were used to assess expression stability and select the most stable genes for gene normalization. Two reference genes, RPL17 and EF1A showed high stability in black rockfish tissue analysis, while GAPDH was the least stable gene. During larvae developmental stages, EF1A, RPL17 and ACTB were identified as the optimal reference genes for data normalization, whereas B2M appeared unsuitable as the reference gene. In summary, our results could provide a useful guideline for reference gene selection and enable more accurate normalization of gene expression data in gene expression studies of black rockfish. PMID:24007945

Liman, Ma; Wenji, Wang; Conghui, Liu; Haiyang, Yu; Zhigang, Wang; Xubo, Wang; Jie, Qi; Quanqi, Zhang

2013-09-01

235

Deep RNA sequencing analysis of readthrough gene fusions in human prostate adenocarcinoma and reference samples  

PubMed Central

Background Readthrough fusions across adjacent genes in the genome, or transcription-induced chimeras (TICs), have been estimated using expressed sequence tag (EST) libraries to involve 4-6% of all genes. Deep transcriptional sequencing (RNA-Seq) now makes it possible to study the occurrence and expression levels of TICs in individual samples across the genome. Methods We performed single-end RNA-Seq on three human prostate adenocarcinoma samples and their corresponding normal tissues, as well as brain and universal reference samples. We developed two bioinformatics methods to specifically identify TIC events: a targeted alignment method using artificial exon-exon junctions within 200,000 bp from adjacent genes, and genomic alignment allowing splicing within individual reads. We performed further experimental verification and characterization of selected TIC and fusion events using quantitative RT-PCR and comparative genomic hybridization microarrays. Results Targeted alignment against artificial exon-exon junctions yielded 339 distinct TIC events, including 32 gene pairs with multiple isoforms. The false discovery rate was estimated to be 1.5%. Spliced alignment to the genome was less sensitive, finding only 18% of those found by targeted alignment in 33-nt reads and 59% of those in 50-nt reads. However, spliced alignment revealed 30 cases of TICs with intervening exons, in addition to distant inversions, scrambled genes, and translocations. Our findings increase the catalog of observed TIC gene pairs by 66%. We verified 6 of 6 predicted TICs in all prostate samples, and 2 of 5 predicted novel distant gene fusions, both private events among 54 prostate tumor samples tested. Expression of TICs correlates with that of the upstream gene, which can explain the prostate-specific pattern of some TIC events and the restriction of the SLC45A3-ELK4 e4-e2 TIC to ERG-negative prostate samples, as confirmed in 20 matched prostate tumor and normal samples and 9 lung cancer cell lines. Conclusions Deep transcriptional sequencing and analysis with targeted and spliced alignment methods can effectively identify TIC events across the genome in individual tissues. Prostate and reference samples exhibit a wide range of TIC events, involving more genes than estimated previously using ESTs. Tissue specificity of TIC events is correlated with expression patterns of the upstream gene. Some TIC events, such as MSMB-NCOA4, may play functional roles in cancer.

2011-01-01

236

Bone to pick: the importance of evaluating reference genes for RT-qPCR quantification of gene expression in craniosynostosis and bone-related tissues and cells  

PubMed Central

Background RT-qPCR is a common tool for quantification of gene expression, but its accuracy is dependent on the choice and stability (steady state expression levels) of the reference gene/s used for normalization. To date, in the bone field, there have been few studies to determine the most stable reference genes and, usually, RT-qPCR data is normalised to non-validated reference genes, most commonly GAPDH, ACTB and 18?S rRNA. Here we draw attention to the potential deleterious impact of using classical reference genes to normalise expression data for bone studies without prior validation of their stability. Results Using the geNorm and Normfinder programs, panels of mouse and human genes were assessed for their stability under three different experimental conditions: 1) disease progression of Crouzon syndrome (craniosynostosis) in a mouse model, 2) proliferative culture of cranial suture cells isolated from craniosynostosis patients and 3) osteogenesis of a mouse bone marrow stromal cell line. We demonstrate that classical reference genes are not always the most ‘stable’ genes and that gene ‘stability’ is highly dependent on experimental conditions. Selected stable genes, individually or in combination, were then used to normalise osteocalcin and alkaline phosphatase gene expression data during cranial suture fusion in the craniosynostosis mouse model and strategies compared. Strikingly, the expression trends of alkaline phosphatase and osteocalcin varied significantly when normalised to the least stable, the most stable or the three most stable genes. Conclusion To minimise errors in evaluating gene expression levels, analysis of a reference panel and subsequent normalization to several stable genes is strongly recommended over normalization to a single gene. In particular, we conclude that use of single, non-validated “housekeeping” genes such as GAPDH, ACTB and 18?S rRNA, currently a widespread practice by researchers in the bone field, is likely to produce data of questionable reliability when changes are 2 fold or less, and such data should be interpreted with due caution.

2012-01-01

237

Selection of stable reference genes in heat stressed peripheral blood mononuclear cells of tropically adapted Indian cattle and buffaloes.  

PubMed

Eleven reference genes from different functional categories were assessed for their stable expression pattern in heat stressed PBMCs across Indian buffalo and cattle using GeNorm, NormFinder and BestKeeper algorithms. As the first report, we suggest B2M, RPS9 and RPS15a as suitable reference genes for accurate normalization of PBMC transcript data while the ACTB gene is not recommended. PMID:23499765

Kishore, Amit; Sodhi, Monika; Khate, Keviletsu; Kapila, Neha; Kumari, Parvesh; Mukesh, Manishi

2013-01-01

238

Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress  

Microsoft Academic Search

We examined eight putative consistently expressed genes—actin (ACT), ?-tubulin, elongation factor 1? (EF1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), ribosomal protein L2 (RPL2), ubiquitin (UBI), and a catalytic subunit of protein phosphatase 2A (PP2Acs)—for their potential as references for the normalization of gene expression in tomato leaves. Expression stability of candidate reference genes was tested during growth conditions of nitrogen

Trond Løvdal; Cathrine Lillo

2009-01-01

239

A simple analytical and experimental procedure for selection of reference genes for reverse-transcription quantitative PCR normalization data  

Microsoft Academic Search

Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set

R. Manjarin; N. L. Trottier; P. S. Weber; J. S. Liesman; N. P. Taylor; J. P. Steibel

2011-01-01

240

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

Microsoft Academic Search

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,

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

2012-01-01

241

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

242

Accuracy in Copy Number Calling by qPCR and PRT: A Matter of DNA  

PubMed Central

The possible implication of copy number variation (CNV) in the genetic susceptibility to human disease needs to be assessed using robust methods that can be applied at a population scale. In this report, we analyze the performance of the two major techniques, quantitative PCR (qPCR) and paralog ratio test (PRT), and investigate the influence of input DNA amount and template integrity on the reliability of both methods. Analysis of three genes (PRELID1, SYNPO and DEFB4) in a large sample set showed that both methods are prone to false copy number assignments if sufficient attention is not paid to DNA concentration and quality. Accurate normalization of samples is essential for reproducible qPCR because it avoids the effect of differential amplification efficiencies between target and control assays, whereas PRT is generally more sensitive to template degradation due to the fact that longer amplicons are usually needed to optimize sensitivity and specificity of paralog sequence PCR. The use of normalized, high quality genomic DNA yields comparable results with both methods.

Fernandez-Jimenez, Nora; Castellanos-Rubio, Ainara; Plaza-Izurieta, Leticia; Gutierrez, Galder; Irastorza, Inaki; Castano, Luis; Vitoria, Juan Carlos; Bilbao, Jose Ramon

2011-01-01

243

Real-time PCR (qPCR) primer design using free online software.  

PubMed

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 commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. PMID:21445907

Thornton, Brenda; Basu, Chhandak

2011-01-01

244

Variation in stability of endogenous reference genes in fallopian tubes and endometrium from healthy and ectopic pregnant women.  

PubMed

RT-qPCR is commonly employed in gene expression studies in ectopic pregnancy. Most use RN18S1, ?-actin or GAPDH as internal controls without validation of their suitability as reference genes. A systematic study of the suitability of endogenous reference genes for gene expression studies in ectopic pregnancy is lacking. The aims of this study were therefore to evaluate the stability of 12 reference genes and suggest those that are stable for use as internal control genes in fallopian tubes and endometrium from ectopic pregnancy and healthy non-pregnant controls. Analysis of the results showed that the genes consistently ranked in the top six by geNorm and NormFinder algorithms, were UBC, GAPDH, CYC1 and EIF4A2 (fallopian tubes) and UBC and ATP5B (endometrium). mRNA expression of NAPE-PLD as a test gene of interest varied between the groups depending on which of the 12 reference genes was used as internal controls. This study demonstrates that arbitrary selection of reference genes for normalisation in RT-qPCR studies in ectopic pregnancy without validation, risk producing inaccurate data and should therefore be discouraged. PMID:22489127

Gebeh, Alpha K; Marczylo, Emma L; Amoako, Akwasi A; Willets, Jonathon M; Konje, Justin C

2012-01-01

245

A combined strategy of “in silico” transcriptome analysis and web search engine optimization allows an agile identification of reference genes suitable for normalization in gene expression studies  

Microsoft Academic Search

Traditionally housekeeping genes have been employed as endogenous reference (internal control) genes for normalization in\\u000a gene expression studies. Since the utilization of single housekeepers cannot assure an unbiased result, new normalization\\u000a methods involving multiple housekeeping genes and normalizing using their mean expression have been recently proposed. Moreover,\\u000a since a gold standard gene suitable for every experimental condition does not exist,

Primetta Faccioli; Gian Paolo Ciceri; Paolo Provero; Antonio Michele Stanca; Caterina Morcia; Valeria Terzi

2007-01-01

246

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  

Microsoft Academic Search

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

Ard M Nijhof; Jesper A Balk; Milagros Postigo; Frans Jongejan

2009-01-01

247

UBC and YWHAZ as suitable reference genes for accurate normalisation of gene expression using MCF7, HCT116 and HepG2 cell lines.  

PubMed

Relative quantification of in vitro gene expression using real-time PCR requires stably expressed reference gene for normalisation. In this study, total RNA from MCF7, HCT116 and HepG2 cells were extracted and converted to cDNA using commercially available kit, and real-time PCR was then performed to analyse the expression levels of twelve reference genes to select the most ideal reference gene for accurate normalisation in gene expression study. geNorm and NormFinder software were used to analyse the stabilities of the reference genes, which showed a wide range of C(t) values. The geNorm analysis showed the following ranking for stability of genes: UBC, YWHAZ > RPLP > TBP > ACTB > HPRT1 > PPIA > GAPDH > GUSB > B2M > TUBB > RRN18S. A similar ranking of reference genes was obtained by NormFinder, and the four most stable reference genes were identical using both approaches. UBC and YWHAZ were proposed to be the two most suitable reference genes based on the above analyses. To further assess the stabilities of the UBC and YWHAZ in a formal experiment, MCF7, HCT116 and HepG2 cell lines were subjected to treatments with 5-aza-dC and TSA. Both UBC and YWHAZ exhibited stable expression levels across control and treatment groups. Therefore, we propose that UBC and YWHAZ are the two most suitable reference genes for our gene expression studies using MCF7, HCT116 and HepG2 cell lines. PMID:21850463

Chua, Siang Ling; See Too, Wei Cun; Khoo, Boon Yin; Few, Ling Ling

2011-12-01

248

[Selection of reliable reference genes for qRT-PCR analysis in human non-cancerous gastric tissue].  

PubMed

Real-time PCR (qRT-PCR) is the standard method for studying changes in relative gene expression in complex diseases like obesity and gastritis. However, variations in amount of starting material, enzymatic efficiency and presence of amplification inhibitors can lead to quantification errors. Hence, the need for accurate data normalization is vital. Among several known strategies for data normalization, the use of reference genes as an internal control is the most common approach. Human gastric tissue has been the least investigated for stability of reference gene expression. In this study, three popular algorithms, GeNorm, NormFinder and BestKeeper were used to evaluate the reference gene stability. Conclusion: HPRT1 and GAPDH are the best performing pair of reference genes for qRT-PCR profiling experiments involving non-malignant gastric tissue samples. PMID:22642115

Birerdinc, A; Mehta, R; Alhussain, R; Afendi, A; Chandhoke, V; Younossi, Z; Baranova, A

2012-01-01

249

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

250

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.

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

2012-01-01

251

Identification of Suitable Reference Genes for Real-Time PCR Analysis of Statin-Treated Human Umbilical Vein Endothelial Cells  

PubMed Central

Proper data normalization in quantitative real-time reverse-transcription polymerase chain reaction (RT-qPCR) is of critical importance for reliable mRNA expression analysis. Due to a diversity in putative reference genes expression stability in different in vitro models, a validation of an internal control gene should be made for each particular tissue or cell type and every specific experimental design. A few approaches have been proposed for reference gene selection, including pair-wise comparison approach and model-based approach. In this article we have assessed the expression stability of eight putative reference genes: ACTB, B2M, GADD45A, GAPDH, HPRT1, PES1, PSMC4, YWHAZ, in human umbilical vein endothelial cells (HUVEC) treated with different statins and with TNF-?. The analysis was performed with three reference gene validation programs: geNorm, NormFinder and BestKeeper. We have shown that hypoxanthine phosphoribosyltransferase 1 gene (HPRT1) and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide gene (YWHAZ) are the most stably expressed genes among the analyzed ones. Furthermore, our results show that ?-actin gene (ACTB) is downregulated by statins and thus should not be used as a normalizing gene in a discussed experimental setup. A ranking of candidate reference genes stability values is provided and might serve as a valuable guide for future gene expression studies in endothelial cells. This is the first report on reference gene selection for RT-qPCR applications in statin-treated HUVEC model.

Zyzynska-Granica, Barbara; Koziak, Katarzyna

2012-01-01

252

Evaluation of putative internal reference genes for gene expression normalization in Nannochloropsis sp. by quantitative real-time RT-PCR.  

PubMed

Quantitative real-time reverse transcription PCR (RT-qPCR), a sensitive technique for quantifying gene expression, depends on the stability of the reference gene(s) used for data normalization. To date, few studies on reference genes have been undertaken for Nannochloropsis sp. In this study, 12 potential reference genes were evaluated for their expression stability using the geNorm and NormFinder statistical algorithms by RT-qPCR. The results showed that the best reference genes differed depending on the treatments: different light intensities (DL), the diurnal cycle (DC), high light intensity (HL) and low temperature treatments (LT). A combination of ACT1, ACT2 and TUA would be appropriate as a reference panel for normalizing gene expression data across all the treatments. ACT2 showed the most stable expression across all tested samples but was not the most stable one for individual treatments. Though 18S showed the least stable expression considering all tested samples, it is the most stable one for LT using geNorm. The expression of Lhc confirmed that the appropriate reference genes are crucial. These results provide a foundation for more accurate use of RT-qPCR under different experimental conditions in Nannochloropsis sp. gene analysis. PMID:22732401

Cao, Shaona; Zhang, Xiaowen; Ye, Naihao; Fan, Xiao; Mou, Shanli; Xu, Dong; Liang, Chengwei; Wang, Yitao; Wang, Wenqi

2012-07-20

253

Selection and reliability of internal reference genes for quantitative PCR verification of transcriptomics during the differentiation process of porcine adult mesenchymal stem cells  

Microsoft Academic Search

Introduction  The objective of this study was to find highly reliable internal-control genes (ICGs) for normalization of qPCR data from\\u000a porcine adult mesenchymal stem cells induced to differentiate toward adipogenic and osteogenic lineages.\\u000a \\u000a \\u000a \\u000a \\u000a Methods  Stem cells were acquired from subcutaneous back fat and bone marrow of three castrated Yorkshire crossbred male pigs. Adipose\\u000a and bone marrow-derived stem cells (ADSCs and BMSCs) were

Elisa Monaco; Massimo Bionaz; Alecsandra Sobreira de Lima; Walter L Hurley; Juan J Loor; Matthew B Wheeler

2010-01-01

254

Identification of suitable reference genes in buffalo grass for accurate transcript normalization under various abiotic stress conditions.  

PubMed

Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a sensitive technique for normalization of the gene expression level of target genes. Buffalograss (Buchloe dactyloides), a warm-season turfgrass with strong abiotic stress resistance, is widely used in North China. Up to now, no work was performed to evaluate the reference genes in buffalograss. In this study, the expression profiles of ten potential reference genes were examined by qRT-PCR in 24 buffalograss samples, which were subjected to a different treatment (salt, osmotic, cold and heat). Three qRT-PCR analysis methods (GeNorm, NormFinder, and Bestkeeper) were used to evaluate the stability of gene expression. The results indicated that DNAJ and ?-ACTIN were the optimal reference genes for salt-treated leaves, and the combination of PP2A and GAPDH was better reference genes for PEG-treated leaves. Under cold stress, DNAJ and ?-ACTIN showed less variety of expression level in leaves. DNAJ and GAPDH exhibited the most stable expression in heat-treated samples. To sum up, glyceral-dehyde-3-phosphate dehydrogenase (GAPDH), ?-ACTIN, DNAJ-like protein (DNAJ) and protein phosphatase 2A (PP2A) were selected as the most stable reference gene among all tested samples. To further validate the suitability of these reference genes, the expression levels of DREB2 (homologs of AtDREB2) were analyzed in parallel. Our results show that the best reference genes differed across different experimental conditions, and these results should enable better normalization and quantification of transcript levels in buffalograss in the future. PMID:24914494

Li, Wei; Qian, Yong-Qiang; Han, Lei; Liu, Jun-Xiang; Sun, Zhen-Yuan

2014-08-15

255

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.

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

256

Validation of novel reference genes for reverse transcription quantitative real-time PCR in drought-stressed sugarcane.  

PubMed

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, Manassés Daniel; Ferreira Neto, José Ribamar Costa; de Nardi, Claudia Huerta; Chabregas, Sabrina Moutinho; Burnquist, William Lee; Kahl, Günter; Benko-Iseppon, Ana Maria; Kido, Ederson Akio

2014-01-01

257

Selection of Reference Genes for Expression Analysis in Diuraphis noxia (Hemiptera: Aphididae) Fed on Resistant and Susceptible Wheat Plants  

PubMed Central

The Russian wheat aphid (RWA), Diuraphis noxia Kurdjumov, is a major global pest of wheat and barley production that causes enormous economic damage. Few studies have been conducted to explore and decipher the molecular basis of RWA strategies to evade plant defense mechanisms. Gene expression studies of RWA in response to wheat genotypes carrying different RWA resistance genes have been initiated in our group; however, a secure and accurate understanding of RWA gene expression is dependent on identification of suitable reference genes. This study analyzed expression profiles of five potential reference genes selected and sequenced during RNA sequencing experiments. The expression of genes coding for actin and ribosomal protein L27 was comparatively less variable in RWA fed on different wheat hosts. Results of geNorm, NormFinder, and BestKeeper expression analyses support the use of actin and ribosomal protein L27 in RT-qPCR studies of RWA gene expression in studies involving RWA-wheat interactions.

Sinha, Deepak K.; Smith, C. Michael

2014-01-01

258

Validation of Reference Genes for Normalization Gene Expression in Reverse Transcription Quantitative PCR in Human Normal Thyroid and Goiter Tissue  

PubMed Central

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been recognized as the most accurate method for quantifying mRNA transcripts, but normalization of samples is a prerequisite for correct data interpretation. So, this study aimed to evaluate the most stable reference gene for RT-qPCR in human normal thyroid and goiter tissues. Beta-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); succinate dehydrogenase, subunit A, flavoprotein (Fp) (SDHA); hypoxanthine phosphoribosyltransferase I (HPRTI); tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ); and beta-2-microglobulin (B2M) were evaluated in 14 thyroid tissue samples (7 normal and 7 goiter tissues) by RT-qPCR. The mean Cq and the maximum fold change (MFC) and NormFinder software were used to assess the stability of the genes. As a result, ACTB gene was more stable than GAPDH, SDHA, HPRTI, YWHAZ, and B2M. In conclusion, ACTB could be used to normalize RT-qPCR data in normal thyroid and goiter tissues.

Bertoni, Ana Paula Santin; Bessestil, Laura Walter; Brasil, Beatriz Maria de Azevedo Assis; Brum, llma Simoni; Furlanetto, Tania Weber

2014-01-01

259

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.

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

2014-01-01

260

Development of plasmid calibrators for absolute quantification of miRNAs by using real-time qPCR.  

PubMed

MicroRNAs (miRNAs) are small noncoding RNAs of approximately 18 to 25 nucleotides in length that negatively regulate gene expression via either the degradation or translational inhibition of their target mRNAs. Because miRNAs are essential for the regulation of critical physiological processes as well as a variety of pathological events, they have emerged as a novel class of molecular diagnostic biomarkers and therapeutic agents or targets. Accordingly, the need for novel methods for the quantification of miRNA has increased due to interest in their clinical implications. Currently, real-time quantitative polymerase chain reaction (qPCR) is considered the most robust technology for nucleic acid quantification. Different tools for miRNA quantification by using qPCR are now commercially available, but only relative quantification strategies have been reported. This situation may be partly due to the difficulty in obtaining an appropriate molecule with which to establish an miRNA calibration range. Here, we describe a rapid and convenient strategy for the development of a calibrator, which enables the absolute quantification of miRNAs by using qPCR and allows the cloning of a synthetic sequence of interest instead of a PCR product into a plasmid. PMID:22642897

Formisano-Tréziny, Christine; de San Feliciano, Marina; Gabert, Jean

2012-07-01

261

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

262

Reliability of gene expression ratios for cDNA microarrays in multiconditional experiments with a reference design  

Microsoft Academic Search

In a typical gene expression profiling experiment with multiple conditions, a common reference sample is used for co-hybridization with the samples to yield expression ratios. Differential expression for any other sample pair can then be calculated by assembling the ratios from their hybridizations with the reference. In this study we test the validity of this approach. Differential expres- sion of

Rainer Konig; Danila Baldessari; Nicolas Pollet; Christof Niehrs; Roland Eils

2004-01-01

263

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.

Silbermayr, Katja; Li, Fuyong; Soudre, Albert; Muller, Simone; Solkner, Johann

2013-01-01

264

Quantitative evaluation of reference genes for real-time PCR during in vitro maturation of ovine oocytes.  

PubMed

Quantitative reverse transcription PCR (RT-qPCR) is a powerful molecular technique that enables gene expression studies to be performed on extremely small samples such as oocytes and pre-implantation embryos. However, the high sensitivity of this technique requires that to prevent bias in expression data interpretation, the reference genes used for normalization are fully validated before use. Difficulties in choosing a reference gene are further compounded by the variable RNA content of the maturing oocyte. In the present study, we evaluated eight commonly used reference genes such as ACTB, GAPDH, H2AFZ, HPRT1, PPIA, SDHA, TUBB and YWHAZ and for sheep oocyte RT-qPCR before and after in vitro maturation. We have also compared different cDNA priming strategies using random hexamers or oligo-dT. GeNorm analysis of the results identified the most reliable genes for normalization to be SDHA, TUBB and PPIA when oocyte cDNA was made with random hexamers, and YWHAZ, TUBB and SDHA when oligo-dT primers were used (H2AFZ and HPRT1 were excluded from the geNorm analysis). Interestingly, the analysis revealed that the least stable genes were ACTB and GAPDH, which are the conventional 'housekeeping' genes used in many studies. We recommend the use of three reference genes to calculate a normalization factor to accurately quantify transcript abundance in sheep oocytes and these vary with the cDNA priming strategy employed. PMID:23066791

O'Connor, T; Wilmut, I; Taylor, J

2013-06-01

265

Selection of internal reference genes for SYBR green qRT-PCR studies of rhesus monkey (Macaca mulatta) tissues  

Microsoft Academic Search

BACKGROUND: The rhesus monkey (Macaca mulatta) is a valuable and widely used model animal for biomedical research. However, quantitative analyses of rhesus gene expression profiles under diverse experimental conditions are limited by a shortage of suitable internal controls for the normalization of mRNA levels. In this study, we used a systematic approach for the selection of potential reference genes in

Kung Ahn; Jae-Won Huh; Sang-Je Park; Dae-Soo Kim; Hong-Seok Ha; Yun-Ji Kim; Ja-Rang Lee; Kyu-Tae Chang; Heui-Soo Kim

2008-01-01

266

Selection of reference genes for qRT-PCR examination of wild populations of Atlantic cod Gadus morhua  

Microsoft Academic Search

BACKGROUND: Extensive sequencing efforts have been taking place for the Atlantic cod (Gadus morhua) in recent years, the number of ESTs in the Genbank has reached more than 140.000. Despite its importance in North Atlantic fisheries and potential use in aquaculture, relatively few gene expression examination exists for this species, and systematic evaluations of reference gene stability in quantitative real-time

Pål A Olsvik; Liv Søfteland; Kai K Lie

2008-01-01

267

Manual annotation and analysis of the defensin gene cluster in the C57BL/6J mouse reference genome  

PubMed Central

Background Host defense peptides are a critical component of the innate immune system. Human alpha- and beta-defensin genes are subject to copy number variation (CNV) and historically the organization of mouse alpha-defensin genes has been poorly defined. Here we present the first full manual genomic annotation of the mouse defensin region on Chromosome 8 of the reference strain C57BL/6J, and the analysis of the orthologous regions of the human and rat genomes. Problems were identified with the reference assemblies of all three genomes. Defensins have been studied for over two decades and their naming has become a critical issue due to incorrect identification of defensin genes derived from different mouse strains and the duplicated nature of this region. Results The defensin gene cluster region on mouse Chromosome 8 A2 contains 98 gene loci: 53 are likely active defensin genes and 22 defensin pseudogenes. Several TATA box motifs were found for human and mouse defensin genes that likely impact gene expression. Three novel defensin genes belonging to the Cryptdin Related Sequences (CRS) family were identified. All additional mouse defensin loci on Chromosomes 1, 2 and 14 were annotated and unusual splice variants identified. Comparison of the mouse alpha-defensins in the three main mouse reference gene sets Ensembl, Mouse Genome Informatics (MGI), and NCBI RefSeq reveals significant inconsistencies in annotation and nomenclature. We are collaborating with the Mouse Genome Nomenclature Committee (MGNC) to establish a standardized naming scheme for alpha-defensins. Conclusions Prior to this analysis, there was no reliable reference gene set available for the mouse strain C57BL/6J defensin genes, demonstrating that manual intervention is still critical for the annotation of complex gene families and heavily duplicated regions. Accurate gene annotation is facilitated by the annotation of pseudogenes and regulatory elements. Manually curated gene models will be incorporated into the Ensembl and Consensus Coding Sequence (CCDS) reference sets. Elucidation of the genomic structure of this complex gene cluster on the mouse reference sequence, and adoption of a clear and unambiguous naming scheme, will provide a valuable tool to support studies on the evolution, regulatory mechanisms and biological functions of defensins in vivo.

2009-01-01

268

Genome-wide identification and characterization of a panel of house-keeping genes in Schistosoma japonicum.  

PubMed

Quantitative real-time polymerase chain reaction (qPCR), as one of the most sensitive and precise gene expression analysis methods, is frequently used to validate data obtained in high-through-put assays. qPCR requires reference genes with stable transcription for accurate normalization. However, no systematic studies on such genes have been performed in the genus Schistosoma japonicum. In this study, eight novel candidate genes selected from a microarray analysis and four commonly used reference genes were systematically validated in a series of qPCR experiments. Based on the results of geNorm, Normfinder, BestKeeper, and the comparative delta-cycle threshold (?CT) integrated analysis, the genes PSMD4, NDUFV2, and TPC2L were found to be most stably expressed in all S. japonicum developmental stages; meanwhile, ACTB and TUBA were found as the least stably expressed genes. This study provided, at the first time, data for genes that can be explored as reliable references in transcriptomic analysis of S. japonicum. PMID:22245333

Liu, Shuai; Cai, Pengfei; Hou, Nan; Piao, Xianyu; Wang, Heng; Hung, Tao; Chen, Qijun

2012-01-01

269

Evaluation of reference genes for qRT-PCR gene expression studies in whole blood samples from healthy and leukemia-virus infected cattle.  

PubMed

Real-time quantitative reverse transcription PCR (qRT-PCR) is the method of choice to investigate the alterations in gene expression involved with BLV pathogenesis. However, the reliability of qRT-PCR data critically depends on proper normalization to a set of stably expressed reference genes. The aim of the study was to validate the expression stability of ten candidate reference genes in RNA isolated from whole blood cells of BLV-negative and BLV-positive cows with hematological abnormalities. The rankings of the candidate genes according to their expression stability were calculated using BestKeeper, NormFinder and qBase(PLUS) software with implemented geNorm(PLUS) algorithm. The results showed that two genes are sufficient for normalization of qRT-PCR studies in whole blood RNA isolated from cows infected with BLV. According to geNorm, UCHL5 and RPLP0 were the best choice, but taking into account possible intergroup variation, NormFinder recommended RPLP0 and B2M as a most suitable pair. The overall ranking based on the geometric mean of the ranking numbers from each method separately showed UCHL5, RPLP0 and TBP as the most stable candidate reference genes. In addition, all three methods unanimously pointed at the commonly used ACTB and GAPDH as the least stable genes. These results further emphasize the need to accurately validate candidate reference genes before use in gene expression qRT-PCR studies. PMID:23548864

Brym, P; Ru??, A; Kami?ski, S

2013-06-15

270

A simple analytical and experimental procedure for selection of reference genes for reverse-transcription quantitative PCR normalization data.  

PubMed

Variation in cellular activity in a tissue induces changes in RNA concentration, which affects the validity of gene mRNA abundance analyzed by reverse transcription quantitative PCR (RT-qPCR). A common way of accounting for such variation consists of the use of reference genes for normalization. Programs such as geNorm may be used to select suitable reference genes, although a large set of genes that are not co-regulated must be analyzed to obtain accurate results. The objective of this study was to propose an alternative experimental and analytical protocol to assess the invariance of reference genes in porcine mammary tissue using mammary RNA and DNA concentrations as correction factors. Mammary glands were biopsied from 4 sows on d 110 of gestation (prepartum), on d 5 (early) and 17 (peak) of lactation, and on d 5 after weaning (postweaning). Relative expression of 7 potential reference genes, API5, MRPL39, VAPB, ACTB, GAPDH, RPS23, and MTG1, and one candidate gene, SLC7A1, was quantified by RT-qPCR using a relative standard curve approach. Variation in gene expression levels, measured as cycles to threshold at each stage of mammary physiological activity, was tested using a linear mixed model fitting RNA and DNA concentrations as covariates. Results were compared with those obtained with geNorm analysis, and genes selected by each method were used to normalize SLC7A1. Quantified relative mRNA abundance of GAPDH and MRPL39 remained unchanged across stages of mammary physiological activity after accounting for changes in tissue RNA and DNA concentration. In contrast, geNorm analysis selected MTG1, MRPL39, and VAPB as the best reference genes. However, when target gene SLC7A1 was normalized with genes selected either based on our proposed protocol or by geNorm, fold changes in mRNA abundance did not differ. In conclusion, the proposed analytical protocol assesses expression invariance of potential reference genes by accounting for variation in tissue RNA and DNA concentrations and thus represents an alternative method to select suitable reference genes for RT-qPCR analysis. PMID:21943746

Manjarin, R; Trottier, N L; Weber, P S; Liesman, J S; Taylor, N P; Steibel, J P

2011-10-01

271

Stability of reference genes for real-time PCR analyses in channel catfish (Ictalurus punctatus) tissues under varying physiological conditions.  

PubMed

Real-time PCR is a highly sensitive, relatively easy to perform assay for quantifying mRNA abundance. However, there are several complexities built into the assay that can affect data interpretation. Most notably, the selection of an appropriate internal control for normalization is essential for expression data interpretation. In this study we investigated the suitability of seven commonly used genes [18S ribosomal RNA (18S), alpha tubulin (TUBA), beta actin (ACTB), beta-2 microglobulin (B2M), embryonic elongation factor-1 alpha (EEF1A), glyceraldehyde phosphate dehydrogenase (GAPDH), and RNA polymerase II polypeptide B (POLR2B)] as potential quantitative references for normalizing real-time PCR data generated in the study of channel catfish physiology. Gene expression and stability were evaluated among 15 channel catfish tissues and within physiologically-relevant tissues in response to experimental manipulation (i.e. LHRH injection, fasting, and acute stress). Expression of the seven candidate reference genes varied across all tissue types tested, indicating that none of the genes could suitably serve as reference genes for cross tissue comparisons. Experimentally altering the physiological state of the fish differentially affected expression of the various reference genes depending on experimental design and tissue type, with 18S unaffected by the experimental treatment in all tissues examined. For example, the selection of a differentially expressed gene, GAPDH, as opposed to 18S, to normalize hepatic growth hormone receptor during fasting resulted in misinterpretation of the data. These results reveal the importance of providing comprehensive details of reference gene validation when publishing real-time PCR results, with this manuscript serving as a basic guideline for reference gene selection in channel catfish research. PMID:18692590

Small, Brian C; Murdock, Christopher A; Bilodeau-Bourgeois, A Lelania; Peterson, Brian C; Waldbieser, Geoffrey C

2008-11-01

272

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.

2014-01-01

273

Identification of new reference genes for the normalisation of canine osteoarthritic joint tissue transcripts from microarray data  

Microsoft Academic Search

BACKGROUND: Real-time reverse transcriptase quantitative polymerase chain reaction (real-time RT-qPCR) is the most accurate measure of gene expression in biological systems. The comparison of different samples requires the transformation of data through a process called normalisation. Reference or housekeeping genes are candidate genes which are selected on the basis of constitutive expression across samples, and allow the quantification of changes

Lindsey J Maccoux; Dylan N Clements; Fiona Salway; Philip JR Day

2007-01-01

274

Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut  

PubMed Central

Background Wild peanut species (Arachis spp.) are a rich source of new alleles for peanut improvement. Plant transcriptome analysis under specific experimental conditions helps the understanding of cellular processes related, for instance, to development, stress response, and crop yield. The validation of these studies has been generally accomplished by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) which requires normalization of mRNA levels among samples. This can be achieved by comparing the expression ratio between a gene of interest and a reference gene which is constitutively expressed. Nowadays there is a lack of appropriate reference genes for both wild and cultivated Arachis. The identification of such genes would allow a consistent analysis of qRT-PCR data and speed up candidate gene validation in peanut. Results A set of ten reference genes were analyzed in four Arachis species (A. magna; A. duranensis; A. stenosperma and A. hypogaea) subjected to biotic (root-knot nematode and leaf spot fungus) and abiotic (drought) stresses, in two distinct plant organs (roots and leaves). By the use of three programs (GeNorm, NormFinder and BestKeeper) and taking into account the entire dataset, five of these ten genes, ACT1 (actin depolymerizing factor-like protein), UBI1 (polyubiquitin), GAPDH (glyceraldehyde-3-phosphate dehydrogenase), 60S (60S ribosomal protein L10) and UBI2 (ubiquitin/ribosomal protein S27a) emerged as top reference genes, with their stability varying in eight subsets. The former three genes were the most stable across all species, organs and treatments studied. Conclusions This first in-depth study of reference genes validation in wild Arachis species will allow the use of specific combinations of secure and stable reference genes in qRT-PCR assays. The use of these appropriate references characterized here should improve the accuracy and reliability of gene expression analysis in both wild and cultivated Arachis and contribute for the better understanding of gene expression in, for instance, stress tolerance/resistance mechanisms in plants.

2011-01-01

275

RefPrimeCouch--a reference gene primer CouchApp.  

PubMed

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

276

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

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

2013-01-01

277

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.

Duhoux, Arnaud; Delye, Christophe

2013-01-01

278

Reference genes to study herbicide stress response in Lolium sp.: up-regulation of P450 genes in plants resistant to acetolactate-synthase inhibitors.  

PubMed

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; Délye, Christophe

2013-01-01

279

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

280

Screening suitable reference genes for normalization in reverse transcription quantitative real-time PCR analysis in melon.  

PubMed

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

281

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.

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

2014-01-01

282

Gene transfer technologies for the production of enzyme and protein reference materials.  

PubMed

To maintain the success of recommended methods and to allow comparison among various methods of enzyme analysis, enzyme reference materials are required, having catalytic properties as close as possible to those of the corresponding human enzymes. Though human sources are preferable, ethical reasons require the extraction and purification from animal tissues. By providing theoretically unlimited amounts of material, gene transfer technologies and mass culture can overcome the need of human or mammalian tissues. We have used these technologies to produce human gamma-glutamyltransferase (GGT) and pancreatic lipase (PL) in various types of host cells. Different strategies were tested, especially for GGT, depending on the inherent properties and requirements of the human enzyme. Expression and purification protocols were optimized, yielding good amounts of recombinant enzymes which share many physico-chemical and catalytic features with their natural counterparts. Kinetic constants and catalytic behavior were very similar, demonstrating the usefulness of these products as reference materials. We assume recombinant DNA technologies could be successfully applied to most enzymes or proteins assayed in clinical chemistry laboratories. PMID:9028623

Oster, T; Visvikis, A; Schiele, F; Wellman-Rousseau, M; Siest, G

1997-01-01

283

The CIN4 Chromosomal Instability qPCR Classifier Defines Tumor Aneuploidy and Stratifies Outcome in Grade 2 Breast Cancer  

PubMed Central

Purpose Quantifying chromosomal instability (CIN) has both prognostic and predictive clinical utility in breast cancer. In order to establish a robust and clinically applicable gene expression-based measure of CIN, we assessed the ability of four qPCR quantified genes selected from the 70-gene Chromosomal Instability (CIN70) expression signature to stratify outcome in patients with grade 2 breast cancer. Methods AURKA, FOXM1, TOP2A and TPX2 (CIN4), were selected from the CIN70 signature due to their high level of correlation with histological grade and mean CIN70 signature expression in silico. We assessed the ability of CIN4 to stratify outcome in an independent cohort of patients diagnosed between 1999 and 2002. 185 formalin-fixed, paraffin-embedded (FFPE) samples were included in the qPCR measurement of CIN4 expression. In parallel, ploidy status of tumors was assessed by flow cytometry. We investigated whether the categorical CIN4 score derived from the CIN4 signature was correlated with recurrence-free survival (RFS) and ploidy status in this cohort. Results We observed a significant association of tumor proliferation, defined by Ki67 and mitotic index (MI), with both CIN4 expression and aneuploidy. The CIN4 score stratified grade 2 carcinomas into good and poor prognostic cohorts (mean RFS: 83.8±4.9 and 69.4±8.2 months, respectively, p?=?0.016) and its predictive power was confirmed by multivariate analysis outperforming MI and Ki67 expression. Conclusions The first clinically applicable qPCR derived measure of tumor aneuploidy from FFPE tissue, stratifies grade 2 tumors into good and poor prognosis groups.

Szasz, Attila Marcell; Li, Qiyuan; Eklund, Aron C.; Sztupinszki, Zsofia; Rowan, Andrew; Tokes, Anna-Maria; Szekely, Borbala; Kiss, Andras; Szendroi, Miklos; Gyorffy, Balazs; Szallasi, Zoltan; Swanton, Charles; Kulka, Janina

2013-01-01

284

Finding the joker among the maize endogenous reference genes for genetically modified organism (GMO) detection.  

PubMed

The comparison of five real-time polymerase chain reaction (PCR) methods targeted at maize ( Zea mays ) endogenous sequences is reported. PCR targets were the alcohol dehydrogenase (adh) gene for three methods and high-mobility group (hmg) gene for the other two. The five real-time PCR methods have been checked under repeatability conditions at several dilution levels on both pooled DNA template from several genetically modified (GM) maize certified reference materials (CRMs) and single CRM DNA extracts. Slopes and R(2) coefficients of all of the curves obtained from the adopted regression model were compared within the same method and among all of the five methods, and the limit of detection and limit of quantitation were analyzed for each PCR system. Furthermore, method equivalency was evaluated on the basis of the ability to estimate the target haploid genome copy number at each concentration level. Results indicated that, among the five methods tested, one of the hmg-targeted PCR systems can be considered equivalent to the others but shows the best regression parameters and a higher repeteability along the dilution range. Thereby, it is proposed as a valid module to be coupled to different event-specific real-time PCR for maize genetically modified organism (GMO) quantitation. The resulting practicability improvement on the analytical control of GMOs is discussed. PMID:19902949

Paternò, Annalisa; Marchesi, Ugo; Gatto, Francesco; Verginelli, Daniela; Quarchioni, Cinzia; Fusco, Cristiana; Zepparoni, Alessia; Amaddeo, Demetrio; Ciabatti, Ilaria

2009-12-01

285

Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder ( Paralichthys olivaceus)  

Microsoft Academic Search

Japanese flounder (Paralichthys olivaceus) is an important economic fish species cultured worldwide. In this report, we compared the potentials of ten housekeeping genes as quantitative real time RT-PCR (qRT-PCR) references for the study of gene expression in Japanese flounder under normal physiological conditions and during bacterial infection. For this purpose, the expression of the ten genes in eight flounder tissues

Wen-jiang Zheng; Li Sun

2011-01-01

286

Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus).  

PubMed

Quantitative real-time reverse-transcriptase polymerase chain reaction (RT-qPCR) has been used frequently to study gene expression related to fish immunology. In such studies, a stable reference gene should be selected to correct the expression of the target gene. In this study, seven candidate reference genes (glyceraldehyde-3-phosphate dehydrogenase (GADPH), ubiquitin-conjugating enzyme (UBCE), 18S ribosomal RNA (18S rRNA), beta-2-microglobulin (B2M), elongation factor 1 alpha (EF1A), tubulin alpha chain-like (TUBA) and beta actin (ACTB)), were selected to analyze their stability and normalization in seven tissues (liver, spleen, kidney, brain, heart, muscle and intestine) of Nile tilapia (Oreochromis niloticus) challenged with Streptococcus agalactiae or Streptococcus iniae, respectively. The results showed that all the candidate reference genes exhibited tissue-dependent transcriptional variations. With PBS injection as a control, UBCE was the most stable and suitable single reference gene in the intestine, liver, brain, kidney, and spleen after S. iniae infection, and in the liver, kidney, and spleen after S. agalactiae infection. EF1A was the most suitable in heart and muscle after S. iniae or S. agalactiae infection. GADPH was the most suitable gene in intestine and brain after S. agalactiae infection. In normal conditions, UBCE and 18S rRNA were the most stably expressed genes across the various tissues. These results showed that for RT-qPCR analysis of tilapia, selecting two or more reference genes may be more suitable for cross-tissue analysis of gene expression. PMID:23792389

Yang, Chang Geng; Wang, Xian Li; Tian, Juan; Liu, Wei; Wu, Fan; Jiang, Ming; Wen, Hua

2013-09-15

287

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.

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

2013-01-01

288

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.

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

289

Identification by Q-PCR of Trypanosoma cruzi lineage and determination of blood meal sources in triatomine gut samples in México.  

PubMed

Triatomine vectors were collected on human dwellings in Michoacán México. Blood meal sources were identified by real time polymerase chain reaction (Q-PCR) using DNA extracted from triatomine guts. The assay was performed with one only specific primer set to amplify a fragment of the mitochondrial 12S ribosomal gene from vertebrate species. Also Trypanosoma cruzi parasites were detected in triatomine gut samples by microscopy and the positive infection was tested in mice. In addition T. cruzi discrete taxonomic units (DTUs) were identified by Q-PCR with two sets of primers that amplify the mini-circle region (miniexon) and 18S ribosomal mitochondrial gene. The sequences obtained from 18S ribosomal gene amplifications confirmed the presence of T. cruzi I and II lineages, and provide evidence of the presence of lineage TcIII and TcIV. PMID:22995149

Ibáñez-Cervantes, Gabriela; Martínez-Ibarra, Alejandro; Nogueda-Torres, Benjamín; López-Orduña, Eduardo; Alonso, Ana L; Perea, Cynthia; Maldonado, Teresa; Hernández, José Manuel; León-Avila, Gloria

2013-02-01

290

Identification of suitable reference genes for quantitative RT-PCR during 3T3-L1 adipocyte differentiation.  

PubMed

Quantitative reverse transcription PCR (qRT-PCR) is becoming increasingly important in the effort to gain insight into the molecular mechanisms underlying adipogenesis. However, the expression profile of a target gene may be misinterpreted due to the unstable expression of the reference genes under different experimental conditions. Therefore, in this study, we investigated the expression stability of 10 commonly used reference genes during 3T3-L1 adipocyte differentiation. The mRNA expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and transferrin receptor (TFRC) significantly increased during the course of 3T3-L1 adipocyte differentiation, which was decreased by berberine, an inhibitor of adipogenesis. Three popular algorithms, GeNorm, NormFinder and BestKeeper, identified 18 ribosomal RNA and hydroxymethylbilane synthase (HMBS) as the most stable reference genes, while GAPDH and TFRC were the least stable ones. Peptidylprolyl isomerase A [PIPA (cyclophilin A)], ribosomal protein, large, P0 (36-B4), beta-2-microglobulin (B2M), ?1-tubulin, hypoxanthine-guanine phosphoribosyltransferase (HPRT) and ?-actin showed relatively stable expression levels. The choice of reference genes with various expression stabilities exerted a profound influence on the expression profiles of 2 target genes, peroxisome proliferator-activated receptor (PPAR)?2 and C/EBP?. In addition, western blot analysis revealed that the increased protein expression of GAPDH was markedly inhibited by berberine during adipocyte differentiation. This study highlights the importance of selecting suitable reference genes for qRT-PCR studies of gene expression during the process of adipogenesis. PMID:24626784

Zhang, Juan; Tang, Hongju; Zhang, Yuqing; Deng, Ruyuan; Shao, Li; Liu, Yun; Li, Fengying; Wang, Xiao; Zhou, Libin

2014-05-01

291

Sequencing and validation of reference genes to analyze endogenous gene expression and quantify yellow dwarf viruses using RT-qPCR in viruliferous Rhopalosiphum padi.  

PubMed

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

292

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.

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

2014-01-01

293

Validation of reference genes as internal control for studying viral infections in cereals by quantitative real-time RT-PCR  

Microsoft Academic Search

BACKGROUND: Reference genes are commonly used as the endogenous normalisation measure for the relative quantification of target genes. The appropriate application of quantitative real-time PCR (RT-qPCR), however, requires the use of reference genes whose level of expression is not affected by the test, by general physiological conditions or by inter-individual variability. For this purpose, seven reference genes were investigated in

Jana Jarošová; Jiban K Kundu

2010-01-01

294

Effective Alu Repeat Based RT-Qpcr Normalization in Cancer Cell Perturbation Experiments  

PubMed Central

Background Measuring messenger RNA (mRNA) levels using the reverse transcription quantitative polymerase chain reaction (RT-qPCR) is common practice in many laboratories. A specific set of mRNAs as internal control reference genes is considered as the preferred strategy to normalize RT-qPCR data. Proper selection of reference genes is a critical issue, especially in cancer cells that are subjected to different in vitro manipulations. These manipulations may result in dramatic alterations in gene expression levels, even of assumed reference genes. In this study, we evaluated the expression levels of 11 commonly used reference genes as internal controls for normalization of 19 experiments that include neuroblastoma, T-ALL, melanoma, breast cancer, non small cell lung cancer (NSCL), acute myeloid leukemia (AML), prostate cancer, colorectal cancer, and cervical cancer cell lines subjected to various perturbations. Results The geNorm algorithm in the software package qbase+ was used to rank the candidate reference genes according to their expression stability. We observed that the stability of most of the candidate reference genes varies greatly in perturbation experiments. Expressed Alu repeats show relatively stable expression regardless of experimental condition. These Alu repeats are ranked among the best reference assays in all perturbation experiments and display acceptable average expression stability values (M<0.5). Conclusions We propose the use of Alu repeats as a reference assay when performing cancer cell perturbation experiments.

Rihani, Ali; Van Maerken, Tom; Pattyn, Filip; Van Peer, Gert; Beckers, Anneleen; De Brouwer, Sara; Kumps, Candy; Mets, Evelien; Van der Meulen, Joni; Rondou, Pieter; Leonelli, Carina; Mestdagh, Pieter; Speleman, Frank; Vandesompele, Jo

2013-01-01

295

Spatial and temporal consistency of putative reference genes for real-time PCR in a model tapeworm.  

PubMed

Relative quantification of gene expression by real-time PCR relies on the use of reference genes whose expressed levels remain consistent across experimental conditions. Here we compare expression levels of commonly employed endogenous housekeeping genes against a developmental regulatory gene in the model tapeworm Hymenolepis microstoma, examining variation both spatially across regions of the adult worm and temporally through the course of larval metamorphosis. ?-Tubulin, RNA polymerase II and 60S ribosomal subunit L28 showed the most variance among candidate reference genes when comparing changes in expression along the anteroposterior gradient of development represented by the adult body, whereas expression of 18S rDNA and cyclic AMP were highly consistent and could be used reliably for relative quantification. The transcription factor Hox4, referenced to either 18S or cAMP, showed 3-fold higher expression levels in the neck region than in more mature regions of the strobila. In contrast, variance among samples representing progressive stages of larval metamorphosis were greater for candidate reference genes than for Hox4, indicating that none of the candidates can be used reliably for quantifying relative changes in gene expression during metamorphosis. PMID:21907248

Pouchkina-Stantcheva, Natalia N; Cunningham, Lucas J; Olson, Peter D

2011-12-01

296

Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons.  

PubMed

Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicate that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons. PMID:15726375

Yang, Litao; Chen, Jianxiu; Huang, Cheng; Liu, Yuhui; Jia, Shirong; Pan, Liangwen; Zhang, Dabing

2005-06-01

297

Expression Stabilities of Candidate Reference Genes for RT-qPCR under Different Stress Conditions in Soybean  

PubMed Central

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.

Liu, Chunji; Zhang, Jie; Hou, Chunyan; Wang, Dongmei

2013-01-01

298

Reference gene selection for real-time quantitative PCR analysis of the mouse uterus in the peri-implantation period.  

PubMed

The study of uterine gene expression patterns is valuable for understanding the biological and molecular mechanisms that occur during embryo implantation. Real-time quantitative RT-PCR (qRT-PCR) is an extremely sensitive technique that allows for the precise quantification of mRNA abundance; however, selecting stable reference genes suitable for the normalization of qRT-PCR data is required to avoid the misinterpretation of experimental results and erroneous analyses. This study employs several mouse models, including an early pregnancy, a pseudopregnancy, a delayed implantation and activation, an artificial decidualization and a hormonal treatment model; ten candidate reference genes (PPIA, RPLP0, HPRT1, GAPDH, ACTB, TBP, B2M, 18S, UBC and TUBA) that are found in uterine tissues were assessed for their suitability as internal controls for relative qRT-PCR quantification. GeNorm(PLUS), NormFinder, and BestKeeper were used to evaluate these candidate reference genes, and all of these methods identified RPLP0 and GAPDH as the most stable candidates and B2M and 18S as the least stable candidates. However, when the different models were analyzed separately, the reference genes exhibited some variation in their expression levels. PMID:23638092

Lin, Pengfei; Lan, Xiangli; Chen, Fenglei; Yang, Yanzhou; Jin, Yaping; Wang, Aihua

2013-01-01

299

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.

2011-01-01

300

Selection of Reference Genes for Gene Expression Studies in Porcine Whole Blood and Peripheral Blood Mononuclear Cells under Polyinosinic:Polycytidylic Acid Stimulation  

PubMed Central

Investigating gene expression of immune cells of whole blood or peripheral blood mononuclear cells (PBMC) under polyinosinic:polycytidylic acid (poly I:C) stimulation is valuable for understanding the immune response of organism to RNA viruses. Quantitative real-time PCR (qRT-PCR) is a standard method for quantification of gene expression studies. However, the reliability of qRT-PCR data critically depends on proper selection of reference genes. In the study, using two different analysis programs, geNorm and NormFinder, we systematically evaluated the gene expression stability of six candidate reference genes (GAPDH, ACTB, B2M, RPL4, TBP, and PPIA) in samples of whole blood and PBMC with or without poly I:C stimulation. Generally, the six candidate genes performed a similar trend of expression stability in the samples of whole blood and PBMC, but more stably expressed in whole blood than in PBMC. geNorm ranked B2M and PPIA as the best combination for gene expression normalization, while according to NormFinder, TBP was ranked as the most stable reference gene, followed by B2M and PPIA. Comprehensively considering the results from the two programs, we recommended using the geometric mean of the three genes, TBP, PPIA and B2M, to normalize the gene expression of whole blood and PBMC with poly I:C stimulation. Our study is the first detailed survey of the gene expression stability in whole blood and PBMC with or without poly I:C stimulation and should be helpful for investigating the molecular mechanism involved in porcine whole blood and PBMC in response to poly I:C stimulation.

Wang, Jiying; Wang, Yanping; Wang, Huaizhong; Hao, Xiaojing; Wu, Ying; Guo, Jianfeng

2014-01-01

301

Evaluation of potential reference genes for qRT-PCR studies in human hepatoma cell lines treated with TNF-?.  

PubMed

In this study, the expression of eight candidate reference genes, B2M, ACTB, GAPDH, HMBS, HPRT1, TBP, UBC, and YWHAZ, was examined to identify optimal reference genes by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis in two human hepatoma cell lines, BEL-7402 and SMMC-7721, treated with tumor necrosis factor-? (TNF-?) for different time periods. The expression stability of these genes was analyzed by three independent algorithms: geNorm, NormFinder, and BestKeeper. Results showed that TBP was the most stably expressed gene in BEL-7402 and SMMC-7721 cell lines under current experimental conditions, and that the optimal set of reference genes required for accurate normalization was TBP and HMBS, based on the pairwise variation value determined with geNorm. UBC and ACTB were ranked as the least stable genes by same algorithms. Our findings provide evidence that using TBP alone or in combination with HMBS as endogenous controls could be a reliable method for normalizing qRT-PCR data in human hepatoma cell lines treated with TNF-?. PMID:23811755

Wu, Chang; Wang, Xiang; Zhong, Ming; Liu, Hailing; He, Qiongqiong; Yang, Xiaojing; Wen, Jifang; Feng, Deyun

2013-09-01

302

Reference Gene Selection for Insect Expression Studies Using Quantitative Real-Time PCR: The Head of the Honeybee, Apis mellifera , After a Bacterial Challenge  

Microsoft Academic Search

In this study an important and often neglected aspect of gene expression studies in insects, the validation of appropriate reference genes with stable expression levels between sample groups, is addressed. Although in this paper the reference gene selection for the honeybee, Apis mellifera L. (Hymenoptera: Apidae) head was tested in the context of bacterial challenge with Escherichia coli, this work

Bieke Scharlaken; Dirk C. de Graaf; Karen Goossens; Marleen Brunain; Luc J. Peelman; Frans J. Jacobs

2008-01-01

303

Exploiting a Reference Genome in Terms of Duplications: The Network of Paralogs and Single Copy Genes in Arabidopsis thaliana  

PubMed Central

Arabidopsis thaliana became the model organism for plant studies because of its small diploid genome, rapid lifecycle and short adult size. Its genome was the first among plants to be sequenced, becoming the reference in plant genomics. However, the Arabidopsis genome is characterized by an inherently complex organization, since it has undergone ancient whole genome duplications, followed by gene reduction, diploidization events and extended rearrangements, which relocated and split up the retained portions. These events, together with probable chromosome reductions, dramatically increased the genome complexity, limiting its role as a reference. The identification of paralogs and single copy genes within a highly duplicated genome is a prerequisite to understand its organization and evolution and to improve its exploitation in comparative genomics. This is still controversial, even in the widely studied Arabidopsis genome. This is also due to the lack of a reference bioinformatics pipeline that could exhaustively identify paralogs and singleton genes. We describe here a complete computational strategy to detect both duplicated and single copy genes in a genome, discussing all the methodological issues that may strongly affect the results, their quality and their reliability. This approach was used to analyze the organization of Arabidopsis nuclear protein coding genes, and besides classifying computationally defined paralogs into networks and single copy genes into different classes, it unraveled further intriguing aspects concerning the genome annotation and the gene relationships in this reference plant species. Since our results may be useful for comparative genomics and genome functional analyses, we organized a dedicated web interface to make them accessible to the scientific community

Sangiovanni, Mara; Vigilante, Alessandra; Chiusano, Maria Luisa

2013-01-01

304

Characterization of reference genes for quantitative real-time PCR analysis in various tissues of Anoectochilus roxburghii.  

PubMed

Accurate quantification of transcript profiling with quantitative real time polymerase chain reaction (qRT-PCR) relies on the reliable normalization of an appropriate reference gene. This study reported the identification and validation of nine reference genes, including ?-tubulin (?-TUB), elongation factor 1 alpha (EF-1?), elongation factor 1 beta (EF-1?), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (UBQ), actin 1/2(ACT-1 and ACT-2), 18S rRNA, and 26S rRNA, from Anoectochilus roxburghii (Wall.) Lindl., a valuable herb remedy widely used for various diseases treatment in traditional Chinese medicine. Transcriptional levels of the candidate reference genes were examined using qRT-PCR analysis and revealed differential expression of the genes in the leaf, stem, root, flower, and peduncle tissues. The relative quantities data were subjected to geNorm software for ranking the expression stability of the reference genes and the results showed that EF-1? and ACT-2 were the two best stable genes whereas GAPDH and 26S rRNA did not favor normalization of qRT-PCR in these tissues. The expression pattern of a squalene synthase encoding gene (SS) was also determined in parallel. The analyses were in great consistency when the qRT-PCR data was normalized to the expression of each or both of EF-1? and ACT-2 as the internal control, further confirming the reliability of EF-1? and ACT-2 as the best internal control. The present study provided the first important clues for accurate data normalization in transcript profiling in A. roxburghii, which will be essential to further functional genomics study in the valuable medicinal plant. PMID:22201024

Zhang, Gang; Zhao, Mingming; Song, Chao; Luo, Anxiong; Bai, Jianfa; Guo, Shunxing

2012-05-01

305

Exploiting a Reference Genome in Terms of Duplications: The Network of Paralogs and Single Copy Genes in Arabidopsis thaliana.  

PubMed

Arabidopsis thaliana became the model organism for plant studies because of its small diploid genome, rapid lifecycle and short adult size. Its genome was the first among plants to be sequenced, becoming the reference in plant genomics. However, the Arabidopsis genome is characterized by an inherently complex organization, since it has undergone ancient whole genome duplications, followed by gene reduction, diploidization events and extended rearrangements, which relocated and split up the retained portions. These events, together with probable chromosome reductions, dramatically increased the genome complexity, limiting its role as a reference. The identification of paralogs and single copy genes within a highly duplicated genome is a prerequisite to understand its organization and evolution and to improve its exploitation in comparative genomics. This is still controversial, even in the widely studied Arabidopsis genome. This is also due to the lack of a reference bioinformatics pipeline that could exhaustively identify paralogs and singleton genes. We describe here a complete computational strategy to detect both duplicated and single copy genes in a genome, discussing all the methodological issues that may strongly affect the results, their quality and their reliability. This approach was used to analyze the organization of Arabidopsis nuclear protein coding genes, and besides classifying computationally defined paralogs into networks and single copy genes into different classes, it unraveled further intriguing aspects concerning the genome annotation and the gene relationships in this reference plant species. Since our results may be useful for comparative genomics and genome functional analyses, we organized a dedicated web interface to make them accessible to the scientific community. PMID:24833233

Sangiovanni, Mara; Vigilante, Alessandra; Chiusano, Maria Luisa

2013-01-01

306

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

Microsoft Academic Search

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)

Kohei Tatsumi; Kazuo Ohashi; Sanae Taminishi; Teruo Okano; Akira Yoshioka; Midori Shima

2008-01-01

307

Normalizing genes for real-time polymerase chain reaction in epithelial and nonepithelial cells of mouse small intestine.  

PubMed

Gene expression studies in intestinal epithelial and stromal cells are a common tool for investigating the mechanisms by which the homeostasis of the small intestine is regulated under normal and pathological conditions. Quantitative real-time PCR (qPCR) is a sensitive and highly reproducible method of gene expression analysis, with expression levels quantified by normalization against reference genes in most cases. However, the lack of suitable reference genes for epithelial cells with different differentiation states and nonepithelial tissue cells has limited the application of qPCR in gene expression studies of small intestinal samples. In this study, 13 housekeeping genes, ACTB, B2M, GAPDH, GUSB, HPRT1, HMBS, HSP90AB1, RPL13A, RPS29, RPLP0,PPIA, TBP, and TUBA1, were analyzed to determine their applicability for isolated crypt cells, villus cells, deepithelialized mucosa, and whole mucosa of the mouse small intestine. Using geNorm and NormFinder software, GUSB and TBP were identified as the most stably expressed genes, whereas the expressions of the commonly used reference genes GAPDH, B2M, and ACTB, and ribosomal protein genes RPL13A, RPS29, and RPLP0 were relatively unstable. Thus, this study demonstrates that GUSB and TBP are the optimal reference genes for the normalization of gene expression in the mouse small intestine. PMID:20036209

Wang, Fengchao; Wang, Junping; Liu, Dengqun; Su, Yongping

2010-04-15

308

Selection of Reference Genes for Expression Analysis in Diuraphis noxia (Hemiptera: Aphididae) Fed on Resistant and Susceptible Wheat Plants.  

PubMed

The Russian wheat aphid (RWA), Diuraphis noxia Kurdjumov, is a major global pest of wheat and barley production that causes enormous economic damage. Few studies have been conducted to explore and decipher the molecular basis of RWA strategies to evade plant defense mechanisms. Gene expression studies of RWA in response to wheat genotypes carrying different RWA resistance genes have been initiated in our group; however, a secure and accurate understanding of RWA gene expression is dependent on identification of suitable reference genes. This study analyzed expression profiles of five potential reference genes selected and sequenced during RNA sequencing experiments. The expression of genes coding for actin and ribosomal protein L27 was comparatively less variable in RWA fed on different wheat hosts. Results of geNorm, NormFinder, and BestKeeper expression analyses support the use of actin and ribosomal protein L27 in RT-qPCR studies of RWA gene expression in studies involving RWA-wheat interactions. PMID:24862828

Sinha, Deepak K; Smith, C Michael

2014-01-01

309

Assessment of reference genes for real-time quantitative PCR gene expression normalization during C2C12 and H9c2 skeletal muscle differentiation.  

PubMed

Skeletal muscle differentiation occurs during muscle development and regeneration. To initiate and maintain the differentiated state, a multitude of gene expression changes occur. Accurate assessment of these differentiation-related gene expression changes requires good quality template, but more specifically, appropriate internal controls for normalization. Two cell line-based models used for in vitro analyses of muscle differentiation incorporate mouse C2C12 and rat H9c2 cells. In this study, we set out to identify the most appropriate controls for mRNA expression normalization during C2C12 and H9c2 differentiation. We assessed the expression profiles of Actb, Gapdh, Hprt, Rps12 and Tbp during C2C12 differentiation and of Gapdh and Rps12 during H9c2 differentiation. Using NormFinder, we validated the stability of the genes individually and of the geometric mean generated from different gene combinations. We verified our results using Myogenin. Our study demonstrates that using the geometric mean of a combination of specific reference genes for normalization provides a platform for more precise test gene expression assessment during myoblast differentiation than using the absolute expression value of an individual gene and reinforces the necessity of reference gene validation. PMID:24146429

Masilamani, Twinkle J; Loiselle, Julie J; Sutherland, Leslie C

2014-04-01

310

Selection of reference genes for quantitative real-time PCR expression studies in the apomictic and sexual grass Brachiaria brizantha  

PubMed Central

Background Brachiaria brizantha is an important forage grass. The occurrence of both apomictic and sexual reproduction within Brachiaria makes it an interesting system for understanding the molecular pathways involved in both modes of reproduction. Quantitative real time PCR (qRT-PCR) has emerged as an important technique to compare expression profile of target genes and, in order to obtain reliable results, it is important to have suitable reference genes. In this work, we evaluated eight potential reference genes for B. brizantha qRT-PCR experiments, isolated from cDNA ovary libraries. Vegetative and reproductive tissues of apomictic and sexual B. brizantha were tested to validate the reference genes, including the female gametophyte, where differences in the expression profile between sexual and apomictic plants must occur. Results Eight genes were selected from a cDNA library of ovaries of B. brizantha considering the similarity to reference genes: EF1 (elongation factor 1 alpha), E1F4A (eukaryotic initiation factor 4A), GAPDH (glucose-6-phosphate dehydrogenase), GDP (glyceroldehyde-3-phosphate dehydrogenase), SUCOA (succinyl-CoA ligase), TUB (tubulin), UBCE (ubiquitin conjugating enzyme), UBI (ubiquitin). For the analysis, total RNA was extracted from 22 samples and raw Ct data after qRT-PCR reaction was analyzed for primer efficiency and for an overall analysis of Ct range among the different samples. Elongation factor 1 alpha showed the highest expression levels, whereas succinyl-CoA ligase showed the lowest within the chosen set of samples. GeNorm application was used for evaluation of the best reference genes, and according to that, the least stable genes, with the highest M values were tubulin and succinyl-CoA ligase and the most stable ones, with the lowest M values were elongation factor 1 alpha and ubiquitin conjugating enzyme, when both reproductive and vegetative samples were tested. For ovaries and spikelets of both sexual and apomictic B. brizantha the genes with the lowest M values were BbrizUBCE, BbrizE1F4A and BbrizEF1. Conclusion In total, eight genes belonging to different cellular processes were tested. Out of them, BbrizTUB was the less stable while BbrizEF1 followed by BbrizUBCE were the more stable genes considering male and female reproductive tissues, spikelets, roots and leaves. Regarding the best reference genes for ovary tissues, where apomictic and sexual reproduction must occur, the best reference genes were BbrizUBCE, BbrizE1F4A and BbrizEF1. Our results provide crucial information for transcriptional analysis in the Brachiaria ssp, helping to improve the quality of gene expression data in these species, which constitute an excellent plant system for the study of apomixis.

Silveira, Erica Duarte; Alves-Ferreira, Marcio; Guimaraes, Larissa Arrais; da Silva, Felipe Rodrigues; Carneiro, Vera Tavares de Campos

2009-01-01

311

Selection of reference genes for quantitative real-time PCR in a rat asphyxial cardiac arrest model  

PubMed Central

Background Cardiac arrest, and the associated arrest of blood circulation, immediately leads to permanent brain damage because of the exhaustion of oxygen, glucose and energy resources in the brain. Most hippocampal CA1 neurons die during the first week post the insult. Molecular data concerning the recovery after resuscitation are sparse and limited to the early time period. Expression analysis of marker genes via quantitative real-time RT-PCR enables to follow up the remodeling process. However, proper validation of the applied normalization strategy is a crucial prerequisite for reliable conclusions. Therefore, the present study aimed to determine the expression stability of ten commonly used reference genes (Actb, actin, beta; B2m, beta-2 microglobulin;CypA, cyclophilin A; Gapdh, glyceraldehyde-3-phosphate dehydrogenase; Hprt, hypoxanthine guanine phosphoribosyl transferase; Pgk1, phosphoglycerate kinase 1; Rpl13a, ribosomal protein L13A; Sdha, succinat dehydrogenase complex, subunit a, flavoprotein (Fp); Tbp, TATA box binding protein; Ywhaz, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide) in the rat hippocampus four, seven and twenty-one days after cardiac arrest. Moreover, experimental groups treated with the anti-inflammatory and anti-apoptotic drug minocycline have been included in the study as well. Results The microglial marker Mac-1, used as a target gene to validate the experimental model, was found to be upregulated about 10- to 20-fold after cardiac arrest. Expression stability of candidate reference genes was analyzed using geNorm and NormFinder software tools. Several of these genes behave rather stable. CypA and Pgk1 were identified by geNorm as the two most stable genes 4 and 21 days after asphyxial cardiac arrest, CypA and Gapdh at 7 days post treatment. B2m turned out to be the most variable candidate reference gene, being about 2-fold upregulated in the cardiac arrest treatment groups. Conclusion We have validated endogenous control genes for qRT-PCR analysis of gene expression in rat hippocampus after resuscitation from cardiac arrest. For normalization purposes in gene profiling studies a combination of CypA and Pgk1 should be considered 4 and 21 days post injury, whereas CypA and Gapdh is the best combination at 7 days. CypA is most favorable if restriction to a single reference gene for all time points is required.

Langnaese, Kristina; John, Robin; Schweizer, Hannes; Ebmeyer, Uwe; Keilhoff, Gerburg

2008-01-01

312

Selection of reference genes for quantitative real-time PCR normalisation in adipose tissue, muscle, liver and mammary gland from ruminants.  

PubMed

The reliability of reverse transcription quantitative real-time PCR (RT-qPCR) depends on normalising the mRNA abundance using carefully selected, stable reference genes. Our aim was to propose sets of reference genes for normalisation in bovine or caprine adipose tissue (AT), mammary gland, liver and muscle. All of these tissues contribute to nutrient partitioning and metabolism and, thus, to the profitability of ruminant productions (i.e. carcasses, meat and milk). In this study, eight commonly used reference genes that belong to different functional classes (CLN3, EIF3K, MRPL39, PPIA, RPLP0, TBP, TOP2B and UXT) were analysed using the geNorm procedure to determine the most stable reference genes in bovine and/or caprine tissues. Abundances and rankings of reference genes varied between tissues, species and the combination of tissues and/or species. Therefore, we proposed 29 sets of reference genes that differed depending on the tissue and/or species. As examples of the 29 sets, EIF3K, TOP2B and UXT were proposed as the most stable reference genes in bovine AT; UXT, EIF3K and RPLP0 were the most stable reference genes in bovine and caprine AT. The optimal number of reference genes for data normalisation was 3 for 27 of the proposed 29 sets. In two of the 29 sets, four to five reference genes were necessary for data normalisation when the number of studied tissues was increased. For example, UXT, EIF3K, TBP, TOP2B and CLN3 were required for data normalisation in bovine mammary gland, AT, muscle and liver. We have evaluated some of our proposed sets of reference genes for the normalisation of CD36 gene expression. Normalisation using the three most stable reference genes has revealed downregulation of CD36 gene expression in bovine mammary gland by a concentrate-based diet that is supplemented with sunflower oil and upregulation of CD36 gene expression in caprine liver by including a rapidly degradable starch in the diet. The dietary regulation of the gene expression of CD36 has been erased by normalisation with the least stable reference genes, which may result in misinterpretation of CD36 gene regulation. To conclude, our results provide valuable reference gene sets for other studies that aim to measure tissue and/or species-specific mRNA abundance in ruminants. PMID:23552195

Bonnet, M; Bernard, L; Bes, S; Leroux, C

2013-08-01

313

Validation of reference genes of grass carp Ctenopharyngodon idellus for the normalization of quantitative real-time PCR.  

PubMed

Expression of four reference genes of grass carp, including beta-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA (18S) and elongation factor-1 alpha (EF1alpha), was studied in tissues of normal individuals and bacteria-infected individuals. EF1alpha had the most stable expressions followed by 18S rRNA then GAPDH; ACTB had the least stability. After being infected with bacteria, the grass carp showed minimal changes in expression levels of EF1alpha in the liver and head kidney, while ACTB had the most stable expressions in spleen but the least stable in liver. EF1alpha is thus the optimal reference gene in quantitative real-time PCR analysis to quantitate the expression levels of target genes in tissues of grass carp. PMID:20361234

Ye, Xing; Zhang, Lili; Dong, Haiyan; Tian, Yuanyuan; Lao, Haihua; Bai, Junjie; Yu, Lingyun

2010-08-01

314

Optimizing qPCR for the Quantification of Periodontal Pathogens in a Complex Plaque Biofilm  

PubMed Central

Quantitative PCR (qPCR) has recently been used to quantify microorganisms in complex communities, including dental plaque biofilms. However, there is variability in the qPCR protocols being used. This study was designed to evaluate the validity of two of these variables with the intent of developing a more standardized qPCR protocol. The two variables evaluated were (1) the use of DNA content versus actual cell counts to estimate bacterial numbers in mixed plaque samples and (2) the effectiveness of three different universal primers versus species specific primers in amplifying specific target pathogens in these samples. Results lead to the development of a standardized protocol that was shown to be highly reproducible as demonstrated by low coefficients of variation. The results also confirmed that this standardized qPCR protocol can be used as a sensitive method for quantifying specific bacterial species in human plaque samples.

Kirakodu, S.S.; Govindaswami, M.; Novak, M.J.; Ebersole, J.L.; Novak, K.F.

2008-01-01

315

Quantitative PCR (QPCR) technique for detecting lymphocytic choriomeningitis virus (LCMV) in vivo  

PubMed Central

Summary Quantitative PCR (QPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models. We have developed a QPCR assay to accurately quantify lymphocytic choriomeningitis virus (LCMV) in infected mice. We first validated this assay using plasmid DNA and LCMV viral stocks. We then demonstrated that the LCMV QPCR assay can detect LCMV in serum and tissues of chronically infected mice (LCMV-clone 13), with greater sensitivity than conventional plaque assay. Subsequently, we demonstrated that the QPCR assay can detect LCMV in tissues of CD40L-/- mice during a low grade chronic infection with LCMV armstrong. Finally, we improved the assay further such that it was approximately 1000-fold more sensitive than plaque assay for detection of the presence of LCMV in tissue.

McCausland, Megan M.; Crotty, Shane

2008-01-01

316

Stable Internal Reference Genes for the Normalization of Real-Time PCR in Different Sweetpotato Cultivars Subjected to Abiotic Stress Conditions  

PubMed Central

Reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most widely used methods for gene expression analysis, but its successful application depends on the stability of suitable reference genes used for data normalization. In plant studies, the choice and optimal number of reference genes must be experimentally determined for the specific conditions, plant species, and cultivars. In this study, ten candidate reference genes of sweetpotato (Ipomoea batatas) were isolated and the stability of their expression was analyzed using two algorithms, geNorm and NormFinder. The samples consisted of tissues from four sweetpotato cultivars subjected to four different environmental stress treatments, i.e., cold, drought, salt and oxidative stress. The results showed that, for sweetpotato, individual reference genes or combinations thereof should be selected for use in data normalization depending on the experimental conditions and the particular cultivar. In general, the genes ARF, UBI, COX, GAP and RPL were validated as the most suitable reference gene set for every cultivar across total tested samples. Interestingly, the genes ACT and TUB, although widely used, were not the most suitable reference genes in different sweetpotato sample sets. Taken together, these results provide guidelines for reference gene(s) selection under different experimental conditions. In addition, they serve as a foundation for the more accurate and widespread use of RT-qPCR in various sweetpotato cultivars.

Ji, Chang Yoon; Park, Seyeon; Jeong, Jae cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

2012-01-01

317

Stable internal reference genes for the normalization of real-time PCR in different sweetpotato cultivars subjected to abiotic stress conditions.  

PubMed

Reverse transcription quantitative real-time PCR (RT-qPCR) has become one of the most widely used methods for gene expression analysis, but its successful application depends on the stability of suitable reference genes used for data normalization. In plant studies, the choice and optimal number of reference genes must be experimentally determined for the specific conditions, plant species, and cultivars. In this study, ten candidate reference genes of sweetpotato (Ipomoea batatas) were isolated and the stability of their expression was analyzed using two algorithms, geNorm and NormFinder. The samples consisted of tissues from four sweetpotato cultivars subjected to four different environmental stress treatments, i.e., cold, drought, salt and oxidative stress. The results showed that, for sweetpotato, individual reference genes or combinations thereof should be selected for use in data normalization depending on the experimental conditions and the particular cultivar. In general, the genes ARF, UBI, COX, GAP and RPL were validated as the most suitable reference gene set for every cultivar across total tested samples. Interestingly, the genes ACT and TUB, although widely used, were not the most suitable reference genes in different sweetpotato sample sets. Taken together, these results provide guidelines for reference gene(s) selection under different experimental conditions. In addition, they serve as a foundation for the more accurate and widespread use of RT-qPCR in various sweetpotato cultivars. PMID:23251557

Park, Sung-Chul; Kim, Yun-Hee; Ji, Chang Yoon; Park, Seyeon; Jeong, Jae Cheol; Lee, Haeng-Soon; Kwak, Sang-Soo

2012-01-01

318

Regulation of gene expression by fatty acids: Special reference to fatty acid-binding protein (FABP)  

Microsoft Academic Search

During the last years, the direct involvement of lipidic nutrients in the regulation of genes has been established. Fatty acids may induce or repress the transcription rate of several genes involved in both lipid and carbohydrate metabolisms. Gene up-regulation has been found in various tissues including liver, adipose tissue and small intestine. It is only triggered by saturated and unsaturated

I. Niot; H. Poirier; Ph. Besnard

1997-01-01

319

Reference genes for high-throughput quantitative reverse transcription-PCR analysis of gene expression in organs and tissues of Eucalyptus grown in various environmental conditions.  

PubMed

Interest in the genomics of Eucalyptus has skyrocketed thanks to the recent sequencing of the genome of Eucalyptus grandis and to a growing number of large-scale transcriptomic studies. Quantitative reverse transcription-PCR (RT-PCR) is the method of choice for gene expression analysis and can now also be used as a high-throughput method. The selection of appropriate internal controls is becoming of utmost importance to ensure accurate expression results in Eucalyptus. To this end, we selected 21 candidate reference genes and used high-throughput microfluidic dynamic arrays to assess their expression among a large panel of developmental and environmental conditions with a special focus on wood-forming tissues. We analyzed the expression stability of these genes by using three distinct statistical algorithms (geNorm, NormFinder and ?Ct), and used principal component analysis to compare methods and rankings. We showed that the most stable genes identified depended not only on the panel of biological samples considered but also on the statistical method used. We then developed a comprehensive integration of the rankings generated by the three methods and identified the optimal reference genes for 17 distinct experimental sets covering 13 organs and tissues, as well as various developmental and environmental conditions. The expression patterns of Eucalyptus master genes EgMYB1 and EgMYB2 experimentally validated our selection. Our findings provide an important resource for the selection of appropriate reference genes for accurate and reliable normalization of gene expression data in the organs and tissues of Eucalyptus trees grown in a range of conditions including abiotic stresses. PMID:23161857

Cassan-Wang, Hua; Soler, Marçal; Yu, Hong; Camargo, Eduardo Leal O; Carocha, Victor; Ladouce, Nathalie; Savelli, Bruno; Paiva, Jorge A P; Leplé, Jean-Charles; Grima-Pettenati, Jacqueline

2012-12-01

320

Selection of suitable reference genes for normalization of quantitative real-time polymerase chain reaction in human cartilage endplate of the lumbar spine.  

PubMed

The quantitative real-time polymerase chain reaction (qRT-PCR) is one of the most widely used methods to study gene expression profiles, and it requires appropriate normalization for accurate and reliable results. Although several genes are commonly used as reference genes (such as GAPDH, ACTB, and 18S rRNA), they are also regulated and can be expressed at varying levels. In this study, we evaluated twelve well-known reference genes to identify the most suitable housekeeping gene for normalization of qRT-PCR in human lumbar vertebral endplate with Modic changes, by using the geNorm, NormFinder, and BestKeeper algorithms. Our results showed that the rarely-used SDHA was the most stable single reference gene, and a combination of three, SDHA, B2M, and LDHA, was the most suitable gene set for normalization in all samples. In addition, the commonly-used genes, GAPDH, ACTB and 18S rRNA, were all inappropriate as internal standards. The rankings of reference genes for the three types of Modic change differed, although SDHA and RPL13A uniformly ranked in the first and last position, respectively. Further simulated expression analysis validated that the arbitrary use of a reference gene could lead to the misinterpretation of data. Our study confirmed the necessity of exploring the expression stability of potential reference genes in each specific tissue and experimental situation before quantitative evaluation of gene expression by qRT-PCR. PMID:24558443

Zhou, Zhi-Jie; Zhang, Jian-Feng; Xia, Ping; Wang, Ji-Ying; Chen, Shuai; Fang, Xiang-Qian; Fan, Shun-Wu

2014-01-01

321

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

322

Laser Capture Microdissection Revisited as a Tool for Transcriptomic Analysis: Application of an Excel-Based qPCR Preparation Software (PREXCEL-Q)  

PubMed Central

The ability to reliably analyze cellular and molecular profiles of normal or diseased tissues is frequently complicated by the inherent heterogeneous nature of tissues. Laser Capture Microdissection (LCM) is an innovative technique that allows the isolation and enrichment of pure subpopulations of cells from tissues under direct microscopic examination. Material obtained by LCM can be used for downstream assays including gene microarrays, western blotting, cDNA library generation and DNA genotyping. We describe a series of LCM protocols for cell collection, RNA extraction and qPCR gene expression analysis. Using reagents we helped develop commercially, we focus on two LCM approaches: laser cutting and laser capture. Reagent calculations have been pre-determined for 10 samples using the new PREXCEL-Q assay development and project management software. One can expect the entire procedure for laser cutting coupled to qPCR to take approximately 12.5-15 h, and laser capture coupled to qPCR to take approximately 13.5-17.5 h.

Sow, Fatoumata B.; Gallup, Jack M.; Sacco, Randy E.; Ackermann, Mark R.

2009-01-01

323

Reference genes for the relative quantification of microRNAs in renal cell carcinomas and their metastases  

Microsoft Academic Search

To obtain accurate results in miRNA expression changes between different sample sets using real-time quantitative polymerase chain reaction (RT-qPCR) analyses, normalization to reference genes that are stably expressed across the sample sets is generally used. A literature search of miRNA expression studies in renal cell carcinoma (RCC) proved that non-miRNAs such as small RNAs or mRNAs have most frequently been

Zofia Wotschofsky; Helmuth-Alexander Meyer; Monika Jung; Annika Fendler; Ina Wagner; Carsten Stephan; Jonas Busch; Andreas Erbersdobler; Alexander C. Disch; Hans-Joachim Mollenkopf; Klaus Jung

2011-01-01

324

Proteomic and targeted qPCR analyses of subsurface microbial communities for presence of methane monooxygenase  

SciTech Connect

The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultraperformance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.

Andrzej J. Paszczynski; Ravindra Paidisetti; Andrew K. Johnson; Ronald L. Crawford; Frederick S. Colwell; Tonia Green; Mark Delwiche; Hope Lee; Deborah Newby; Eoin L. Brodie; Mark Conrad

2011-11-01

325

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.

Rutledge, Robert G.

2011-01-01

326

Evaluation of gene expression data generated from expired Affymetrix GeneChip® microarrays using MAQC reference RNA samples  

Microsoft Academic Search

BACKGROUND: The Affymetrix GeneChip® system is a commonly used platform for microarray analysis but the technology is inherently expensive. Unfortunately, changes in experimental planning and execution, such as the unavailability of previously anticipated samples or a shift in research focus, may render significant numbers of pre-purchased GeneChip® microarrays unprocessed before their manufacturer’s expiration dates. Researchers and microarray core facilities wonder

Zhining Wen; Charles Wang; Quan Shi; Ying Huang; Zhenqiang Su; Huixiao Hong; Weida Tong; Leming Shi

2010-01-01

327

Differentiating microbial forensic qPCR target and control products by electrospray ionization mass spectrometry.  

PubMed

Molecular bioforensic research is dependent on rapid and sensitive methods such as real-time PCR (qPCR) for the identification of microorganisms. The use of synthetic positive control templates containing small modifications outside the primer and probe regions is essential to ensure all aspects of the assay are functioning properly, including the primers and probes. However, a typical qPCR or reverse transcriptase qPCR (qRT-PCR) assay is limited in differentiating products generated from positive controls and biological samples because the fluorescent probe signals generated from each type of amplicon are indistinguishable. Additional methods used to differentiate amplicons, including melt curves, secondary probes, and amplicon sequencing, require significant time to implement and validate and present technical challenges that limit their use for microbial forensic applications. To solve this problem, we have developed a novel application of electrospray ionization mass spectrometry (ESI-MS) to rapidly differentiate qPCR amplicons generated with positive biological samples from those generated with synthetic positive controls. The method has sensitivity equivalent to qPCR and supports the confident and timely determination of the presence of a biothreat agent that is crucial for policymakers and law enforcement. Additionally, it eliminates the need for time-consuming methods to confirm qPCR results, including development and validation of secondary probes or sequencing of small amplicons. In this study, we demonstrate the effectiveness of this approach with microbial forensic qPCR assays targeting multiple biodefense agents (bacterial, viral, and toxin) for the ability to rapidly discriminate between a positive control and a positive sample. PMID:23675878

Motley, S Timothy; Redden, Cassie L; Sannes-Lowery, Kristin A; Eshoo, Mark W; Hofstadler, Steven A; Burans, James P; Rosovitz, M J

2013-06-01

328

Microarray-driven validation of reference genes for quantitative real-time polymerase chain reaction in a rat vocal fold model of mucosal injury  

Microsoft Academic Search

Relative quantification by normalization against a stably expressed reference gene is a widely used data analysis method in microarray and quantitative real-time polymerase chain reaction (qRT-PCR) platforms; however, recent evidence suggests that many commonly utilized reference genes are unstable in certain experimental systems and situations. The primary aim of this study, therefore, was to screen and identify stably expressed reference

Zhen Chang; Changying Ling; Masaru Yamashita; Nathan V. Welham

2010-01-01

329

Selection of reference genes for quantitative real-time PCR analysis in chicken embryo fibroblasts infected with avian leukosis virus subgroup J  

PubMed Central

Background The selection of stably expressed reference genes is a prerequisite when evaluating gene expression, via real-time PCR, in cells in response to viral infections. The objective of our study was to identify suitable reference genes for mRNA expression analysis in chicken embryonic fibroblasts (CEF) after infection with avian leukosis virus subgroup J (ALV-J). Findings The expression levels of 11 potential reference genes in CEF infected with ALV-J were determined by real-time PCR. The expression stability of these genes were analyzed and ranked using the geNorm tool. Analysis indicated that the genes RPL30 (ribosomal protein L30) and SDHA (succinate dehydrogenase complex, subunit A) were the most stably expressed genes in the ALV-J infected CEF. Conclusions The RPL30 and SDHA were deemed suitable for use as reference genes for real-time PCR analysis of mRNA gene expression during ALV-J infection, whereas commonly used ACTB and GAPDH are unsuitable to be reference genes.

2013-01-01

330

De novo characterization of the gene-rich transcriptomes of two color-polymorphic spiders, Theridion grallator and T. californicum (Araneae: Theridiidae), with special reference to pigment genes  

PubMed Central

Background A number of spider species within the family Theridiidae exhibit a dramatic abdominal (opisthosomal) color polymorphism. The polymorphism is inherited in a broadly Mendelian fashion and in some species consists of dozens of discrete morphs that are convergent across taxa and populations. Few genomic resources exist for spiders. Here, as a first necessary step towards identifying the genetic basis for this trait we present the near complete transcriptomes of two species: the Hawaiian happy-face spider Theridion grallator and Theridion californicum. We mined the gene complement for pigment-pathway genes and examined differential expression (DE) between morphs that are unpatterned (plain yellow) and patterned (yellow with superimposed patches of red, white or very dark brown). Results By deep sequencing both RNA-seq and normalized cDNA libraries from pooled specimens of each species we were able to assemble a comprehensive gene set for both species that we estimate to be 98-99% complete. It is likely that these species express more than 20,000 protein-coding genes, perhaps 4.5% (ca. 870) of which might be unique to spiders. Mining for pigment-associated Drosophila melanogaster genes indicated the presence of all ommochrome pathway genes and most pteridine pathway genes and DE analyses further indicate a possible role for the pteridine pathway in theridiid color patterning. Conclusions Based upon our estimates, T. grallator and T. californicum express a large inventory of protein-coding genes. Our comprehensive assembly illustrates the continuing value of sequencing normalized cDNA libraries in addition to RNA-seq in order to generate a reference transcriptome for non-model species. The identification of pteridine-related genes and their possible involvement in color patterning is a novel finding in spiders and one that suggests a biochemical link between guanine deposits and the pigments exhibited by these species.

2013-01-01

331

Model of gene expression in extreme cold - reference transcriptome for the high-Antarctic cryopelagic notothenioid fish Pagothenia borchgrevinki  

PubMed Central

Background Among the cold-adapted Antarctic notothenioid fishes, the high-latitude bald notothen Pagothenia borchgrevinki is particularly notable as the sole cryopelagic species, exploiting the coldest and iciest waters of the Southern Ocean. Because P. borchgrevinki is a frequent model for investigating notothenioid cold-adaptation and specialization, it is imperative that “omic” tools be developed for this species. In the absence of a sequenced genome, a well annotated reference transcriptome of the bald notothen will serve as a model of gene expression in the coldest and harshest of all polar marine environments, useful for future comparative studies of cold adaptation and thermal responses in polar teleosts and ectotherms. Results We sequenced and annotated a reference transcriptome for P. borchgrevinki, with added attention to capturing the transcriptional responses to acute and chronic heat exposures. We sequenced by Roche 454 a normalized cDNA library constructed from pooled mRNA encompassing multiple tissues taken from environmental, warm acclimating, and acute heat stressed specimens. The resulting reads were assembled into 42,620 contigs, 17,951 of which could be annotated. We utilized this annotated portion of the reference transcriptome to map short Illumina reads sequenced from the gill and liver of environmental specimens, and also compared the gene expression profiles of these two tissue transcriptomes with those from the temperate model fish Danio rerio. From this, we identified a conserved group of 58 GO terms, in which terms related to transcription and its regulation, ubiquitin-protein ligase activity, protein ubiquitination, and protein binding among others are more prevalent in the bald notothen, suggesting the pertinent genes play essential roles in cold temperature functioning. Conclusion We sequenced multiple tissue transcriptomes from native and heat-exposed experimental specimens of the high Antarctic, cryopelagic notothenioid P. borchgrevinki to construct a reference transcriptome. In a proof of concept, we utilized the annotated reference transcriptome to profile the gene expression patterns of gill and liver, and identified a suite of over and under-represented GO terms when compared to the tropical water zebrafish suggesting these functions may be important for surviving in freezing waters. The transcriptome resource from this study will aid future investigations of cold adaptation and thermal response of polar ectothermic species.

2013-01-01

332

PTPN22 gene polymorphisms in autoimmune diseases with special reference to systemic lupus erythematosus disease susceptibility.  

PubMed

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease. SLE is a result of one or more immune mechanisms, like autoantibody production, complement activation, multiple inflammation and immune complex deposition leading to organ tissue damage. SLE affected patients are susceptible to common and opportunistic infections. There are several reports suggesting that Mycobacterium tuberculosis infection precipitates SLE in patients from endemic areas. Genetic factors and environmental factors also play an important role in the overall susceptibility to SLE pathophysiology. Recently, protein tyrosine phosphatase, non-receptor type 22 (PTPN22) gene, has been found to be associated with several autoimmune diseases like SLE, Grave's disease and Hashimoto thyroiditis. The missense R620W polymorphism, rs 2476601, in PTPN22 gene at the nucleotide 1858 in codon 620 (620Arg > Trp) has been associated with autoimmune diseases. The PTPN22 locus is also found to be responsible for development of pulmonary tuberculosis in certain populations. The PTPN22 1858C/T gene locus will be ideal to look for SLE susceptibility to tuberculosis in the Indian population. In this review, we focus on human PTPN22 gene structure and function as well as the association of PTPN22 gene polymorphisms with SLE susceptibility. PMID:20739780

Pradhan, V; Borse, V; Ghosh, K

2010-01-01

333

Suitable reference genes for the analysis of direct hyperplasia in mice  

SciTech Connect

The liver is capable of undergoing a proliferative growth, known as direct hyperplasia, in which the naive liver increases in size due to stimulation with primary mitogens. To produce accurate gene expression data, housekeeping genes (HKGs) that are stably expressed need to be determined. In the present study, liver regeneration was promoted via the direct hyperplasia mode by inducing mice with 1,4-bis[2-(3,5-dichloropyridyloxy)]benzene. Gene expression levels of nine commonly used HKGs were analyzed in the liver of different timing during the regeneration. The stability of gene expression was assessed using two different analysis programs, geNorm and NormFinder. Using these analyses, we identified that PPIA and RPL4 showed the most stable expression regardless of the status of the liver regeneration. In conclusion, the present study demonstrated that the use of PPIA and RPL4 were the most optimal in providing reliable normalization of gene expression when assessing liver regeneration attributed to direct hyperplasia.

Takagi, Soichi [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (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; Utoh, Rie [Institute of Advanced Biomedical Engineering and Science, Tokyo Women's Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666 (Japan); Tatsumi, Kohei; Shima, Midori [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)

2008-12-26

334

Addressing fluorogenic real-time qpcr inhibition using the novel custom excel file system ‘FocusField2-6GallupqPCRSet-upTool-001’ to attain consistently high fidelity qPCR reactions  

Microsoft Academic Search

The purpose of this manuscript is to discuss fluorogenic real-time quantitative polymerase chain reaction (qPCR) inhibition\\u000a and to introduce\\/define a novel Microsoft Excel-based file system which provides a way to detect and avoid inhibition, and\\u000a enables investigators to consistently design dynamically-sound, truly LOG-linear qPCR reactions very quickly. The qPCR problems\\u000a this invention solves are universal to all qPCR reactions, and

Jack M. Gallup; Mark R. Ackermann

2006-01-01

335

EF1? is a useful internal reference for studies of gene expression regulation in amphioxus Branchiostoma japonicum.  

PubMed

Amphioxus is a well-known model organism widely used for interspecies comparative genome study, developmental homology analysis and comparative immunological investigation. However, no study has been performed so far to evaluate the internal reference for quantitative RT-PCR (qRT-PCR) studies of gene expression in this important species. In this study, two software applications (geNorm and NormFinder) were used to evaluate the expression stability of 4 housekeeping genes (ACTB, GAPDH, 18S rRNA and EF1?) in 8 different normal tissues (whole body, gut, gut-free body, hepatic caecum, gill, hind-gut, notochord and muscle) and 2 tissues (gut and gut-free body) challenged with LPS and LTA in amphioxus Branchiostoma japonicum. Our results showed that in the normal tissues, the expression of 18S rRNA was most abundant, whereas the expression levels of the other three genes were close to each other, with the expression of ACTB being most unstable. Following challenge with LPS and LTA, all the four genes exhibited varied degrees of expression changes in the different tissues and the expression stabilities of the genes were also affected by the different experimental conditions. Yet, the overall ranking results produced by the two algorithms consistently indicated that the expression of EF1? showed the most least variation in the different tissues, suggesting that EF1? is a suitable internal control for qRT-PCR studies in amphioxus B. japonicum. PMID:22554576

Wang, Yanfeng; Zhang, Shicui

2012-06-01

336

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.

2009-01-01

337

Selection and Validation of Reference Genes for Quantitative Real-Time PCR in Buckwheat (Fagopyrum esculentum) Based on Transcriptome Sequence Data  

PubMed Central

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications – geNorm, NormFinder and BestKeeper - were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

Demidenko, Natalia V.; Logacheva, Maria D.; Penin, Aleksey A.

2011-01-01

338

Multiplex qPCR for Detection and Absolute Quantification of Malaria  

PubMed Central

We describe development of an absolute multiplex quantitative real-time PCR for detection of Plasmodium spp., P. falciparum and P. vivax targets in order to produce an assay amenable to high throughput but with reduced costs. Important qPCR experimental details and information that is critical to performance and reliability of assay results were investigated. Inhibition studies were performed to test and compare co-purification of PCR inhibitors in samples extracted from whole blood using either the manual or automated methods. To establish the most optimal qPCR reaction volume, volume titration of the reaction master mix was performed starting at 10 µl to 1 µl reaction master mix with 1 µl of template DNA in each reaction. As the reaction volume decreased, qPCR assays became more efficient with 1 µl reaction master mix being the most efficient. For more accurate quantification of parasites in a sample, we developed plasmid DNAs for all the three assay targets for absolut