Dataset Information

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

ABSTRACT:

Background

The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.

Results

The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard deviation computations with the hkgFinder tool. Our data revealed that the mRNA expression levels of three ribosomal RNAs (RDN5.8, RDN18, and RDN25) remained stable in response to fluconazole, while PGK1, UBC7, and UBC13 mRNAs showed only approximately 2.9-, 3.0-, and 2.5-fold induction by azole, respectively. By contrast, mRNA levels of the other 10 reference genes (ACT1, EF1?, GAPDH, PPIA, RPL2A, RPL10, RPL13A, SDHA, TUB1, and UBC4) were dramatically increased in C. glabrata following antifungal treatment, exhibiting changes ranging from 4.5- to 32.7-fold. We also assessed the expression stability of these reference genes using the 2(-??CT) method and three other software packages. The stability rankings of the reference genes by geNorm and the 2(-??CT) method were identical to those by hkgFinder, whereas the stability rankings by BestKeeper and NormFinder were notably different. We then validated the suitability of six candidate reference genes (ACT1, PGK1, RDN5.8, RDN18, UBC7, and UBC13) as internal controls for ten target genes in this system using the comparative CT method. Our validation experiments passed for all six reference genes analyzed except RDN18, where the amplification efficiency of RDN18 was different from that of the ten target genes. Finally, we demonstrated that the relative quantification of target gene expression varied according to the endogenous control used, highlighting the importance of the choice of internal controls in such experiments.

Conclusions

We recommend the use of RDN5.8, UBC13, and PGK1 alone or the combination of RDN5.8 plus UBC13 or PGK1 as reference genes for RT-qPCR analysis of gene expression in C. glabrata following azole treatment. In contrast, we show that ACT1 and other commonly used reference genes (GAPDH, PPIA, RPL13A, TUB1, etc.) were not validated as good internal controls in the current model.

SUBMITTER: Li QQ

PROVIDER: S-EPMC3482582 | biostudies-literature | 2012 Jun

REPOSITORIES: biostudies-literature

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Publications

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

Li Qingdi Quentin QQ Skinner Jeff J Bennett John E JE

BMC molecular biology 20120629

<h4>Background</h4>The selection of stable and suitable reference genes for real-time quantitative PCR (RT-qPCR) is a crucial prerequisite for reliable gene expression analysis under different experimental conditions. The present study aimed to identify reference genes as internal controls for gene expression studies by RT-qPCR in azole-stimulated Candida glabrata.<h4>Results</h4>The expression stability of 16 reference genes under fluconazole stress was evaluated using fold change and standard ...[more]

PMID: 22747760

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Project description:BackgroundQuantitative real-time PCR (qPCR) is a widely used technique for gene expression analysis. Its reliability is highly dependent upon selection of the appropriate reference genes for accurate gene expression normalization. In this study, we investigated the expression stability of 10 commonly used reference genes in a mouse myocardial infarction model.Methods & resultsThe expression stability of the 10 reference genes (Actb, B2m, Eef1a1, Gapdh, Hprt, Polr2a, Ppia, Rpl13a, Tbp, Tpt1) was analyzed using the geNorm software. Overall, the combination of Hprt, Rpl13a and Tpt1 was the most stable reference gene set in our experiments. Gapdh, Polr2a and Actb consistently showed the highest gene expression variability and the expression levels of Gapdh, Polr2a, Actb, B2m and Eef1a1 were found to be selectively up- or downregulated after myocardial infarction. We normalized the expression of Nppb and Vcam1, using different reference gene strategies and demonstrated that their induction after myocardial infarction was most clearly revealed with the optimal reference gene combination. However, the use of suboptimal reference gene combinations resulted in detrimental effects on gene expression levels and variability with a gradual loss of the expression differences and a significant reduction in statistical power.ConclusionsHprt, Rpl13a and Tpt1 are a set of stably expressed reference genes for accurate gene expression normalization in myocardial infarction studies in mice. We found that Gapdh, Polr2a and Actb display high expression variability in mouse myocardial infarction tissues and that loss of statistical power and increase in sample size are the evident consequences of choosing suboptimal combinations of reference genes. We furthermore caution against the use of Gapdh, Polr2a, Actb, B2m and Eef1a1 for gene expression normalization in myocardial infarction studies because of selective up- or downregulation after myocardial infarction, which could potentially lead to biased study outcomes.

Project description: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, EF1α, 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.

Dataset Information

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

Background

Results

Conclusions

Publications

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

Similar Datasets

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