Dataset Information

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses.

ABSTRACT: Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stability in various samples, including different tissues and under different abiotic stress treatments. The expression of these genes was determined using five programs-geNorm, NormFinder, BestKeeper, ?Ct, and RefFinder. The results showed that ?-TUB2 (alpha-tubulin 2) and DnaJ (chaperone protein DnaJ 49) were the most stable reference genes across all the tested samples. We measured the expression profiles of the defense response gene SmCAT (catalase) using the two most stable and one least stable reference genes in all samples of S. matsudana. The relative quantification of SmCAT varied greatly according to the different reference genes. We propose that ?-TUB2 and DnaJ should be the preferred reference genes for normalization and quantification of transcript levels in future gene expression studies in willow species under various abiotic stress conditions.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC5264508 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses.

Zhang Yunxing Y Han Xiaojiao X Chen Shuangshuang S Zheng Liu L He Xuelian X Liu Mingying M Qiao Guirong G Wang Yang Y Zhuo Renying R

Scientific reports 20170125

Salix matsudana is a deciduous, rapidly growing willow species commonly cultivated in China, which can tolerate drought, salt, and heavy metal stress conditions. Selection of suitable reference genes for quantitative real-time PCR is important for normalizing the expression of the key genes associated with various stresses. To validate suitable reference genes, we selected 11 candidate reference genes (five traditional housekeeping genes and six novel genes) and analyzed their expression stabili ...[more]

PMID: 28120870

Similar Datasets

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.

Dataset Information

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses.

Publications

Selection of suitable reference genes for quantitative real-time PCR gene expression analysis in Salix matsudana under different abiotic stresses.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets