Abstract
Relative quantitative real-time PCR (qPCR) assays serve as important tools for validating differential gene expression data. A reference gene that is stably expressed across sample types and experimental treatments is crucial for accuracy in interpretation of relative qPCR data. Twelve previously validated reference genes were evaluated in this study to identify a most suitable reference gene that can be used for gene expression study of bovine tuberculosis (bTB) infected and bTB test-false positive cattle, in peripheral blood mononuclear cells after a 4 hour or after an overnight stimulation with bovine tuberculin antigen. Stability of the candidate reference genes were evaluated using the BestKeeper, geNorm and NormFinder programs. The SDHA was found to be the most stably expressed reference gene, regardless of infection status and varying length (4 hours or overnight) of antigen stimulation, while expression of many widely used reference genes are not stable under the studied experimental conditions. We also confirmed that the geNorm and NormFinder programs yielded similar findings in determining the stability of reference genes, which differ largely from the BestKeeper program. This finding stresses the importance of validating the reference gene(s) chosen for each experimental study, and the need for using multiple programs for the evaluation.
Highlights
Quantitative real-time PCR has become the method of choice for quantification of gene expression levels
Use of a reference gene with an expression level that fluctuates randomly can lead to increased non-specific variation, and use of a reference gene with an expression level that changes with the sample type or with experimental treatments can lead to erroneous interpretation of data [1, 5, 6]
The Quantitative real-time PCR (qPCR) data (Ct value) of the 12 reference genes were generated for each animal using RNA that was subjected to 3 different treatments
Summary
Quantitative real-time PCR (qPCR) has become the method of choice for quantification of gene expression levels. In relative qPCR, a reference gene is used to normalize disparities in RNA recovery and cDNA synthesis efficiency. This permits true comparisons of gene regulation between samples from within a group, and between samples among different groups [1, 3, 4]. The underlying assumption is that the reference gene is expressed at a constant level, and the level of expression remains unchanged across sample types and experimental treatments. Use of a reference gene with an expression level that fluctuates randomly can lead to increased non-specific variation, and use of a reference gene with an expression level that changes with the sample type or with experimental treatments can lead to erroneous interpretation of data [1, 5, 6]
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