Computational protocol: An overview of technical considerations when using quantitative real-time PCR analysis of gene expression in human exercise research

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Protocol publication

[…] Several traditional reference genes have been widely used in the qPCR analysis. A review article has reported that 33% and 32% of the expression analysis from 6 high-impact journals used glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and actin beta (ACTB) as reference genes respectively for papers published in 1999 []. However, the same review pointed out that the expression of both GAPDH and ACTB varies considerably under different experimental settings in a range of tissues []. GAPDH was originally identified as an intermediate in glycolysis pathway and expected to be stably present in all cells; thus it was selected as a reference gene. However, other activities of GAPDH, including functions in endocytosis, translational control, and DNA replication [], were not recognised until later [].Various reference genes have been used in different exercise studies. Mahoney et al. -[] reported that β-2-microglobulin (B2M) and ACTB were the most stable reference genes following 300 eccentric contractions, whereas B2M and GAPDH were the most stable following 75 min of high-intensity intermittent cycling. In another study, muscle biopsies were taken before, immediately after, and 4 h following 30 min of treadmill running at 70% of VO2max, and RNA was extracted from 40 single fibres. GAPDH was found to be stably expressed in all samples []. Thus, when using reference genes as internal controls for an exercise study, the stability of each reference gene needs to be evaluated carefully, and there is no ‘one-size-fits-all’ gene that can be used in all studies and with all exercise protocols.In order to reduce the variability of internal control, it is recommended to use multiple genes for normalisation [, ]. Using samples obtained from neuroblastoma cell lines, Vandesompele and his colleagues showed that normalisation using a single reference gene led to differences of 3.0-fold in 25%, and 6.4-fold in 10%, of the cases analysed []. The evaluation of reference genes can be achieved by running a statistical analysis on the Cq value or using available software. Several software programs are available for reference gene evaluation using different analytical approaches, such as BestKeeper [], NormFinder [], and GeNorm [].In our laboratory, we have six commonly-used reference genes, ACTB, TATA-box binding protein (TBP), Cyclophilin, GAPDH, B2M, and 18S rRNA (). There are a few reasons why these candidate reference genes were chosen. First of all, these six genes are potentially stably-expressed reference genes, and are widely used in the qPCR analysis of skeletal muscle samples obtained in human exercise studies [, , ]. Second, they belong to different functional classes; thus it is unlikely they are co-regulated. However, using comparative Cq method, it is difficult to qualify small differences in gene expression (i.e., less than 2-fold) unless multiple stably-expressed reference genes are used for normalisation [, ]. […]

Pipeline specifications

Software tools BestKeeper, NormFinder
Application qPCR
Organisms Homo sapiens