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

Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experim...

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Bibliographic Details
Published in:	PloS one 2018-05, Vol.13 (5), p.e0196438-e0196438
Main Authors:	Kuang, Jujiao, Yan, Xu, Genders, Amanda J, Granata, Cesare, Bishop, David J
Format:	Article
Language:	eng
Subjects:	Biology and life sciences Biopsy Breast cancer Data processing Deoxyribonucleic acid DNA DNA polymerase Enzymes Exercise Gene expression Genetic engineering Health aspects Laboratory tests Medicine and Health Sciences Methods Muscles Musculoskeletal system Polymerase chain reaction Proteins Quality Quality control Reproducibility Research and analysis methods Ribonucleic acid RNA Skeletal muscle Workflow
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Summary:	Gene expression analysis by quantitative PCR in skeletal muscle is routine in exercise studies. The reproducibility and reliability of the data fundamentally depend on how the experiments are performed and interpreted. Despite the popularity of the assay, there is a considerable variation in experimental protocols and data analyses from different laboratories, and there is a lack of consistency of proper quality control steps throughout the assay. In this study, we present a number of experiments on various steps of quantitative PCR workflow, and demonstrate how to perform a quantitative PCR experiment with human skeletal muscle samples in an exercise study. We also tested some common mistakes in performing qPCR. Interestingly, we found that mishandling of muscle for a short time span (10 mins) before RNA extraction did not affect RNA quality, and isolated total RNA was preserved for up to one week at room temperature. Demonstrated by our data, use of unstable reference genes lead to substantial differences in the final results. Alternatively, cDNA content can be used for data normalisation; however, complete removal of RNA from cDNA samples is essential for obtaining accurate cDNA content.
ISSN:	1932-6203 1932-6203