Repeatability of exercise‐induced changes in mRNA expression and technical considerations for qPCR analysis in human skeletal muscle

Abstract

New Findings What is the central question of this study?Are individual changes in exercise‐induced mRNA expression repeatable (i.e. representative of the true response to exercise rather than random error)? What is the main finding and its importance?Exercise‐induced changes in mRNA expression are not repeatable even under identical experimental conditions, thereby challenging the use of mRNA expression as a biomarker of adaptive potential and/or individual responsiveness to exercise. Abstract It remains unknown if (1) the observed change in mRNA expression reflects an individual's true response to exercise or random (technical and/or biological) error, and (2) the individual responsiveness to exercise is protocol‐specific. We examined the repeatability of skeletal muscle PGC‐1α, PDK4, NRF‐1, VEGF‐A, HSP72 and p53 mRNA expression following two identical endurance exercise (END) bouts (END‐1, END‐2; 30 min of cycling at 65% of peak work rate (WRpeak), n = 11) and inter‐individual variability in PGC‐1α and PDK4 mRNA expression following END and sprint interval training (SIT; 8 × 20 s cycling intervals at ∼170% WRpeak, n = 10) in active young males. The repeatability of key gene analysis steps (RNA extraction, reverse transcription, qPCR) and within‐sample fibre‐type distribution (n = 8) was also determined to examine potential sources of technical error in our analyses. Despite highly repeatable exercise bout characteristics (work rate, heart rate, blood lactate; ICC > 0.71; CV < 10%; r > 0.85, P < 0.01), gene analysis steps (ICC > 0.73; CV < 24%; r > 0.75, P < 0.01), and similar group‐level changes in mRNA expression, individual changes in PGC‐1α, PDK4, VEGF‐A and p53 mRNA expression were not repeatable (ICC < 0.22; CV > 20%; r < 0.21). Fibre‐type distribution in two portions of the same muscle biopsy was highly variable and not significantly related (ICC = 0.39; CV = 26%; r = 0.37, P = 0.37). Since individual changes in mRNA expression following identical exercise bouts were not repeatable, inferences regarding individual responsiveness to END or SIT were not made. Substantial random error exists in changes in mRNA expression following acute exercise, thereby challenging the use of mRNA expression for analysing individual responsiveness to exercise.

Investigating the reproducibility of maximal oxygen uptake responses to high-intensity interval training

“…Further, we recently found poor reproducibility for acute changes in skeletal muscle mRNA expression following identical exercise bouts in a similar population. 27 Because changes in mRNA expression following acute exercise are thought to represent the initiation of adaptive processes that contribute to chronic changes in aerobic capacity, 28 the lack of reproducibility in mRNA responses may partly explain the lack of reproducibility in observed VO 2 max responses to training.…”

The Development of a Personalised Training Framework: Implementation of Emerging Technologies for Performance

“…Interestingly, there have been wide differences in measured mRNA expression within muscle between trained and untrained subjects in response to exercise [149,150], suggesting that transcriptomics may hold promise as a "predictor" of training outcomes. More recently, however, authors have suggested that the association between increased mRNA expression and increased gene expression may not be as strong as once thought [151], and, indeed, may be due to technical error or random biological variation [152]; as a result, transcriptomics may not be as useful as proteomics within the personalised medicine approach to athlete preparation.…”

Interpretation of exercise-induced changes in human skeletal muscle mRNA expression depends on the timing of the post-exercise biopsies

“…This is consistent with previous research showing that not all individuals respond the same way to a standardised exercise dose [39][40][41] . This individual response could be due to genetic background, non-genetic biological and behavioral factors, day-to-day fluctuations, and technical or biological variability associated with the sampling of human skeletal muscle and the analysis of exercise-induced mRNA 42,43 .…”