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Physiol. Genomics (May 25, 2004). doi:10.1152/physiolgenomics.00067.2004
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Submitted on March 18, 2004
Accepted on May 12, 2004

Real-time RT-PCR analysis of housekeeping genes in human skeletal muscle following acute exercise

Douglas J Mahoney1, Kate Carey2, Ming-Hua Fu1, Rodney Snow2, David Cameron Smith2, Gianni Parise3, and Mark A Tarnopolsky4*

1 Department of Medical Sciences, McMaster University, Hamilton, ON, Canada
2 Department of Health Sciences, Deakin University, Burwood, Victoria, Australia
3 Department of Kinesiology, McMaster University, Hamilton, ON, Canada
4 Department of Pediatrics, McMaster University, Hamilton, ON, Canada

* To whom correspondence should be addressed. E-mail: tarnopol{at}mcmaster.ca.

Studies examining gene expression with RT-PCR typically normalize their mRNA data to a constitutively expressed housekeeping gene. The validity of a particular housekeeping gene must be determined for each experimental intervention. We examined the expression of various housekeeping genes following an acute bout of endurance (END) or resistance (RES) exercise. Twenty-four healthy subjects performed either an interval-type cycle ergometry workout to exhaustion (~75 minutes; END) or 300 single leg eccentric contractions (RES). Muscle biopsies were taken before, and 3 h and 48 h following exercise. Real-time RT-PCR was performed on {beta}-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and {beta}2-microglobulin ({beta}2M). In a second study, 10 healthy subjects performed 90 minutes of cycle ergometry at ~65% of VO2max, and we examined a fifth housekeeping gene, 28S ribosomal RNA (rRNA), and re-examined {beta}2M, from muscle biopsy samples taken immediately post-exercise. We showed that CYC increased 48 h following both END and RES exercise (3 and 5 fold, respectively; p < 0.01), and 28S rRNA increased immediately following END exercise (2 fold; p = 0.02). {beta}-actin trended toward an increase following END exercise (1.85 fold collapsed across time; p = 0.13), and GAPDH trended toward a small yet robust increase at 3 h following RES exercise (1.4 fold; p = 0.067). In contrast, {beta}2M was not altered at any time point post-exercise. We conclude that {beta}2M and {beta}-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, while {beta}2M and GAPDH are the most stably expressed following END exercise.




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