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

Abstract

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 a interval-type cycle ergometry workout to exhaustion ( approximately 75 min; END) or 300 single-leg eccentric contractions (RES). Muscle biopsies were taken before exercise 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 beta2-microglobulin (beta2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at approximately 65% of Vo(2 max), and we examined a fifth housekeeping gene, 28S rRNA, and reexamined beta2M, from muscle biopsy samples taken immediately postexercise. 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, beta2M was not altered at any time point postexercise. We conclude that beta2M and beta-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas beta2M and GAPDH are the most stably expressed following END exercise.