Effect of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR

The present study examined the validity and reliability of measuring the expression of various genes in human skeletal muscle using quantitative real-time RT-PCR on a GeneAmp 5700 sequence detection system with SYBR Green 1 chemistry. In addition, the validity of using some of these genes as endogenous controls (i.e., housekeeping genes) when human skeletal muscle was exposed to elevated total creatine levels and exercise was also examined. For all except 28S, linear relationships between the logarithm of the starting RNA concentrations and the cycle threshold (C_T) values were established for ß-actin, ß2-microglobulin (ß₂M), cyclophilin (CYC), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). We found a linear response between C_T values and the logarithm of a given amount of starting cDNA for all the genes tested. The overall intra-assay coefficient of variance for these genes was 1.3% and 21% for raw C_T values and the linear value of 2^-C_T, respectively. Interassay variability was 2.3% for raw C_T values and 34% for the linear value of 2^-C_T. We also examined the expression of various housekeeping genes in human skeletal muscle at days 0, 1, and 5 following oral supplementation with either creatine or a placebo employing a double-blind crossover study design. Treatments were separated by a 5-wk washout period. Immediately following each muscle sampling, subjects performed two 30-s all-out bouts on a cycle ergometer. Creatine supplementation increased (P < 0.05) muscle total creatine content above placebo levels; however, there were no changes (P > 0.05) in C_T values across the supplementation periods for any of the genes. Nevertheless, 95% confidence intervals showed that GAPDH was variable, whereas ß-actin, ß₂M, and CYC were the least varying genes. Normalization of the data to these housekeeping genes revealed variable behavior for ß₂M with more stable expressions for both ß-actin and CYC. We conclude that, using real-time RT-PCR, ß-actin or CYC may be used as housekeeping genes to study gene expression in human muscle in experiments employing short-term creatine supplementation combined with high-intensity exercise.

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

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 (~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 ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M). In a second study, 10 healthy subjects performed 90 min of cycle ergometry at ~65% of O2 max, and we examined a fifth housekeeping gene, 28S rRNA, and reexamined ß2M, 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). ß-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, ß2M was not altered at any time point postexercise. We conclude that ß2M and ß-actin are the most stably expressed housekeeping genes in skeletal muscle following RES exercise, whereas ß2M and GAPDH are the most stably expressed following END exercise.

Frequency distribution of TATA box and extension sequences on human promoters

TATA box is one of the most important transcription factor binding sites. But the exact sequences of TATA box are still not very clear yet. In this study, we conducted a dedicated analysis on the frequency distribution of TATA Box and its extension sequences on human promoters. Sixteen TATA elements derived from TATA Box motif, TATAWAWN, were classified into three distribution patterns: peak, bottom-peak and bottom. Fourteen TATA extension sequences (up to two base extensions) were predicted to be the new TATA Box elements because of their high motif factors, which indicate their statistical significance. Statistical analysis on the promoters of mouse, zebrafish and drosophila melanogaster verified seven of these elements. It was also observed that the distribution of TATA elements on the promoters of housekeeping genes are very similar with their distribution on the promoters of tissue specific genes in human.