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

¹ Department of Medical Sciences, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
³ Department of Kinesiology, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
⁴ Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada L8N 3Z5
² School of Health Sciences, Deakin University, Burwood, Victoria 3125, Australia

	ABSTRACT

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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 O_{2 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.

gene expression; endogenous controls; validation

	INTRODUCTION

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ANALYSIS of mRNA expression in skeletal muscle has become increasingly prevalent in exercise studies (1, 4, 8, 12, 15, 17, 19, 22). Although numerous techniques are available to estimate mRNA expression, real-time RT-PCR has gained favor among scientists as it is a highly sensitive, accurate, and fast technique that offers high-throughput and the ability to quantify mRNA copy number (10). However, there is substantial technical variability associated with RT-PCR, arising from RNA degradation, differences in the quantity and purity of the input RNA, differences in reverse transcription (RT) and polymerase chain reaction (PCR) efficiency, and pipetting error. PCR exponentially amplifies a small starting quantity of cDNA to a large amount of DNA product; as such, even seemingly insignificant technical variability prior to amplification can lead to considerable error in the end product (7), although this is less of a concern using real-time vs. conventional RT-PCR (10). To overcome this limitation, researchers typically normalize the mRNA expression of their genes of interest to a constitutively expressed internal endogenous control, or housekeeping gene (9). In choosing an appropriate housekeeping gene, several considerations must be taken into account: 1) the expression of the housekeeping gene must remain constant throughout the given intervention; 2) the amplification efficiency of the housekeeping gene should be similar to that of the genes of interest; and 3) the abundance of the housekeeping gene should be similar to that of the genes of interest (6). It is important that a given housekeeping gene be validated for each experimental intervention in which it is used.

There are several common housekeeping genes that have been used to normalize mRNA expression in skeletal muscle following acute exercise, including ß-actin (13, 21, 23), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (12, 15, 22), cyclophilin (CYC) (4, 17), and 28S ribosomal RNA (rRNA) (14). Although numerous studies have systematically evaluated the validity of these housekeeping genes following different experimental conditions (11, 18, 26, 27, 29), we are not aware of such a study following acute exercise. Thus the purpose of the present study was to examine whether the genes ß-actin, GAPDH, 28S rRNA, CYC, and ß2-microglobulin (ß2M) are appropriate housekeeping genes following an acute bout of either endurance (END) or resistance (RES) exercise.

	MATERIALS AND METHODS

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Subjects
The present study used previously collected muscle samples from two larger studies. In study 1, 24 healthy males volunteered to participate (age: RES, 23.4 ± 3.7 yr; END, 21.8 ± 2.5 yr). Subjects were assigned to an acute exercise protocol based on their training history, either 300 high-intensity eccentric contractions (RES; n = 12) or high-intensity cycle ergometry to exhaustion (75 min; END; n = 12). Subjects in the RES group had not participated in lower body resistance training within 6 mo of the study, and those in the END group had not regularly participated in endurance activities within 6 mo of the study. In study 2, 10 healthy, recreationally active men and women (age: 21.9 ± 2.2 yr) were recruited to perform a bout of moderate intensity cycle ergometry (90 min; END; n = 6 males, 4 females). Before inclusion into either study, potential subjects were required to complete a health questionnaire to ensure that they were healthy and fit to participate. Each subject was given an information sheet describing all testing procedures, was informed of the purposes and associated risks, and gave written consent prior to participation. The projects were approved by the McMaster University Hamilton Health Sciences Human Research Ethics Board and conformed to the Declaration of Helsinki guidelines.

Testing Protocol
Prior to testing, each subject underwent a baseline muscle biopsy collection (see details below). Prior to each tissue collection, subjects were required to abstain from any form of physical exertion (within 72 h), avoid alcohol (within 48 h), eat their habitual diet (within 48 h), and abstain from caffeine (within 12 h). In study 1, subjects were given a 355-kcal defined formula diet (Ensure) to be consumed 2 h before each collection, whereas subjects in study 2 were postabsorptive.

Cycle ergometry protocols (END).
Approximately 1 wk following the baseline biopsy, subjects performed a maximal oxygen uptake test (O_{2 max}) using an incremental cycle ergometry test to exhaustion. In study 1, no sooner than 72 h following their O_{2 max} test, subjects performed a high-intensity endurance exercise protocol on the cycle ergometer. Briefly, subjects began pedaling at 60% of their predetermined O_{2 max} for 30 min at greater than 70 rpm. Following the initial 30 min, the intensity was increased to 65% O_{2 max} for 5 min, then dropped back to 60% for 5 min, increased to 70% O_{2 max} for 5 min, dropped to 60% for 5 min, etc., to a maximum of 85% O_{2 max}. If 85% O_{2 max} was attained, then subjects continued with intervals of 85% O_{2 max} for 2 min followed by 60% O_{2 max} for 2 min, etc., until test completion. The test ended when subjects were unable to maintain 70 rpm, which lasted 75 min for most subjects. In study 2, no sooner than 72 h following their O_{2 max} test, subjects performed 90 min of cycle ergometry at 65% of O_{2 max} in the postabsorptive state.

Eccentric contraction protocol (RES).
Approximately 1 wk following the baseline biopsy, subjects in the RES group were given a familiarization session with the testing apparatus, a Biodex isokinetic dynamometer (System 3; Biodex Medical Systems, Ronkonkoma, NY). Approximately 1 wk following this session, subjects performed an eccentrically biased isokinetic protocol adapted from one previously described (3). Briefly, following a short warm-up (10 min of light cycle ergometry), subjects were seated in the dynamometer, and their nondominant leg was strapped into a lever arm. The lever arm was programmed to extend the subject’s leg to 150° of flexion (where 180° is full extension) at a moderate speed (30°/s), and then flex their leg to 90° of flexion at a fast speed (120°/s). Subjects were not required to contract during the extension phase. During the flexion phase, subjects were instructed to maximally resist the descending lever arm throughout the entire range of motion (i.e., eccentric contraction) and were verbally encouraged to oppose the movement of the lever arm throughout the duration of each contraction. The entire test consisted of 30 sets of 10 repetitions, with a 1-min rest between each set.

Tissue Collection
In study 1, muscle biopsies were taken 1–2 wk before (baseline) and at 3 h and 48 h following the completion of the exercise protocol. In study 2, muscle biopsies were taken immediately before and after the exercise protocol. Needle biopsy samples were taken from the vastus lateralis of the nondominant leg 15 cm proximal to the lateral knee joint of each subject under local anesthesia (1% lidocaine) using manual suction. All biopsies were taken from separate incisions 6 cm apart, to minimize the potential effect of the biopsy itself on gene expression (20). Approximately 150 mg of muscle was taken from each biopsy, and this was immediately dissected from excess fat and connective tissue. The muscle was quickly sectioned, and 25 mg was placed in an RNase-free polyethylene tube, flash-frozen in liquid nitrogen, and stored at –86°C until being processed for analysis. The remaining muscle was stored at –86°C for subsequent studies.

Analysis of RNA Expression
RNA extraction.
Total RNA was extracted from muscle using a commercially available TRIzol Reagent (Life Technologies, catalog no. 15596) following the manufacturer’s instructions. Briefly, 25 mg of muscle was removed from the freezer and immediately immersed in 1 ml of TRIzol reagent. The muscle was homogenized on ice using a glass homogenizer, and the aqueous and organic phases were separated using 200 µl of chloroform. Total RNA was precipitated using 500 µl of isopropyl alcohol, washed three times with 75% ethanol, redissolved in 15 µl of DEPC-treated H₂O, aliquoted, and stored at –86°C. The concentration and purity of the RNA was determined using a UV spectrophotometer (Shimadzu UV-1201; Mandel Scientific, Guelph, Ontario) by measuring the absorbance at 260 (OD₂₆₀) and 280 (OD₂₈₀) nm. Measurements were done in duplicate and had an average coefficient of variation (CV) of <10%. The average purity (OD₂₆₀/OD₂₈₀) of the samples was 1.4. RNA integrity was assessed in a randomly chosen subset of samples using agarose gel electrophoresis, and the OD ratio of 28S to 18S rRNA was consistently greater than 1 for each sample checked, indicating high-quality RNA.

RT and real-time PCR.
For study 1, RT was performed on 1 µg of total RNA using a commercially available kit (1st Strand cDNA Synthesis Kit for RT-PCR, Roche, catalog no. 1483188). Prior to RT, RNA samples were treated with DNase I for 30 min at 37°C to remove any contaminating DNA (DNA-free, Ambion, catalog no. 1906) according to the manufacturer’s instructions. DNase I was inactivated by a commercially available inactivation solution (DNase inactivation solution, Ambion, catalog no. 1906) according to the manufacturer’s instructions. Total RNA was converted to cDNA using AMV reverse transcriptase in a reaction volume of 20 µl containing 1x reaction buffer, 5 mM MgCl₂, 1 mM dNTP mixture, 3.2 µg of random primer p(dN)6, 50 U of RNase inhibitor, 0.8 µl of AMV reverse transcriptase, and 1 µg of DNA-free RNA. RT was performed in a thermal cycler (GeneAmp PCR system 9700, Applied Biosystems) with a profile of 25°C for 10 min, 42°C for 60 min, and 99°C for 5 min. All samples were run together, and several negative controls were run simultaneously that did not contain either RNA (no template controls) or the reverse transcriptase enzyme (RT negative), to control for RNA and genomic DNA contamination, respectively. Following RT, samples were individually aliquoted and stored at –80°C until analysis.

For study 1, real-time PCR for ß-actin, CYC, ß2M, and GAPDH was performed using a GeneAmp 5700 sequence detection system (Applied Biosystems, Foster City, CA) using SYBR Green 1 chemistry, as previously described (18). This technique has been successfully used to validate housekeeping gene expression levels following serum starvation (25). All samples were run in duplicate. For study 2, 28S rRNA and ß2M were reverse transcribed and amplified in a single reaction tube and amplification was detected in real time using TaqMan chemistry (TaqMan One-Step RT-PCR Master Mix Reagents, part no. 4309169, Applied Biosystems) according to the manufacturer’s instructions. RT (same details as above) and real-time PCR (details in Table 1) were done using an iCycler real-time PCR system (Bio-Rad Laboratories, Hercules, CA). All samples were run in tetraplicates, fluorescence emission was detected through a Tamra 548 filter, and C_T was automatically calculated. These transcripts were extensively optimized, run simultaneously with RNA- and RT-negative controls, and agarose gel electrophoresis was used to confirm the specificity of the priming.

–CT

	RESULTS

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Real-Time RT-PCR Quality Control and Performance Characteristics
After optimization, the amplification for all genes was shown to be specific and highly efficient. Quality control data and performance characteristics for ß-actin, CYC, ß2M, and GAPDH have been presented elsewhere (18). For 28S and ß2M analysis using TaqMan chemistry and a one-step protocol, real-time RT-PCR was optimized and monitored to have combined RT and PCR efficiencies of 110% (r² = 0.98) and 113% (r² = 0.97), respectively, and intra-assay CV values of 16% and 17%, respectively (using linear 2^–CT values). Agarose gel electrophoresis confirmed that a single, specific product of appropriate size was being amplified. After 40 cycles of PCR, there was no observable amplification in the RNA or RT-negative control. For quality control, we compared the data generated using real-time RT-PCR with conventional agarose gel detection RT-PCR (using ß-actin) and found a correlation coefficient of 0.99 (P < 0.01; not shown).

Analysis of Housekeeping Genes
From study 1, we analyzed the housekeeping gene data using both nonnormalized C_T values (2^–CT) and C_T values of each individual housekeeping gene normalized to each other (2^–CT). The latter approach was taken to observe the relationship between the various housekeeping genes. Using nonnormalized 2^–CT values, we showed that CYC increased at 48 h following both END and RES exercise (3- and 5-fold, respectively; P < 0.01; Figs. 1 and 2) and was variable in the RES group at 3 h. ß-Actin trended toward an increase following END exercise (1.85-fold collapsed across time; P = 0.13; Fig. 1), whereas GAPDH trended toward a small but constitutive increase at 3 h following RES exercise (1.4-fold; P = 0.067; Fig. 2). In contrast, ß2M was not significantly altered post-END or post-RES exercise, and ß-actin and GAPDH were very stable in the RES and END groups, respectively (Fig. 1 and 2). When we examined each housekeeping gene normalized to one another (2^–CT), we found similar results (data not shown). Finally, from study 2 we found that 28S rRNA significantly increased immediately following END exercise (2-fold; P = 0.02; Fig. 3), whereas ß2M was stable (P > 0.05; Fig. 3).

View larger version (16K): [in this window] [in a new window]   Fig. 1. mRNA expression of ß-actin, cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and ß2-microglobulin (ß2M) following endurance (END) exercise. CYC increased 48 h following END exercise (3-fold; *P < 0.01), whereas ß-actin trended toward an increase at 3 h and 48 h (1.85-fold collapsed across time; P = 0.13). GAPDH and ß2M were not altered at either time point. Values are means ± SE.

View larger version (16K): [in this window] [in a new window]   Fig. 2. mRNA expression of ß-actin, CYC, GAPDH, and ß2M following resistance (RES) exercise. CYC increased 48 h following RES exercise (5-fold; *P < 0.01), whereas GAPDH trended toward a small yet robust increase at 3 h (1.4-fold; P = 0.067). ß2M and ß-actin were not altered at either time point. Values are means ± SE.

View larger version (10K): [in this window] [in a new window]   Fig. 3. Expression of 28S ribosomal RNA (rRNA) and ß2M mRNA immediately following a 90 min bout of moderate intensity (65% O2max) exercise. 28S rRNA increased 2-fold immediately following END exercise (*P = 0.02; n = 10), whereas ß2M expression remained stable immediately post-END exercise. Values are means ± SE.

	DISCUSSION

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Several investigators have used CYC as a housekeeping gene following acute exercise (4, 17), as well as ß-actin following acute END exercise (13, 21, 23) and GAPDH following acute RES exercise (15, 22). Our data suggest that these may not have been appropriate housekeeping genes to use following such interventions. Given that we observed increases in each of these "housekeeping" genes post-END and/or post-RES exercise, it is possible that small increases in mRNA expression of the genes of interest may have been "masked" in these studies, and observable changes may have, in reality, been larger than reported. However, it should be noted that several issues ought to be considered before making broad extrapolations of our data to the literature. First, the intensity of our exercise protocols were very high, and intensity is likely a determinant of gene expression in skeletal muscle post-acute exercise, as has been shown postexercise training (24). Second, our RES protocol consisted solely of eccentric contractions, and it is possible that CYC and GAPDH may have been more stably expressed following dynamic resistance exercise with a concentric component, as eccentric and concentric contractions are known to induce different molecular responses (5). Finally, our study is limited in the number of time points examined postexercise. As gene expression can be rapidly altered following exercise, our results at 3 h and 48 h may not represent all 48 h following exercise.

Intra- and Interindividual Variability
Recently, two studies have reported large intra- and interindividual variability in skeletal muscle gene expression using DNA microarrays (2, 28). We have not previously observed much intra-individual variability using many of the same genes examined in the present study (18). Furthermore, we used a repeated-measures design, which compared individuals to themselves but not to one another, thereby eliminating the interindividual variability in the resting state. However, it is possible that the genetic or epigenetic differences among our subjects may have affected their response to the exercise bout. To manage these differences, we recruited a relatively homogenous group of subjects and controlled for potentially confounding variables such as training status, medical history, pre-exercise diet, and alcohol consumption. Despite this, we observed a variable response to both protocols between individuals for all genes tested. For example, with 28S rRNA, although 9 of 10 subjects responded in the same direction following END exercise, each individual subject responded to a highly variable degree (CV of the response = 55%; Fig. 4). Such variability strengthens any statistically significant changes that we observed (e.g., CYC), but in general would have made it more difficult to observe a statistically significant difference in gene expression. Interestingly, this variance appeared to be somewhat gene specific, as we were nearly able to significantly detect a 1.4-fold increase in GAPDH (P = 0.067), but were less able to significantly detect a larger 1.85-fold increase in ß-actin (P = 0.13). Large "response variability" in gene expression following exercise has been previously documented (22), and the genetic or epigenetic mechanism behind this phenomenon is beyond the scope of this paper.

View larger version (16K): [in this window] [in a new window]   Fig. 4. 28S rRNA response variability following END exercise. Although the direction of the response was similar for 9 of 10 subjects, there was substantial variance in the degree to which each subject responded.

	GRANTS

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D. J. Mahoney was supported by a Natural Sciences and Engineering Research Council of Canada (NSERC) Post-Graduate Scholarship during this study. This study was supported by an NSERC of Canada operating grant to M. A. Tarnopolsky.

	ACKNOWLEDGMENTS

 
We thank Adeel Safdar for technical assistance with the real-time PCR, Dr. Mazen J. Hamadeh and Michaela C. Devries for providing the tissue samples for study 2, and Jeremy Paikin for critically reading the manuscript.

	FOOTNOTES

 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: M. A. Tarnopolsky, Dept. of Pediatrics, Rm. 4U4, McMaster Univ. Medical Center, 1200 Main St. W., Hamilton, Ontario, Canada L8N 3Z5 (E-mail: tarnopol{at}mcmaster.ca).

10.1152/physiolgenomics.00067.2004

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