The Importance of Internal Controls in mRNA Quantification

The following is the abstract of the article discussed in the subsequent letter:

	ABSTRACT

Kraniou, Yorgos, David Cameron-Smith, Marie Misso, Greg Collier, and Mark Hargreaves. Effects of exercise on GLUT-4 and glycogenin gene expression in human skeletal muscle. J Appl Physiol 88: 794-796, 2001.To investigate the effect of exercise on GLUT-4, hexokinase, and glycogenin gene expression in human skeletal muscle, 10 untrained subjects (6 women and 4 men, 21.4 ± 1.2 yr, 66.3 ± 5.0 kg, peak oxygen consumption = 2.30 ± 0.19 l/min) exercised for 60 min on a cycle ergometer at a power output requiring 73 ± 4% peak oxygen consumption. Muscle samples were obtained by needle biopsy before, immediately after, and 3 h after exercise. Gene expression was quantified, relative to 29S ribosomal protein cDNA, by RT-PCR. GLUT-4 gene expression was increased immediately after exercise (1.7 ± 0.4 vs. 0.9 ± 0.3 arbitrary units; P < 0.05) and remained significantly higher than baseline 3 h after the end of exercise (2.2 ± 0.4 vs. 0.9 ± 0.3 arbitrary units; P < 0.05). Hexokinase II gene expression was significantly higher than the resting value 3 h after the end of exercise (2.9 ± 0.4 vs. 1.3 ± 0.3 arbitrary units; P < 0.05). Exercise increased glycogenin mRNA more than twofold (2.8 ± 0.6 vs. 1.2 ± 0.2 arbitrary units; P < 0.05) 3 h after the end of exercise. For the first time, we report that a single bout of exercise is sufficient to cause upregulation of GLUT-4 and glycogenin gene expression in human skeletal muscle. Whether these increases, together with the associated increase in hexokinase II gene expression, lead to increased expression of these key proteins in skeletal muscle and contribute to the enhanced skeletal muscle glucose uptake, glycogen synthesis, and insulin action observed following exercise remains to be determined.

	LETTER

The Importance of Internal Controls in mRNA Quantification

To the Editor: Studies on the regulation of genes within exercising muscles are an exciting new and expanding field within muscle physiology. However, molecular biology is a new concept for many people within the muscle society, and in the excitement results are often accepted and published uncritically, without consideration for the necessary controls. A clear example of this is demonstrated in the article of Kraniou et al. (5), in which they report increase of GLUT-4, glycogenin, and hexokinase II gene expression after a single bout of exercise. They have quantified the mRNA corresponding to the respective genes by RT-PCR and normalized the figures by quantifying S29 mRNA as well. The rationale behind the normalization is that an internal control is needed to correct for variations in reverse transcription. This internal control, of course, has to be constitutive under the experimental conditions, that is, unaffected by the exercise. The authors have taken biopsies at three different time points and show that the three mRNA all increase in an almost identical manner relative to S29 mRNA. The authors note this similarity and suggest that this may be due to coordinated regulation. This might indeed be the case, but another possible explanation, which is not addressed, is that these mRNA levels are not increased by the single exercise bout but appear so due to degradation of the S29 mRNA.

The use of internal RNA controls in muscle is hampered by the fact that no RNA is known that is always constitutively expressed. In most cases, either 28S rRNA, -actin mRNA, or glyceraldehyde-3-phosphate dehydrogenase mRNA are used, although their levels have all been shown to change in muscle under certain conditions (1-3, 6). The authors choose instead to use S29 mRNA as internal control, although they give no information as to why they believe this to be a suitable internal control. To my knowledge, S29 mRNA has never been used as internal control before. In fact, in the article (4) describing the mRNA sequence used by Kraniou et al., the S29 mRNA level was shown to exhibit considerable variation.

Because no trustworthy internal RNA control exists within muscle tissue, attempts to validate the chosen internal control should always be performed. In the present case, the same amount of total RNA was always used in the reverse transcription reaction. Therefore, we can expect that if the S29 mRNA level is indeed constant, there should be no difference in S29 PCR yield between the time points, although a significant difference may be masked by large variations. However, it should still be possible to comment on the likelihood of the results originating from a decrease in S29 mRNA levels.

	REFERENCES

1.   Cresci, S, Wright LD, Spratt JA, Briggs FN, and Kelly DP. Activation of a novel metabolic gene regulatory pathway by chronic stimulation of skeletal muscle. Am J Physiol Cell Physiol 270: C1413-C1420, 1996[Abstract/Free Full Text].

2.   Cros, N, Muller J, Bouju S, Pietu G, Jacquet C, Leger JJ, Marini JF, and Dechesne CA. Upregulation of M-creatine kinase and glyceraldehyde-3-phosphate dehydrogenase: two markers of muscle disuse. Am J Physiol Regulatory Integrative Comp Physiol 276: R308-R316, 1999[Abstract/Free Full Text].

3.   De Leeuw, WJ, Slagboom PE, and Vijg J. Quantitative comparison of mRNA levels in mammalian tissues: 28S ribosomal RNA level as an accurate internal control. Nucleic Acids Res 17: 10137-10138, 1989[Free Full Text].

4.   Frigerio, JM, Dagorn JC, and Iovanna JL. Cloning, sequencing and expression of the L5, L21, L27a, L28, S5, S9, S10 and S29 human ribosomal protein mRNAs. Biochim Biophys Acta 1262: 64-68, 1995[Medline].

5.   Kraniou, Y, Cameron-Smith D, Misso M, Collier G, and Hargreaves M. Effects of exercise on GLUT-4 and glycogenin gene expression in human skeletal muscle. J Appl Physiol 88: 794-796, 2000[Abstract/Free Full Text].

6.   Neufer, PD, Ordway GA, Hand GA, Shelton JM, Richardson JA, Benjamin IJ, and Williams RS. Continuous contractile activity induces fiber type specific expression of HSP70 in skeletal muscle. Am J Physiol Cell Physiol 271: C1828-C1837, 1996[Abstract/Free Full Text].

Peter Schjerling, Copenhagen Muscle Research Center Rigshospitalet DK-2100 Copenhagen, Denmark E-mail: viridis{at}biobase.dk

	REPLY

We thank Dr. Schjerling for his letter and for highlighting the important issue of internal controls in mRNA quantification. As assumed members of the so-called "muscle society," we readily recognize the great benefits of applying the tools of molecular biology to our studies. Equally, we are aware of the issues raised in the letter. Given the potential for most, if not all, of the experimental interventions, in which we and others have interest (e.g., acute and chronic endurance or resistance exercise, dietary intervention), to alter the expression of a vast number of genes, identification of a constitutively expressed gene as an internal control is fundamental in determining the true significance of any changes in the expression of specific genes of interest. We used 29S rRNA, whereas other studies have used 18S and 28S rRNA, -actin, and glyceraldehyde-3-phosphate dehydrogenase. To date, no study has systematically examined the effects of the above-mentioned interventions on constitutive gene expression in human skeletal muscle, and there may well be a need for the use of different genes depending on the intervention. In our study (3), there was no effect of exercise on the expression of 29S rRNA. Thus, the observed increases in GLUT-4, hexokinase II, and glycogenin gene expression were not due to a decrease in the expression of our internal control. We apologize if omission of this specific point has caused confusion.

We are confident that the changes in the expression of the GLUT-4, hexokinase II, and glycogenin genes we observed (3) are real and of potential functional significance. It has recently been demonstrated that exercise upregulates the expression of hexokinase II (2) and of a number of metabolic genes in human skeletal muscle (5). Furthermore, it has been demonstrated that GLUT-4 protein levels in human skeletal muscle are increased after a single bout of exercise (1), presumably partly due to an increase in GLUT-4 mRNA. Such changes are consistent with changes observed after prolonged exercise in rats (4). These changes in gene and protein expression may be important for enhancing sarcolemmal glucose transport, muscle glycogen synthesis, and insulin sensitivity in the postexercise period.

	REFERENCES

1.   Greiwe, JS, Holloszy JO, and Semenkovich CF. Exercise induces lipoprotein lipase and GLUT-4 protein in muscle independent of adrenergic-receptor signaling. J Appl Physiol 89: 176-181, 2000[Abstract/Free Full Text].

2.   Koval, JA, DeFronzo RA, O'Doherty RM, Printz R, Ardehali H, Granner DK, and Mandarino LJ. Regulation of hexokinase II activity and expression in human skeletal muscle by moderate exercise. Am J Physiol Endocrinol Metab 274: E304-E308, 1998[Abstract/Free Full Text].

3.   Kraniou, Y, Cameron-Smith D, Misso M, Collier G, and Hargreaves M. Effects of exercise on GLUT-4 and glycogenin gene expression in human skeletal muscle. J Appl Physiol 88: 794-796, 2000.

4.   Kuo, C-H, Browning KS, and Ivy JL. Regulation of GLUT4 protein expression and glycogen storage after prolonged exercise. Acta Physiol Scand 165: 193-201, 1999[Web of Science][Medline].

5.   Pilegaard, H, Ordway GA, Saltin B, and Neufer PD. Transcriptional regulation of metabolic genes during recovery from exercise in humans. Am J Physiol Endocrinol Metab 279: E806-E814, 2000[Abstract/Free Full Text].

Mark Hargreaves,

David Cameron-Smith,

Yorgos Kraniou, School of Health Sciences Deakin University Burwood, Victoria 3125, Australia E-mail: mharg{at}deakin.edu.au