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J Appl Physiol 103: 1914, 2007; doi:10.1152/japplphysiol.00862.2007
8750-7587/07 $8.00
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LETTER TO THE EDITOR

Using ribosomal RNA as a reference in mRNA quantification

TO THE EDITOR: Quantification of mRNA expression in skeletal muscle is an important tool for assessing the effect of muscle loading in exercise physiology. A crucial issue related to mRNA quantification is how to normalize the obtained mRNA data. One approach is to present the amount of a specific mRNA transcript relative to the amount of 18S or 28S ribosomal RNA, as these are thought to be present in a relatively constant amount. However, in some cases the level of ribosomal RNA is influenced significantly by the exercise/training intervention, and other ways of normalization should be considered (2).

In the recent paper by Kim et al. (4) an ~50% decrease in myostatin mRNA in human skeletal muscle is presented as an effect of long-term resistance training, while a simultaneous increase is seen in the amount of myostatin protein. The authors argue that this inverse relationship between expression of myostatin mRNA and protein could represent the negative feedback autoregulation of myostatin, which has been indicated by Forbes et al. (1). However, in this specific case, the explanation for the apparent decrease in myostatin mRNA could be relatively simple and possibly related to the choice of 18S ribosomal RNA as an internal standard. The authors report a substantial increase in the total RNA concentration (40%) as a response to the resistance-training intervention (4), and considering that 18S and 28S ribosomal RNA makes up the bulk of total RNA (~80%), it is highly probable that the amount of 18S is increased in a corresponding manner. Therefore, the training-induced decrease in myostatin mRNA, relative to 18S ribosomal RNA, observed by Kim et al. may well be explained largely by an elevated 18S ribosomal RNA concentration. As pointed out by the authors in a previous publication, an increased total RNA concentration is likely to indicate an increased potential for protein synthesis (because of increased ribosomal RNA levels; Ref. 3). Thus, in the case of an unchanged myostatin mRNA concentration (relative to tissue weight or volume) combined with an increased ribosomal RNA content, it is reasonable to think that the potential for synthesizing myostatin protein is either unchanged or increased. If, under these circumstances, myostatin mRNA is presented relative to 18S ribosomal RNA, the level of myostatin mRNA will appear to decrease, while in reality the potential for synthesis of myostatin protein could well be increased. In other words, the choice to use 18S as a reference for myostatin mRNA may explain the seemingly inverse relationship between changes in myostatin mRNA and myostatin protein observed by Kim et al. (4) in this specific study.

In conclusion, the choice of a non-constant normalization factor can result in erroneous apparent changes in mRNA. Therefore, it should always be taken into account if apparent changes might arise from a non-constant normalization factor, like 18S ribosomal RNA in this case.

FOOTNOTES


Address for reprint requests and other correspondence: K. M. Heinemeier, Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen, Denmark 2100 (e-mail: katjaheinemeier{at}hotmail.com)

REFERENCES

  1. Forbes D, Jackman M, Bishop A, Thomas M, Kambadur R, Sharma M. Myostatin auto-regulates its expression by feedback loop through Smad7 dependent mechanism. J Cell Physiol 206: 264–272, 2006.[CrossRef][Web of Science][Medline]
  2. Kadi F, Schjerling P, Andersen LL, Charifi N, Madsen JL, Christensen LR, Andersen JL. The effects of heavy resistance training and detraining on satellite cells in human skeletal muscles. J Physiol 558: 1005–1012, 2004.[Abstract/Free Full Text]
  3. Kim JS, Cross JM, Bamman MM. Impact of resistance loading on myostatin expression and cell cycle regulation in young and older men and women. Am J Physiol Endocrinol Metab 288: E1110–E1119, 2005.[Abstract/Free Full Text]
  4. Kim JS, Petrella JK, Cross JM, Bamman MM. Load-mediated downregulation of myostatin mRNA is not sufficient to promote myofiber hypertrophy in humans: a cluster analysis. J Appl Physiol (August 2, 2007). doi:10.1152/japplphysiol.01194.2006.

Katja M. Heinemeier
 Institute of Sports Medicine, Bispebjerg Hospital, Copenhagen, Denmark





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