European Journal of Applied Physiology

, Volume 95, Issue 4, pp 351–360

Gene expression in human skeletal muscle: alternative normalization method and effect of repeated biopsies

  • Carsten Lundby
  • Nikolai Nordsborg
  • Keiko Kusuhara
  • Kristina Møller Kristensen
  • P Darrell Neufer
  • Henriette Pilegaard
Original Article

DOI: 10.1007/s00421-005-0022-7

Cite this article as:
Lundby, C., Nordsborg, N., Kusuhara, K. et al. Eur J Appl Physiol (2005) 95: 351. doi:10.1007/s00421-005-0022-7

Abstract

The reverse transcriptase-polymerase chain reaction (RT-PCR) method has lately become widely used to determine transcription and mRNA content in rodent and human muscle samples. However, the common use of endogenous controls for correcting for variance in cDNA between samples is not optimal. Specifically, we investigated (1) a new normalization method based on determining the cDNA content by the flourophores PicoGreen and OliGreen, (2) effect of repeated muscle biopsies on mRNA gene expression, and (3) the spatial heterogeneity in mRNA expression across the muscle. Standard curves using oligo standards revealed a high degree of sensitivity and linearity (2.5–45 ng; R2>0.99) with OliGreen reagent, as was the case for OliGreen analyses with standard curves constructed from serial dilutions of representative RT samples (R2 >0.99 for a ten times dilution range of a representative reversed transcribed (RT) sample). Likewise, PicoGreen reagent detected the RNA:DNA hybrid content in RT samples with great sensitivity. Standard curves constructed from both double-stranded lambda DNA (1–10 ng) and from serial dilutions of representative RT samples consistently resulted in linearity with R2 >0.99. The present determination of cDNA content in reversed transcribed human skeletal muscle RNA samples by both PicoGreen and OliGreen analyses suggests that these fluorophores provide a potential alternative normalization procedure for human gene expression studies. In addition, the present study shows that multiple muscle biopsies obtained from the same muscle do not influence the mRNA response induced by an acute exercise bout for any of the genes examined.

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Carsten Lundby
    • 1
    • 2
  • Nikolai Nordsborg
    • 1
    • 3
  • Keiko Kusuhara
    • 1
    • 4
  • Kristina Møller Kristensen
    • 1
    • 2
    • 4
  • P Darrell Neufer
    • 5
  • Henriette Pilegaard
    • 1
    • 2
    • 4
  1. 1.Copenhagen Muscle Research CentreCopenhagenDenmark
  2. 2.Copenhagen Muscle Research CentreCopenhagenDenmark
  3. 3.Institute of Exercise and Sport SciencesUniversity of CopenhagenCopenhagenDenmark
  4. 4.The August Krogh InstituteUniversity of CopenhagenCopenhagenDenmark
  5. 5.The John B. Pierce Laboratory and Yale University School of MedicineNew HavenUSA