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

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.

Expression of growth-related genes in young and older human skeletal muscle following an acute stimulation of protein synthesis

“…We found that there were no significant changes (P Ͼ 0.05) in mRNA expression between the two baseline muscle biopsy samples (i.e., the second biopsy obtained from the same incision had the same mRNA expression pattern as the original biopsy sample for all 3 genes). Although there have been mixed findings in the literature, changes in mRNA expression are dependent on the variable of interest and the biopsy timing, design, and technique (40,54). Therefore, it appears that our findings are a result of the experimental treatment rather than a prior biopsy effect.…”

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergström Technique

“…Thus, during a given test session multiple incisions in the same muscle should be made, each 2-3 cm apart, to prevent trauma-induced changes in gene expression 24 . Obtaining multiple biopsies from the same muscle through separate incisions does not apparently alter the transcriptional response of genes responsive to the stress of exercise 24,25 . However, the expression of certain genes may be influenced when a second biopsy is taken through the same incision several hours later 24 .…”

AMPKα is essential for acute exercise-induced gene responses but not for exercise training-induced adaptations in mouse skeletal muscle

“…Fluorescence-based real-time PCR (ABI PRISM 7900 Sequence Detection System; Applied Biosystems) was used to determine mRNA content of specific genes as described (39). The mRNA content was analyzed using Taqman probes and forward and reverse primers as listed (Table 1), except for FABPpm (Mm00494703_m1), fatty acid transport protein (FATP)1 (Mm00449511_m1), and FATP4 (Mm01327405_m1) mRNA, which were analyzed using premade kits (Applied Biosystems).…”