Evaluation of valid reference genes during stimulation with static magnetic fields in human myoblast cultures.

Investigation of gene expression using real-time PCR (qRT-PCR) requires normalization with genes that are continuously expressed (reference genes; RGs). For accurate measurements, it is exceedingly important that RG expression is invariant under the investigated experimental conditions. It has recently become evident that RG expression may vary considerably under different culture conditions, which results in inaccurate qRT-PCR measurements. Static magnetic fields (SMFs) have been shown to enhance myogenic cell differentiation in the rat cell line L6, and may also induce differentiation in human myoblast cultures. In order to perform precise qRT-PCR measurements in human myoblast cell cultures stimulated with SMFs, one prerequisite is to find the most suitable RG. In this study, qRT-PCR was used to investigate the gene expression of six widely used RGs in human myoblast cell cultures stimulated with SMFs, with the aim of identifying the most stable among them. The mRNA concentration of β-actin (ACTB), β-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO) were quantified, and the most suitable RGs were identified using the geNorm and NormFinder software programs. Results were verified by BestKeeper software. mRNA expression of the following genes of interest was analyzed: myosin, heavy chain 1, skeletal muscle, adult (MYH1); myosin, heavy chain 3, skeletal muscle, embryonic (MYH3); myosin, heavy chain 8, skeletal muscle, perinatal (MYH8), as well as the immunoreactivity of MYH1 (irMYH1). Using geNorm, PPIA and B2M were found to be the most stable genes, followed by GAPDH. NormFinder identified PPIA as the most stable gene, followed by B2M and GAPDH. Finally, BestKeeper revealed TBP and PPIA to be the most stable genes, while B2M was ranked third.