Identification of valid reference genes during the differentiation of human myoblasts

Background  

Analysis of RNA expression using real-time PCR (qRT-PCR) traditionally includes reference genes (RG) as an internal control. This practice is being questioned as it becomes increasingly clear that RG may vary considerably under certain experimental conditions. Thus, the validity of a particular RG must be determined for each experimental setting. We used qRT-PCR to measure the levels of six RG, which have been reported in the literature to be invariant. The RG were analyzed in human myoblast cultures under differentiation conditions. We examined the expression by qRT-PCR of mRNA encoding Beta-actin (ACTB), Beta-2-microglobulin (B2M), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), peptidylprolyl isomerase A (PPIA), TATA box binding protein (TBP) and ribosomal protein, large, P0 (RPLPO). The mRNA expression of the following genes of interest (GOI) were analyzed: skeletal muscle alpha 1 actin (ACTA1), myogenin/myogenic factor 4 (MYOG), embryonic skeletal muscle myosin heavy chain 3 (MYH3) and the activity of creatine phosphokinase (CK). The geNorm, NormFinder and BestKeeper software programs were used to ascertain the most suitable RG to normalize the RNA input.