Abstract
BackgroundAnalysis 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.ResultsUsing the geNorm program, RPLPO and TBP were found to be the most stable genes, additionally a suitable normalization factor (NF) was calculated. The NormFinder software showed that RPLPO was the most stable, whereas TBP ranked second. BestKeeper program also revealed that RPLPO and TBP as stable genes, but PPIA was the most stable reference gene, whereas GAPDH and ACTB were the worst ranked.ConclusionRNA expression analyses including three independent softwares revealed that RPLPO, TBP as reference genes or NF calculated by geNorm software, are suitable to normalize the mRNA expression in myoblast after culture under differentiation conditions. Significant correlations can be identified between the differentiations markers ACTA1, MYOG, MYH3 and creatine phosphokinase (CK) activity, when the expression is normalized with the NF calculated with RPLPO and TBP.
Highlights
Analysis of RNA expression using real-time PCR traditionally includes reference genes (RG) as an internal control
During the selection of an adequate RG, several indications should be taken into consideration: 1) the expression of the RG must remain constant throughout the intervention; 2) the amplification efficiency of the RG should be similar to that of the genes of interest (GOI); and 3) the abundance of the RG should be similar to that of the genes of interest [8]
We initially verified that our samples truly represented differentiating cells by analyzing the creatine phosphokinase (CK) activity level, a well-known and accepted marker of differentiation [11]
Summary
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. If unrecognized, unexpected changes in RG expression could result in erroneous conclusions about real biological effects such as responses to drugs [4,5,6]. Such errors could be introduced at a number of stages throughout the experimental protocol (input sample, RNA extraction, reverse transcription, etc.) [1,2,7]. Variation in presumably stable RG was shown in studies examining their expression in serum-stimulated fibroblasts [9,10]
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