Selection of reference genes for expression studies with fish myogenic cell cultures

Abstract

Background: Relatively few studies have used cell culture systems to investigate gene expression and the regulation of myogenesis in fish. To produce robust data from quantitative real-time PCR mRNA levels need to be normalised using internal reference genes which have stable expression across all experimental samples. We have investigated the expression of eight candidate genes to identify suitable reference genes for use in primary myogenic cell cultures from Atlantic salmon (Salmo salar L.). The software analysis packages geNorm, Normfinder and Best keeper were used to rank genes according to their stability across 42 samples during the course of myogenic differentiation. Results: Initial results showed several of the candidate genes exhibited stable expression throughout myogenic culture while Sdha was identified as the least stable gene. Further analysis with geNorm, Normfinder and Bestkeeper identified Ef1 alpha, Hprt1, Ppia and RNApolII as stably expressed. Comparison of data normalised with the geometric average obtained from combinations of any three of these genes showed no significant differences, indicating that any combination of these genes is valid. Conclusion: The geometric average of any three of Hprt1, Ef1 a, Ppia and RNApolII is suitable for normalisation of gene expression data in primary myogenic cultures from Atlantic salmon.