Evaluation of candidate reference genes for RT‐qPCR analysis during developmental stages in striped murrel ( Channa striata )

Abstract

Real‐time quantitative PCR (RT‐qPCR) is the most advanced and commonly used method for quantification of gene expression levels because of its high sensitivity, specificity and accuracy. The analytical capability of RT‐qPCR, on the other hand, is dependent on precise normalization by using the most stable reference genes. The present study was undertaken to find the best‐suited reference gene for normalization of qPCR data for ontogenetic studies of striped murrel. Four commonly used and constitutively expressed genes, namely beta‐actin (β‐actin), glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), elongation factor 1‐alpha (EF1α) and 18 S ribosomal protein (18 S RNA) were screened and evaluated for their expression stability in developing larvae from 1 to 35 days post hatch (dph). The stability and suitability of these genes were determined by using four different algorithms; namely, delta Ct, BestKeeper, geNorm and NormFinder. The four algorithms together generated a comprehensive ranking EF1α being the most stable, followed by GAPDH and β‐actin as the least stable gene. This is the first report evaluating the most suitable reference gene for mRNA expression studies in C. striata, in the context of ontogenetic development.