Characterisation and validation of housekeeping genes for qRT-PCR expression analysis in Pterophyllum scalare

Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive and advanced method to quantify the expression of target genes of animals including fish. However, the broad variation in the expression patterns of housekeeping genes (HKGs) among tissues and different developmental stages makes it necessary to conduct studies for the selection of the suitable internal control. There is no report available on the reference genes for normalization of gene expression studies in Pterophyllum scalare. In the present study, four reference genes, viz. alpha-actin (α-actin), beta-actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elongation factor-alpha 1 (EF1α), were first characterized and then screened for their efficacy as a suitable HKG at different developmental stages and tissue types of P. scalare. The different statistical algorithms such as Delta-cT, NormFinder, and geNorm portray β-actin to be the most suitable gene among the four genes, whereas BestKeeper revealed EF1α as the most stable reference gene during different developmental stages and tissue types. However, comprehensive gene stability method demonstrated β-actin to be the most stable gene for conducting any gene expression studies. In conclusion, β-actin is recommended as the most suitable reference gene among the four selected genes for qPCR data normalization during different developmental stages and tissue types of P. scalare.