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

Abstract

Quantitative real-time RT-PCR (qRT-PCR) is the most sensitive and advancedmethod to quantify the expression of target genes of animals including fish. How-ever, the broad variation in the expression patterns of housekeeping genes (HKGs)among tissues and different developmental stages makes it necessary to conductstudies for the selection of the suitable internal control. There is no report availableon the reference genes for normalization of gene expression studies in Pterophyllumscalare. In the present study, four reference genes, viz. alpha-actin (α-actin), beta-actin (β-actin), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and elonga-tion factor-alpha 1 (EF1α), were first characterized and then screened for theirefficacy as a suitable HKG at different developmental stages and tissue types ofP. scalare. The different statistical algorithmssuchasDelta-cT,NormFinder,andgeNorm portray β-actin to be the most suitable gene among the four genes, whereasBestKeeper revealed EF1αas the most stable reference gene during differentdevelopmental stages and tissue types. However, comprehensive gene stabilitymethod demonstrated β-actin to be the most stable gene for conducting any geneexpression studies. In conclusion, β-actin is recommended as the most suitablereference gene among the four selected genes for qPCR data normalization duringdifferent developmental stages and tissue types of P. scalare.