Validation of reference genes for immune gene expression studies during early ontogeny of golden mahseer, Tor putitora

Quantitative real-time PCR stands a powerful tool used for functional genomics in numerous animal models, such as fish. However, optimization of reference genes for mRNA quantitation is important to obtain precise results. Appropriate reference genes for normalizing data in real-time quantitative PCR (qPCR) analyis during ontogeny of golden mahseer, an endangered cyprinid (Tor putitora), have so far not been established. Considering this fact, the current research study aimed to evaluate six candidate housekeeping genes (specifically 18S, ef1α, rpn1, rpl8, actb (β-actin), and S11) for their expression stability all through the early embryonic and larval stages of T. putitora to screen valid candidate reference genes. Five different mathematical algorithms were used for their evaluation, namely BestKeeper, geNorm, NormFinder, RefFinder (web-based) and Delta Cq method. After the evaluation, it was evident that actb exhibited the most consistent and stable expression as a reference gene followed by rpn1 and 18S. Furthermore, substantiation of reference genes was done by normalizing immune genes such as complement component factor-3 (c3) and interleukin-1β mRNA expression in golden mahseer during ontogeny and larval development which reflected actb, rpn1 and 18 s to be ideal reference genes. This study marks the initial attempt to screen the optimal reference gene for analyzing mRNA expression in T. putitora all through its ontogeny and early developmental phases.