Characterization and Evaluation of Selected House-keeping Genes for Quantitative RT-PCR in Macrobrachium rosenbergii Morphotypes

Macrobrachium rosenbergii (De Man, 1879) is an important freshwater prawn cultured globally and is known to exhibit three male morphotypes with differential growth rates. Several gene expression studies using qRT-PCR have been undertaken in this species using any one of the popularly used internal control genes. Here we evaluate four house-keeping genes namely EF1a, GAPDH, b-actin and 18S rRNA for transcript stability across various M. rosenbergii tissues in male morphotypes for use as internal control genes in expression studies. Expression of these genes was measured in 5 tissues (androgenic gland, gill, eyestalk, nerve cord and testis) across morphotypes and Ct values were analyzed using four statistical methods (DCt, geNorm, BestKeeper and NormFinder) developed for this purpose. We report that EF1a is the most suited internal control for data normalization in androgenic gland, testis, nerve cord and gills while GAPDH is best for eyestalk tissue of M. rosenbergii irrespective of the morphotype. Wherever two reference genes are required b-actin can be included for all the tissues except in nerve cord, where GAPDH is more suitable second reference gene. M. rosenbergii EF1a and GAPDH partial mRNA sequences were also generated as part of this study and their phylogenetic relation with other decapods is also reported.