Characterization and Evaluation of Selected House-keeping Genes for Quantitative RT-PCR in Macrobrachium rosenbergii Morphotypes
Characterization and Evaluation of Selected House-keeping Genes for Quantitative RT-PCR in Macrobrachium rosenbergii Morphotypes
Date
2015
Authors
Priyadarshi, Himanshu
Das, Rekha
Kumar, A. Pavan
P, Gireesh Babu
Javed, Hasan
Krishna, Gopal
Marappan, Makesh
Chaudhari, Aparna
Journal Title
Journal ISSN
Volume Title
Publisher
Society of Fisheries Technologists (India)Cochin
Abstract
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.
Description
Keywords
Macrobrachium rosenbergii, morphotypes, quantitative RT-PCR, house-keeping gene, normalization, reference genes
Citation
Fishery Tech 52(3):177-183