Validation of reference gene(s) for quantitative gene expression profiling using rice moth, Corcyra cephalonica as a model

Abstract

Many techniques are available for the quantification of specific mRNA from various tissues of an organism. Among them qRT-PCR is a widely used method, as this is a rapid and requires low quantity of mRNA. The reliability of the data depends on accurate normalization using appropriate internal control(s). The use of constitutively expressed genes (house-keeping genes, HKGs) such as α-actin, β-tubulin and GAPDH as internal controls or reference genes is a widely accepted method for qRT-PCR data normalization. However, expression of HKGs may vary at different developmental stages or during hormone manipulation studies. The present study was carried out using a holometabolous lepidopteran stored grain pest Corcyra cephalonica (rice moth). Seven different constitutively expressed genes α-actin, β-tubulin, 18S rRNA, TBP, rS7, GAPDH, and EF1α were analysed as internal reference genes in various larval tissues. Further the stable gene was characterized during various conditions like ontogeny, hormonal depletion and exogenous hormone application. Gene expression stability was obtained by measuring the M-value for HKGs. The findings indicate that 18S rRNA was suitable as reference gene under normal physiological conditions, tissue distribution and also upon methoprene (JH analog) treatment. On the other hand rS7 was suitable for ecdysteroid treatment related analysis. Present study once again reinforces that, reference genes cannot be universal and need to be validated not only for a given organism but also during various conditions.