Identification of reference genes for quantitative expression analysis in Indian catfish,Clarias magur, under physiological and pathological conditions

Abstract

The reverse transcription quantitative real-time PCR (RT-qPCR) is widely preferred tool for expression analysis of target gene at mRNA level. However, in order to obtain significant biological comparison, data normalization to a stable endogenous internal control gene also called reference gene (RG) is of utmost importance. In the present study, we evaluated the expression stability of four commonly used housekeeping genes, that is beta actin, β-actin; glyceraldehyde-3-phosphate dehydrogenase, GAPDH; elongation factor-1 alpha, EF-1α and 18S ribosomal RNA, 18S for qPCR data normalization. This study was conducted for two varying physiological (Group I: fed vs. non-fed and Group II: earthen pond vs. fibre reinforced plastic tank reared) and one pathological condition (Group III: Aeromonas hydrophila injected vs. PBS injected) in seven different tissues of Indian catfish, Clarias magur, an economically valuable food fish of South-East Asia. Tissue wise expression stability analysis using web-based comprehensive tool Reffinder revealed that EF-1α was the most stable RG in kidney, liver, spleen and gills of group I; spleen, intestine, muscle and brain in group II; and kidney, liver, intestine, muscle and brain in group III. β-actin was the most stable RG in intestine of group I; liver and brain in group II; spleen and gill in group III. 18S was the most stable RG in muscle and brain of group I; kidney and gill in group II. GAPDH was the least stable RG among four selected genes under all the experimental conditions. These findings on selection of RGs will lead to precise interpretation of relative gene expression in different tissues by RT-qPCR in C. magur in the context of physiological studies and bacterial infection.