Stability evaluation and validation of appropriate reference genes for real-time PCR expression analysis of immune genes in the rohu (Labeo rohita) skin following argulosis

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Abstract
Argulosis is one of the most unrestrained economically significant freshwater fish ectoparasitic diseases. Proper selection or normalization of the best reference gene governs the accuracy of results of gene expression studies using real-time PCR. Earlier studies in rohu carp (Labeo rohita) have used reference genes without proper validation. Here, seven candidate reference genes viz., acidic ribosomal protein (ARP0), glyceraldehyde 3-phosphate dehydrogenase, RNA polymerase II (RPo), elongation factor1α (EF1α), α- tubulin (AT), ribosomal protein L 10, and β-actin were evaluated using four algorithms (geNorm, BestKeeper, NormFinder and ∆Ct) followed by a comprehensive gene expression analysis using skin tissue of rohu at varied time points of experimental Argulus siamensis infection. ARP0 and EF1α were found to be the most stable whereas RPo and AT were considered as least stable genes based on basal expression level and variation in expression levels. Validation of candidate reference genes was undertaken by looking into the expression of six immune-related genes using the two most stable and two least stable genes as housekeeping genes in Argulus-infected rohu skin at different time points of infection. An increased expression of immune genes indicated the role of inflammation and the immune modulation process at the site of attachment of parasites in governing infection.

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