2.6| RNase protection assay
RNase protection assay was performed according to Sugiyama et al. (Sugiyama et al., 2001). Briefly, total RNA was extracted from the most posterior pair of gills and hybridized with digoxigenin-labeled RNA probes overnight. Next, RNA was treated with RNase A and RNase T1 to digest single-stranded RNA and electrophoresed in 1% agarose gel containing 1x MOPS and 5.5% formalin. Subsequently, RNA was transferred from the gel to a positively charged nylon membrane (Roche) overnight. Finally, the membrane was blocked and then treated with alkaline phosphatase–conjugated anti-digoxigenin antibody (Roche) at a dilution of 1:2000. Signals were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate. The intensity of the signals was quantified and the relative expression levels were determined as in Northern blotting.