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.