For the synthesis of rock bream spleen cDNA, total RNA was isolated
from the spleen of the fish using RNAiso reagent (Takara), and
1 mg of purified total RNAwas incubated with 1 ml of random primer
(0.5 mg/ml) (Promega) at 80 C for 10 min, and further incubated at
42 C for 60min in a reaction mixture containing 4 ml of 5 reaction
buffer, 4 ml of 10 mM dNTP mix (Takara), 1 ml of M-MLV reverse
transcriptase (Promega) and 0.5 ml of RNase inhibitor (Promega) in
a final reaction volume of 20 ml.