Total RNA was primed with 1 μl oligo dT (12–18) primer (50 μM) in a 6‐μl reaction system, and then incubated at 70°C for 10 min and rapidly cooled on ice prior to reverse transcription reaction. Reverse transcription was carried out at 42°C for 1 h in a final volume of 10 μl containing 2 μl of 5× M‐MLV buffer (TAKARA BIO), 0.5 μl of 10 mM dNTP mix (NEW ENGLAND Biolabs), 0.25 μl of RNase Inhibitor (TAKARA BIO), 1 μl of RTase M‐MLV (RNase H‐) (TAKARA BIO) followed by incubation at 70°C for 15 min to terminate the reaction and stored at ‐20°C until use. For real‐time PCR, the cDNA samples were diluted tenfold.
RT‐qPCR was conducted on LightCycler 96 (Roche). Each reaction mixture consisted of 2 μl of cDNA, 0.5 μl each of forward (10 μM) and reverse primers (10 μM), 12.5 μl 2× SYBR Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA BIO), and 9.5 μl of nuclease‐free water (Millipore) in a total reaction volume of 25 μl. The thermal cycler program consisted of 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers were designed using Primer Premier 5.0 (PRIMIER Biosoft). The primer sequences are listed in TableS1 . Data were obtained from three replicate assays for each sample with normalization to ActB, and relative gene abundance was calculated using the method of 2−ΔΔCt as previously described.10
RT‐qPCR was conducted on LightCycler 96 (Roche). Each reaction mixture consisted of 2 μl of cDNA, 0.5 μl each of forward (10 μM) and reverse primers (10 μM), 12.5 μl 2× SYBR Premix Ex TaqTM II (Tli RNaseH Plus) (TAKARA BIO), and 9.5 μl of nuclease‐free water (Millipore) in a total reaction volume of 25 μl. The thermal cycler program consisted of 40 cycles of 95°C for 15 s and 60°C for 1 min. Primers were designed using Primer Premier 5.0 (PRIMIER Biosoft). The primer sequences are listed in Table