Real time PCR
Total RNA was isolated with TRIzol reagent (Invitrogen, Carlsbad, CA,
USA) according to the manufacturer’s instruction. High-quality total RNA
(A260/280 = 2.00) was reverse-transcribed into cDNA. Small RNA qRT-PCR
was conducted using an miRNA First Strand cDNA Synthesis kit (stem-loop
method) (Sangon Biotech, Shanghai, China). A total of 3 μg total RNA was
used for first-strand cDNA synthesis with the stem-loop primer andU6 reverse primer in a 20-μL reaction volume. The U6snoRNA was used as an internal control. For qRT-PCR analysis of coding
genes, 2 µg total RNA treated with DNase I was reserve-transcribed into
cDNA using M-MuLV (Takara, Otsu, Japan) with oligos (dT). The internal
control was ACT7 . qRT-PCR was performed using SYBR mix (YEASEN,
China) on a Bio-CFX96 instrument. Three biological replicates and three
technological replicates were analyzed. Real-time PCR and reverse
transcriptase (RT)-PCR were performed using gene-specific primer pairs
(Table S5).