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 a 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 and U6 reverse primer in a 20-μL reaction volume. TheU6 snoRNA 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).