Total RNA extraction and cDNA synthesis

Total RNA was extracted using the LC sciences Total RNA Purification Kit (#TRK-1001, LC sciences, USA), which purifies all sizes of total RNA, including mRNA, ribosomal RNA, miRNA and other small RNA (20–200 nt), according to the previous methods with some modification [63]. The powder ground from 50 mg sample in liquid nitrogen was immediately transferred into a 2.0 ml RNase-free tube and added 600 μl extraction buffer with 6% Plant RNA Isolation Aid (Ambion, #Am9690). After shaking vigorously, the mixture was incubated in the ice for 15 min and then centrifuged at 12,000 rpm for 10 min at room temperate, by which the yield of total RNA could be improved. The remaining processes followed the manufacturer’s instructions. The integrity of total RNA was further assessed by 1.5% agarose gel electrophoresis, and the RNA concentration and purity were determined by NanoDrop™ 8000 Spectrophotometer (Thermo Fisher Scientific, USA). Only RNA samples with an A260/A280 ratio between 1.9 and 2.1 and A260/A230 greater than 1.80 were used for cDNA synthesis. Then, 1.5 μg of total RNA was polyadenylated with ATP by poly (A) polymerase (PAP) at 37 °C for 1 h in a 20-μl reaction mixture using the Poly(A) Tailing Kit (#AM1350, Invitrogen, USA). Then, 10 μl (750 ng) of the E-PAP-treated total RNA was reverse transcribed with a poly(T) adapter universal reverse transcription (RT)-primer (5′-AACGAGACGACGACAGACTTTTTTTTTTTTTTTV-3′) using SuperScript III reverse transcriptase Kit (#18080-051, Invitrogen, USA) following the manufacturer’s instruction. The cDNA was diluted 20-fold with nuclease-free water for qRT-PCR.