Reverse transcription PCR (RT-PCR) of differentially expressed miRNA

Total RNA was extracted from wheat caryopses under control and drought conditions using Trizol reagent according to the manufacturer's instructions. RNA was reverse transcribed to cDNA using One-Step miRNA RT Kit (SinoGene, China). The reverse transcription reaction mixture contained 12 μL RNA template mix, 1 μL Oligo-dT adapter primer, 4 μL 5 × reaction buffer, 1 μL M-MLV RTase and 2 μL 10 mM of dNTP. The reverse transcription reaction was performed at 37°C for 60 min followed by 85°C for 10 min. RT-PCR was performed for 4 miRNAs using cDNA as template and U6 served as internal control to normalize the cDNA concentrations. PCR amplification was performed in a 25 μL reaction volume containing 0.5 μL Taq enzyme, 2.5 μL cDNA template, 3 μL 10 × buffer (including MgCl₂), 1 μL dNTP, 1 μL of each primer at the concentration of 20 mM, and 11 μL nuclease-free ddH₂O. The condition of PRC was set as follows: 95°C for 3 min, followed by 25–35 cycles of 94°C for 30 s, 52°C for 30 s, and 68°C for 30 s, depending upon the individual miRNA. Final extension was performed at 68°C for 7 min. Amplified products were separated on 1.5% agarose gel and the marker used was NormalRun™ prestained 250 bp-I DNA ladder (Generay, China).