Quantitative RT-PCR for Transcript and miRNA Expression

For cDNA synthesis,1 μg of total RNA was used. First-strand cDNA was synthesized using a PrimeScript RT reagent kit with gDNA eraser (Takara, Dalian, China). Premix Ex TaqTM II(TaKaRa)was used for real-time quantitative PCR (qRT-PCR). Gene-specific primers are shown in Table S1. qRT-PCR was undertaken with a 7500 Fast Real-Time PCR system (Applied Biosystems Inc., Carlsbad, CA, USA). The 25-μL reaction solutions contained 12.5 μL 2XSYBR Premix Ex TaqTM II, 1 μL forward primer, 1 μL reverse primer, 2 μL DNA template and 8.5 μL H₂O. For the mature miRNAs, RT-PCR employed a stem-loop primer to detect miRNAs. The primers were based on those described by Chen et al. and Varkonyi–Gasic et al. [37 (link),38 (link)]. Their 3′-ends, which were complementary to the six nucleotides at the miRNA 3′-end, were combined with the 44-nt sequence to form a stem-loop structure.The stem-loop reverse transcription reactions were performed using M-MLV Reverse Transcriptase (Invitrogen) according to the supplier’s manual. U6 was used as a reference for small RNA expression validation [39 (link)]. The expression levels of target transcripts were normalized using the housekeeping gene UBQ7 as the reference control. Three biological replicates were used for qRT-PCR validation. The 2^−ΔΔT method was used to calculate relative expression levels of salt-responsive miRNAs and target genes.