Total RNA was isolated with Trizol reagent (Invitrogen, USA). For quantitative detection of the primary MIR156s, cDNA was firstly synthesized using M-MLV (Promega, USA) and detected with the TransStart Tip Green qPCR Super Mix (TransGen Biotech, China). For comparison of mature MIR156, total RNA was purified with the miRcute miRNA purification kit (Tiangen Biotech, China) and further cDNA was reverse transcribed with the miRcute miRNA first-strand cDNA synthesis kit (Tiangen Biotech, China). The mature miR156 was detected with the miRcute miRNA qPCR Detection Kit (Tiangen Biotech, China). Quantitative RT-PCR was conducted with the SYBR Premix ExTaq kit (Takara, Japan) in a total volume of 25 μL on the Applied Biosystems 7500 real-time PCR system according to the manufacturer’s manual. The level of PP2A (AT1G13320) transcript was adopted as an internal control. The expression of mature MIR156 or each primary MIR156 (pri-MIR156A~H) in different samples was calculated by the 2ΔCt method, in which: ΔCt = Ct (PP2A)−Ct (target). The expression level of target gene after treatments are the ratio of expression in treated samples compared with the controls by the 2ΔΔCt method, in which ΔΔCt = (CtPP2A−Cttarget) treatment−(CtPP2A−Cttarget) control.
All the primers used for qRT-PCR above are shown in Supplementary Table1 .
All the primers used for qRT-PCR above are shown in Supplementary Table