Mircute mirna sybr green kit

Genomic DNA was extracted from the mature leaves of P. suaveolens with a Plant Genomic DNA Kit (TIANGEN, Beijing, China). Total RNA was isolated from the tested samples using SV Total RNA Isolation System (Promega, Madison, WI, USA), and treated with RNAse-free DNAse I to eliminate the residual genomic DNA, according to the manufacturer’s instructions (Promega, Madison, WI, USA).
Relative quantification of the expressions for Psu-miR475b and its targets, and GUS gene by quantitative real-time PCR (qRT-PCR) were performed on 7500 Real-Time PCR System, by using MiRcute miRNA SYBR Green Kit (TianGen, Beijing, China) and SuperScript^TM III Platinum^® Two-Step qRT-PCR Kit with SYBR^® Green (Invitrogen, Carlsbad, CA, USA), respectively. According to the previous reports59 60 and our generated Solexa sequencing (data not shown), miR167e and miR168a-3p were used as inner references for Psu-miR475b, and poplar ACTIN gene as endogenous reference gene for miR475b-targets27 . Data were from at least three quantitative PCR replicates per sample and three biological replicates. The specific primers of Psu-miR475b and its target genes, GUS gene and reference gene are shown in Supplementary Table S2.

Total miRNA was isolated from the cerebellar granule neuron precursors using the miRcute miRNA kit (Tiangen Biotech), and cDNA was synthesised using the miRcute miRNA cDNA kit (Tiangen Biotech). The miRcute miRNA SYBR Green kit (Tiangen Biotech) was used for quantitative real-time PCR. The miR-9 primers were from Tiangen Biotech, and U6 was used as the internal control.