Mirna sybr green rt qpcr kit

RT-qPCR validation was carried out on the same samples used for transcriptome sequencing (n = 3). Primers were designed from the candidate gene sequences by premier 5.0 software and the online Primer-BLAST program. Primers used in this study are provided in Table S1. One microgram total RNA for RNA sequencing was reverse transcribed into cDNA. Real-time PCR assays were conducted on a CFX96 real-time PCR Detection System (Bio-Rad, Hercules, CA) with 5 μL SYBR Green Master Mix (Takara, China). β-actin was selected as the reference gene according to a previous study (26 (link)). The small RNA of the same samples used for sequencing was extracted using an RNAiso for Small RNA Kit (Takara, China) according to the manufacturer's protocol. Subsequently, the first-strand cDNA was synthesized for mature miRNA expression analysis by a Mir-X™ miRNA First-Strand Synthesis Kit (Code No. 638315, Takara, China). The qPCR was carried out using a miRNA SYBR Green RT-qPCR Kit (Takara, China) with the provided miRNA reference gene (U6). Relative quantitative levels were calculated based on the 2^−ΔΔCT method (44 (link)).

To validate the expression pattern of miRNAs and their target genes, we performed quantitative real-time PCR (qRT-PCR) assays. We first extracted RNA from the same samples with Plant RNA Kit (Omega Bio-Tek, United States) and reverse transcribed using PrimeScript RT Kit (Takara Bio, Japan). The qRT-PCR assays of candidate genes were conducted using TB Green^® Premix Ex Taq™ II (Takara Bio, Japan) on CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Inc., United States) following protocol: 95°C for 5 min, followed by 40 cycles at 95°C for 5 s, 60°C for 30 s and 72°C for 30 s. The relative gene expression level was computed with 2^–ΔΔCt method using PP2A as the reference gene for candidate genes. Small RNA was extracted with Plant miRNA Kit (Omega, China) following the manufacturer’s protocols. Mature miRNAs were reverse transcribed into cDNA using Mir-X™ miRNA First-Strand Synthesis Kit (Code No. 638315, Takara, China). The qPCR was carried out using miRNA SYBR Green RT-qPCR Kit (Takara, China) following similar temperature settings. The relative expression level of miRNA was estimated using 5S rRNA as internal reference. Primer pairs of candidate genes and miRNAs were designed with NCBI primer-blast and were provided (Supplementary Table 1).