Chamq universal sybr qpcr master

For miRNA qPCR, reverse transcription of miRNA was performed by mir-XTM miRNA First-Strand Synthesis Kit (Takara, Tokyo, Japan). Briefly, 1 μg of total RNA was incubated in a thermal cycler for 1 h at 37 °C, then terminating at 85 °C for 5 min to inactivate the enzymes. The qPCR was carried out with mixture of 0.8 μL of cDNA, 0.4 μL primers, 5 μL of Chamq Universal SYBR qPCR Master (Vazyme, Nanjing, China), and 3.8 μL ddH₂O. And the reference gene U6 was used to normalize the expression levels by the 2^-ΔΔCt method. For qPCR of target genes (Table S9), the cDNA of mRNA was obtained by Primer ScriptTM RT reagent Kit (Takara, Tokyo, Japan). The reference gene TBP [53 ] was used to correct gene expression levels. All data are expressed as the mean ± SEM. The one-way ANOVA was used in SPSS 19.0 (IBM, NY, USA), and Duncan’s new multiple range tests were used to analyze statistical significance.

Total RNA were extracted from leaves of treated seedlings using TRIzol Reagent (Invitrogen). After treatment with DNase I, approximately 2 μg of the total RNA were synthesized the first-strand cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) with oligo (dT) as a primer. qPCR reactions labeled by ChamQ Universal SYBR qPCR Master (Vazyme) were performed on a cycler apparatus (Bio-Rad CFX96). The data were analyzed by the 2-DCT method. Rice Actin was used as internal controls. The qPCR primers were listed in Table S7. Two biological replicates were included.