Revertra ace 1 qpcr rt master mix with gdna remover

Total RNA was extracted using the TRIzol reagent (Invitrogen, Waltham, MA, USA) by following the manufacturer’s protocol, after which cDNA was synthesized using the ReverTra Ace 1 qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). The qRT-PCR was performed using 2 × T5 Fast qPCR Mix (SYBR Green I, Tsingke, Beijing, China) according to the manufacturer’s instructions. The rice OsActin gene served as an internal control for the data normalization in the formal analysis. The results are presented as the mean ± SD in triplicate. Bar graphs were generated using GraphPad Prism 9. The specific primers used are listed in Supplementary Table 2.

For gene expression analysis, total RNA was extracted from leaves at different collected time points with or without Xoo treatment using TRIzol reagent (Invitrogen, Waltham, MA, USA) and reverse transcribed to cDNA using the ReverTra Ace 1 qPCR RT Master Mix with gDNA Remover (TOYOBO, Osaka, Japan). To monitor osa-miR1432, the miRNAs were extracted from the aforementioned samples using an miRNA Isolation Kit (OMEGA, Atlanta, GA, USA), and the specific stem-loop RT primer for osa-miR1432 was applied to the reverse transcription protocol [43 (link)]. Quantitative reverse transcription-PCR (qRT-PCR) was performed using indicated primers and TransStart Green qRT-PCR Super Mix (TransGen, Beijing, China). SnRNA U6 and OsACTIN genes separately served as the internal reference to data normalization for miRNAs and mRNAs, and the relative expression levels of miRNAs and target genes were determined using a one-way ANOVA followed by post hoc Tukey HSD analysis with three biological replicates and at least three technical repeats. Specific primers for quantitative real-time PCR are listed in Supplementary Table S1.