Hiscript 2 q rt supermix for qrt pcr kit

Total RNA was isolated from cultured cells using the RNAiso Plus reagent (TakaRa), and reverse transcription (RT) experiment was carried out using the HiScript II Q RT SuperMix for qRT-PCR kit (Vazyme). RT-PCR was carried out using the AceQ qPCR SYBR Green Master Mix (Vazyme) on a RT-PCR system (Roche) with primers as listed in Supplementary Table S3. The expression levels of indicated genes were normalized to an internal control(18 S), and the relative expression levels were evaluated using the 2^−ΔΔCT method. Each target was measured in triplicate.

Field-fresh tissue samples were collected, ground in liquid nitrogen, and used for total RNA extraction using the EASY Spin Plant RNA Rapid Extraction Kit (RN09, Aidlab, Beijing, China). A total of 1 μg of total RNA was used for genomic DNA removal and first-strand cDNA synthesis using the HiScript II Q RT SuperMix for qRT-PCR Kit (R223, Vazyme). The qRT-PCR reaction mixture (20 μL) included 10 μL of Universal SYBR qPCR Master Mix (Q711, Vazyme), 5 μL of diluted cDNA, 0.5 μL of 10 μmol/L forward and reverse primers each, and 4 μL of ddH₂O. Reactions were induced in triplicate, with fluorescence detection enabled via a Bio-Rad CFX Connect Real-Time PCR System, using the ΔΔCq method. GhHistone3 (Accession number: AF024716) served as the reference gene, with specific primer sequences provided in the Supplementary Materials.