Hifair 3 reverse transcriptase

Total RNA from different samples was extracted by Total RNA Extractor (Trizol) (Sangon Biotech, Shanghai, China) and reverse transcribed into cDNA using Hifair^® III; Reverse Transcriptase (Yeasen, Shanghai, China) according to the instruction book.
The RT-PCR was performed using PrimerSTAR MaxDNA Polymerase (TaKaRa, Shiga, Japan), and the appropriate annealing temperature for PCR according to the properties of primer pairs for different genes. The number of amplification reaction cycles in this study was 30. The RT-PCR experiment for each gene was conducted with 3 biological replicates. The PCR products were determined using electrophoresis on the 1% agarose gels. Specific primers for different OsSR genes and the control gene OsActin used for RT-PCR are listed in Table S7.
For the qRT-PCR experiment, SYBR Green qPCR Master Mix (TOROIVD) was used, and the experiment was conducted on LightCycler 480 II (Roche, South San Francisco, CA, USA). The data was analyzed as previously described using OsActin as the internal standard (Livak & Schmittgen, 2001 (link)). The qRT-PCR experiment was carried out using 3 biological replicates, and 3 technical replicates were performed for each biological replicate. The qRT-PCR data was calculated using 2^−ΔΔCT method and Student’s t-test. The primer sequences for qRT-PCR are listed in Table S6.

According to the relative manual, total RNA was isolated using the E.Z.N.A^@ Plant RNA kit (Omega Bio-tek Inc., Norcross, GA, USA). We detected the quality and purity of RNA using K5800C (KAIAO, Beijing, China). According to the manufacturer’s protocols, 0.5 ug RNA of each sample was used for 1st strand cDNA synthesis using Hifair^® III Reverse Transcriptase (YEASEN, Shanghai, China). Subsequently, the cDNA was diluted 7-fold for RT-qPCR analysis.

Total RNA was extracted from bladder tissue with FastPure^® Cell/Tissue Total RNA Isolation Kit V2 (RC112, Vazyme, Nanjing, China) and reverse transcribed to cDNA using Hifair^® III Reverse Transcriptase (11111ES92; YEASEN, Shanghai, China). Then, the Hieff^® qPCR SYBR Green Master Mix (11202ES03, YEASEN) on QuantStudio 6 Flex (Applied Biosystems, Thermofisher, USA) was used to conduct qRT-PCR, and 2^−ΔΔCt was used to calculate relative RNA levels. The primers used in the qRT-PCR assay are demonstrated in Table 1.