Hiscript 2 q rt supermix for qpcr gdna wiper

Total RNA was extracted from different rice tissues or developing grains using an RNAprep Pure Plant kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA was amplified from 1 µg of total RNA using HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Nanjing, China). The RT‒qPCR was carried out with a Roche Light Cycler 480 system using AceQ qPCR SYBR Green Master Mix (Vazyme, Nanjing, China). The OsActin gene (LOC_Os03g50885) was used as an internal control for normalization. The primers used for expression analysis are listed in Table S3. Three biological replicates were performed. The relative expression level was calculated by the relative quantification method as described (Livak and Schmittgen 2001 (link)).

Total RNA was isolated using the TRIzol reagent (Sigma-Aldrich, St. Louis, MO, USA), and cDNA was prepared by removing the residual gDNA and reverse transcribed with HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme) according to the manufacturer’s instructions.
For RT-qPCR, 1000 ng of the RNA sample was reverse transcribed and then the cDNA was diluted at a ratio of 1:2. All qPCR were performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) according to the manufacturer’s instructions.

As previously described [85 (link)], RNA was extracted with Trizol reagent (R401-01, Vazyme, Nanjing, China). cDNA synthesis was achieved by HiScript^® II Q RT SuperMix for qPCR (+gDNA wiper) (R223-01, Vazyme, Nanjing, China). Real-time quantitative PCR was performed by the Bio-Rad CFX Connect Real-Time PCR System with MonAmp™ ChemoHS qPCR Mix (MQ00401S, Monad, Shanghai, China). The primers for this experiment were purchased from Sangon (Shanghai, China), and the sequences of all primers are listed in Table 1.

Total RNA was isolated using TransZol reagent (TransGen Biotech Inc., Beijing, China) in accordance with the manufacturer’s protocol. The extracted total RNA was dissolved in diethylpyrocarbonate-treated water. The cDNA template for gene cloning was synthesized from 2μg of RNA using HiScript II One Step RT-PCR Kit (Vazyme, Piscataway, NJ, United States). While for qRT-PCR, the cDNA was synthesized from 1μg total RNA using HiScript II Q RT SuperMix for qPCR (+g DNA wiper; Vazyme, Piscataway, NJ, United States).

One μg of total RNA was used to synthesize cDNA in a 20 μL reaction mixture using HiScript ^®II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China) according to the manufacturer’s instructions. Real-time PCR was carried out on the ABI StepOnePlus^™ Real-Time PCR systems. Gene-specific primer sequences of the reference and target genes are listed in Table 1 and were synthesized by Invitrogen Biotech Co. Ltd. (Shanghai, China). The following thermal profile was used for qRT-PCR: 95°C for 3 min, followed by 40 cycles of 95°C for 10 sec and 60°C for 30 sec. The relative gene expression was calculated using the 2^-ΔΔCT method. Both GAPDH and HPRT1 genes were used to normalize variations in the amount of starting material.

Two ecological types of S. alfredii were treated by CdCl₂ with different concentrations (HE: 10 and 100 μM, NHE: 10 μM) and times (0, 6, 24 h, 3, and 7 days). The roots, stems, and leaves of each plant were separated and frozen rapidly in liquid nitrogen. The total RNA was extracted using a Spin Column Plant Total RNA Purification Kit (Sangon) and then synthesized to cDNA with a HiScript II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme). Real-time quantitative PCR (RT-qPCR) was performed using a ChamQ SYBR Color qPCR Master Mix (Without ROX) (Vazyme), with LightCycler 480 System (Roche, United States). The RT-qPCR protocol was as follows: 95°C for 3 min, 40 cycles of 95°C for 10 s, 60°C for 30 s. The melting-curve analysis was included to verify the specificity of the primer. The mean amplification efficiency was analyzed with the LinReg software (Ruijter et al., 2009 (link)). The specific primers for RT-qPCR were designed according to the SaPCR2 sequence as follows: SaPCR2 forward 5′-GCGGTGGGATGTGGTCTAC-3′ and SaPCR2 reverse 5′-CGATAATCTCGGCTATTTGGC-3′, SaACTIN1 forward 5′-TGTGCTTTCCCTCTATGCC-3′, and reverse: 5′-CGCTCAGCAGTGGTTGTG-3′ (Chao et al., 2010 (link)). The relative expression levels were calculated using 2^−ΔCt method.