Hiscript 2 q select rt supermix for qpcr gdna wiper kit

Total RNA was isolated utilizing the TRIzol reagent (Vazyme Biotech Co., Nanjing, China). Subsequently, DNase I-treated total RNAs underwent reverse transcription (RT) employing the HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co.). Replicate quantitative analyses were conducted using the 2 × T5 Fast qPCR Mix (SYBRGreenI) kit (TsingKe Co., Beijing, China). The primers employed for the quantitative real-time polymerase chain reaction (qRT-PCR) are provided in Table 1.

Total RNA was extracted using TRIzol reagent (Vazyme Biotech Co, Ltd., Nanjing, China). DNase I-treated total RNAs were used for reverse transcription (RT) with HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co). Quantitative assays were performed in triplicate using the 2 × T5 Fast qPCR Mix (SYBRGreenI) kit (Vazyme Biotech Co). The primers for qRT-PCR are shown in Table S1.

C. goeringii was collected from Yunnan Province, China. Total RNA was extracted using a Plant RNA MiniPrep™ kit (Zymo Research Corporation). Using 500 ng of RNA, complementary DNA was synthesized via reverse transcription based on a HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co., Ltd) and a (dT)15 primer. cDNA (1 μl) was used for subsequent qRT-PCR using the AceQ qPCR SYBR® Green Master Mix kit (Vazyme Biotech Co., Ltd) in an ABI StepOne system (Applied Biosystems) according to the default protocol. Each sample was analysed in triplicate. The primers used in this study are listed in Supplementary Table 39.

Total RNA was extracted using the FastPure® Plant Total RNA Isolation Kit (Vazyme, China) and operated according to the manufacturer’s instructions. The first-strand cDNA was synthesized using a HiScript® II Q Select RT Super Mix for qPCR + gDNA wiper Kit (Vazyme). The housekeeping gene of cassava actin was used as an internal control to normalize the data. The primers were designed using the NCBI-Primer-BLAST tool (Table S5) and the range of the PCR products was set as 80 ~ 200 bp. qRT-PCR was carried out using ChamQ Universal SYBR qPCR Master Mix (Vazyme) in a qTOWER2.2 real-time PCR system (Analytik Jena, Germany) in accordance with the manufacturer’s protocol. The total volume of 10 µL qRT-PCR reaction was used, including 5 µL ChamQ Universal SYBR qPCR Master Mix, 0.4 µL of forward primer (10 µM), 0.4 µL of reverse primer (10 µM), 1 µL of cDNA, and 3.2 µL of ddH₂O. The cycling conditions were 95 ℃ for 30 S, followed by 42 cycles at 95 ℃ for 10 S and 60 ℃ for 30 S. Melting curve analysis was then performed ranging from 60 to 95 ℃. The relative expression levels in CK and drought treatments were calculated with the formulas 2^−ΔCt and 2^−ΔΔCt [53 (link)], respectively. Three independent biological replicates were applied for all treatments. Statistical analysis was calculated using IBM SPSS 25.0.

Total RNA was extracted from leaf tissue using TRIzol reagent (Vazyme Biotech Co, Ltd., People’s Republic of China). DNase I-treated total RNAs were subjected to reverse transcription (RT) with HiScript II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech Co, Ltd., People’s Republic of China) according to the manufacturer’s instructions. Quantitative assays were performed using AceQTM qPCR SYBR Green Master Mix (Vazyme Biotech Co, Ltd., China). Relative expression level is normalized to the amount of OsActin (LOC_Os03g50885) in the same sample and presented as 2^–△CT. All primers used for RT-qPCR are listed in Supplementary Table S6.

The expression level of candidate genes in two Pb-tolerant (HANNA and III-229) and two Pb-sensitive (6024–1 and EH3143) genotypes were evaluated by quantitative real time PCR (qRT-PCR). Total RNA was extracted from seven-day-old radicles grown under normal (0 mg/L) or Pb stress (100 mg/L) condition using TransZol kit (Trans Gene Biotech). A total amount of 500 ng RNA was used to synthesize first strand cDNA using HiScript® II Q Select RT SuperMix for qPCR (+gDNA wiper) kit (Vazyme Biotech). The gene copy specific primers of candidate genes were designed using Primer Premier 5 (Additional file 9: Table S5). The qRT-PCR assay was carried out using LightCycler® 480 SYBR Green I Master kit (Roche Life Science) in LightCycler® 480 qPCR machine (Roche Life Science) according to the manufacturer instructions. Data were collected from three technical replicates. The relative expression level was normalized by BnACTIN7 using a △CT method (Li et al., 2017). The tukey test was employed for differentiation analysis on relative gene expression level between accessions and treatments.