The total genomic DNA was extracted from 4-day-old cultured fungal hyphae using a HiPure fungal DNA kit II (Magen). Extraction of total RNA and first-strand cDNA synthesis were performed using RNA-easy isolation reagent (Vazyme) and a HiScript III first-strand cDNA synthesis kit (+gDNA wiper) (Vazyme), respectively. Genomic DNA and cDNA were used for PCR amplification of the genes or DNA fragments using specific primers (see Table S8 in the supplemental material). Finally, the PCR products were used for agarose gel electrophoresis and sequencing analysis.
Hiscript 3 first strand cdna synthesis kit gdna wiper
Manufactured by Vazyme
Sourced in China
The HiScript III first-strand cDNA synthesis kit (+gDNA wiper) is a tool for the reverse transcription of RNA into cDNA. It includes components to remove genomic DNA contamination.
Automatically generated - may contain errors
Lab products found in correlation
Chamq universal sybr qpcr master mix, by Vazyme (5 mentions)
Rna easy isolation reagent, by Vazyme (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Total rna extraction kit, by Qiagen (2 mentions)
Sybr qpcr master mix, by Vazyme (1 mentions)
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (1 mentions)
Trizol regent, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometry, by Thermo Fisher Scientific (1 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Maxima sybr green qpcr master mix, by Vazyme (1 mentions)
Sybr green mix, by Roche (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Sybr premix ex taq kit, by Takara Bio (1 mentions)
Abi prism 7500 real time pcr system, by Takara Bio (1 mentions)
Trizol kit, by Thermo Fisher Scientific (1 mentions)
Lightcycle 480 2, by Roche (1 mentions)
11 protocols using hiscript 3 first strand cdna synthesis kit gdna wiper
1
Fungal DNA and RNA Isolation
2
Quantitative Gene Expression Analysis
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Total RNA was extracted using Trizol regent (Invitrogen), the manufacturer's protocol. The concentration of total RNA was determined by calculating the absorbance ratio at 260/280 using a Nanodrop Spectrophotometry (Thermo Scientific, USA), and the quality of the RNA was determined by electrophoresis on a 1% agarose gel. To generate the first-strand cDNA for gene amplification and expression analysis, 2 μg of total RNA was reverse-transcribed using the HiScript III first-strand cDNA Synthesis Kit (+ gDNA wiper) (Vazyme Biotech).
Real-time PCR was performed using QuantStudio™ 5 Real-time PCR system (Applied Biosystems). Amplification reactions were carried out in triplicate, and each reaction containing 10 μL of 2 × SYBR qPCR master mix (Vazyme Biotech), 1 μL of the diluted cDNA, 0.4 μL of each primer, and 8.2 μL ddH2O. The Cp value of each sample was computed automatically by software, and relative fold changes were calculated using the 2−ΔΔCt method. The PCR amplification efficiency was calculated using the standard curve method, and optimized to over 95%. Primer specificity was evaluated by examining the melt curves.
Real-time PCR was performed using QuantStudio™ 5 Real-time PCR system (Applied Biosystems). Amplification reactions were carried out in triplicate, and each reaction containing 10 μL of 2 × SYBR qPCR master mix (Vazyme Biotech), 1 μL of the diluted cDNA, 0.4 μL of each primer, and 8.2 μL ddH2O. The Cp value of each sample was computed automatically by software, and relative fold changes were calculated using the 2−ΔΔCt method. The PCR amplification efficiency was calculated using the standard curve method, and optimized to over 95%. Primer specificity was evaluated by examining the melt curves.
3
Rice GRF Expression Analysis under Phytohormones
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The rice cultivar Zhong-Hua-11 (ZH11, Oryza sativa L. ssp. japonica) was used to analysis the expression of GRFs. The rice plants were soaked in 100 µM hormone (IAA, BR, GA3, 6BA, ABA, and MeJ) at the three-leaf-one-heart stage for various time points (0 h, 6 h, 9 h, 12 h, 24 h). All the seedlings were cultured at 28 °C for 12 days with 14-h light/10-h dark and 60% relative humidity (Cheng et al., 2021 (link)).
All RNAs of samples were extracted using TRIzol Reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA), And the first-strand cDNA was synthesized from RNA treated with a 2 µg DNA enzyme using HiScript III First Strand cDNA Synthesis Kit (+gDNA Wiper) (Vazyme, Nanjing, China). The RT-qPCR analysis of GRFs in rice was performed by using the HieffqPCR SYBR Green Master Mix (No Rox) (Yeasen, Shanghai, China), and the relative expression level of the 12 GRFs was analyzed with the 2−ΔΔCT method. GRFs specific primers used for RT-qPCR analysis were listed inSupplementary Table 8
All RNAs of samples were extracted using TRIzol Reagent according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA), And the first-strand cDNA was synthesized from RNA treated with a 2 µg DNA enzyme using HiScript III First Strand cDNA Synthesis Kit (+gDNA Wiper) (Vazyme, Nanjing, China). The RT-qPCR analysis of GRFs in rice was performed by using the HieffqPCR SYBR Green Master Mix (No Rox) (Yeasen, Shanghai, China), and the relative expression level of the 12 GRFs was analyzed with the 2−ΔΔCT method. GRFs specific primers used for RT-qPCR analysis were listed in
4
RNA Extraction and qPCR Analysis
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After differentiation and treatment, genomic RNA was extracted using an RNA extraction kit (NucleoSpin RNA Plus, Macherey, Düren, Germany) in accordance with the manufacturer’s instructions. The concentration and purity of the isolated RNA were evaluated using Nanodrop spectrophotometer (Thermo Scientific Nanodrop, NanoDrop Technologies, Wilmington, DE, USA). The cDNA was synthesized from one μg of RNA using the HiScript III First Strand cDNA Synthesis kit +gDNA wiper (R312-02, Vazyme, Nanjing, China). Meanwhile, the qPCR was conducted using Maxima SYBR green qPCR Master Mix (Q712-02, Vazyme) according to the manufacturer’s instructions. The nucleotide primer sequences used in this study were presented in Table 3 . The primers were synthesized by Bio3 Scientific Sdn. Bhd. (Puchong, Malaysia). The glycerldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, was used as an internal reference (forward 5′-GTCATCCCTGAGCTGAACGG-3′, reverse 5′-AAGTGGTCGTTGAGGGCAAT-3′). Each gene was amplified three times using RT-qPCR. The amplification parameters were as follows: 95 °C for 30 s, 95 °C for 5 s, and 60 °C for 31 s for a total of 40 cycles. Using the 2-∆∆Cq method, the quantification values were subsequently calculated and analyzed. Ratio in untreated cells (negative control) was assigned as 1.
5
Quantitative Analysis of Gene Expression
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Total RNA was extracted from the cell lines using the Total RNA Extraction Kit (Qiagen GmbH). cDNA was prepared using a HiScript®III First-Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech Co., Ltd.) according to the manufacturer's instructions. Subsequently, qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd.). Primers were synthesized by GenScript. The reaction steps were as follows: i) pre-denaturation at 95°C for 30 sec; ii) PCR reaction of 40 cycles at 95°C for 5 sec, followed by incubation at 60°C for 30 sec. The sequences of the β-actin and the ACKR primers are presented in Table I . Cycle thresholds were recorded and the relative expression of target genes was calculated and quantified using the 2−∆∆Cq method with β-actin as the reference gene (24 (link)).
6
Quantitative RT-PCR for Gene Expression
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RNA was extracted from the cell lines using the Total RNA Extraction Kit from Qiagen GmbH. In order to synthesize cDNA, the HiScript®III First-Strand cDNA Synthesis Kit (+gDNA wiper) from Vazyme Biotech Co., Ltd. was used, following the instructions provided by the manufacturer. Subsequently, qPCR was performed using the ChamQ Universal SYBR qPCR Master Mix, also from Vazyme Biotech Co., Ltd. All of the kits/mixes mentioned above were used according to the manufacturer's instructions. The primers utilized in this process were synthesized by Sangon Biotech Co., Ltd. The qPCR program included a pre-denaturation step at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Table II contains the sequences of the β-actin and ARF primers. The cycle thresholds were recorded and the 2−∆∆Cq method (11 (link)), with β-actin serving as the reference gene, was used to determine and measure the relative expression levels of the target genes.
7
RNA Extraction and Reverse Transcription
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Total RNA was extracted with RNA-easy isolation reagent (Vazyme Biotech Co., Ltd.). Reverse transcription was conducted with a HiScript III first-strand cDNA synthesis kit (+gDNA wiper) (Vazyme Biotech Co., Ltd.) according to the manufacturer’s instructions. Afterwards, cDNA samples were analyzed by qPCR using ChamQ universal SYBR qPCR master mix (Vazyme Biotech Co., Ltd.). Primers used for RT-qPCR are listed in Table S1C .
8
Quantitative Real-Time PCR Analysis
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Total RNA was extracted with TRIzol and reverse-transcribed into cDNA using HiScript III First Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme Biotech Co., Ltd, China). RT-qPCR was performed using SYBR Green Mix (KAPA Biosystems, Wilmington, MA, USA) on a 7900HT FAST real-time PCR system (Life Technologies). GAPDH or 18S rRNA expression was used as a normalization control. The primer sequences used are listed in Table S2 .
9
Total RNA Extraction and RT-qPCR Analysis
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Total RNA isolation and RT‐qPCR assays were performed as described previously (Zhang et al., 2022 (link)). Briefly, total RNA was extracted from aphid tissues, tobacco or wheat leaves using TRIzol Reagent (Thermo Fisher, USA) according to the manufacturer's protocols. First‐strand cDNA synthesis was conducted using the HiScript III First‐Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, China), and RT‐qPCR was performed on the ABI Prism 7500 Real‐Time PCR System using SYBR Premix Ex Taq Kit (TaKaRa, Japan) and sequence‐specific primers (Table S2 ). Three replicates were conducted for each treatment, and each replicate contains three technical replicates.
10
Quantifying Mtb Gene Expression
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Total RNA from Mtb was converted to cDNA using a commercial kit [HiScript III first-strand cDNA Synthesis Kit (+gDNA wiper); Vazyme], and qPCR of each sample was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The conventional 2–∆∆Ct method was used to determine the relative mRNA expression of candidate genes in drug-resistant Mtb and its parent strain. SYBR Green was used as the fluorescent probe. Mean±sem was calculated from three independent experiments, and significant differences were determined using an unpaired Student’s t-test. The primers used in this study are listed in Table S11.
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