The strains were cultured according to the method described above, and the expression level of related genes in the pyruvate cycle was detected by quantitative real-time PCR (qRT-PCR). Briefly, the total RNA (Dobiothec, Fuzhou, China) of Xanthomonas oryzae was extracted according to the Biospin Total RNA Extraction Kit step. The concentration and purity of total RNA of Xanthomonas oryzae were measured by μDrop plate, and the cDNA of X. oryzae was obtained by One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). qRT-PCR was carried out on a 96-well plate containing 20 μL of liquid per well, including 10 μL 2× Perfect start Green qPCR SuperMix, 0.4 μL 50× Passive Reference Dye optional, 6.8 μL nuclease-free water, 2 μL cDNA template, and 0.4 μL per pair of specific primers. The detailed information of the primers is listed in Table S1 . To analyze the relative expression level of the target gene, we converted the data to a percentage relative to the control group, and the data were calculated following the method mentioned before [16 (link),37 (link)]. At least three biological replicates were carried out.
One step gdna removal and cdna synthesis supermix kit
Manufactured by Transgene
Sourced in China
The One-Step gDNA Removal and cDNA Synthesis SuperMix Kit is a laboratory reagent designed for the simultaneous removal of genomic DNA and synthesis of complementary DNA (cDNA) from RNA samples in a single-step reaction. The kit contains a proprietary enzyme mix that efficiently eliminates genomic DNA contamination and generates high-quality cDNA for downstream applications such as real-time PCR and gene expression analysis.
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Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (4 mentions)
Perfectstar green qpcr supermix kit, by Transgene (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Trizol reagent, by Sangon (2 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (2 mentions)
Chamq sybr qpcr master mix, by Vazyme (2 mentions)
Transstart top green qpcr supermix, by Transgene (2 mentions)
Nanophotometer p330, by Implen (2 mentions)
Perfectstart green qpcr supermix, by Transgene (1 mentions)
Rneasy plant mini kit, by Qiagen (1 mentions)
Total rna kit, by Tiangen Biotech (1 mentions)
Sybr premix ex taq rt pcr kit, by Takara Bio (1 mentions)
Oligo dt primer, by Transgene (1 mentions)
Abi steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions)
Tip green qpcr supermix, by Transgene (1 mentions)
Lightcycler 480, by Roche (1 mentions)
Total rna extraction kit, by Tiangen Biotech (1 mentions)
Cfx96 thermal cycler, by Bio-Rad (1 mentions)
Transstart top green qpcr supermix kit, by Transgene (1 mentions)
33 protocols using one step gdna removal and cdna synthesis supermix kit
1
Quantitative Analysis of Pyruvate Cycle Genes in Xanthomonas oryzae
2
Validating Gene Silencing in Bactrocera cucurbitae
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qRT‒PCR analysis was used to validate gene expression in Sardh-silenced males of B. cucurbitae. Total RNA was extracted. Then, cDNA was synthesized with a One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China) using the extracted RNA. Then, a PerfectStarTM Green qPCR SuperMix Kit (TransGen Biotech, Beijing, China) was used to perform quantitative real-time PCR to compare the gene expression levels. Gene-specific primers (Supplementary Table S1 ) were designed on NCBI with primer blast. The RPL60 gene was used as a reference gene [47 (link)]. The PCR procedure was set according to the manufacturers’ instructions. Five biological replicates were performed.
3
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from T. absoluta using TRIzol reagent (Invitrogen, Carlsbad, CA, United States), according to the manufacturer’s instructions, and the quality of the resulting RNA was evaluated using both spectroscopy (Nano Photometer P330; Implen, Munich, Germany) and agarose gel electrophoresis. First-strand cDNA was then synthesised from 2 μg isolated RNA using the One-Step gDNA Removal and cDNA Synthesis Super Mix kit (TransGen, Beijing, China).
4
Quantifying Sardh and ItaE Genes in Fruit Fly Rectum
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Total RNA was extracted from the rectum of mature B. dorsalis and B. cucurbitae males. Then, cDNA was synthesized with a One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China) using the extracted RNA. Specific primers (Supplementary Table S1 ) for the Sardh and ItaE genes of B. dorsalis and B. cucurbitae were designed to quantify the absolute expression of Sardh and ItaE in the mature male rectum. The cDNA was used for amplification with the primers. The amplified fragment was then cloned into the pMD 18-T vector, which was then transferred into E. coli DH5α to reproduce. The reproduced vector was then extracted with a plasmid extraction kit and diluted in a series of 10-fold dilutions to obtain 5 different plasmid concentrations (measured by a Nanodrop spectrophotometer). A standard curve for qPCR was then generated by amplifying the 16S rRNA of the plasmid. The absolute expression of Sardh and ItaE in B. dorsalis and B. cucurbitae was then determined by referring to the standard curve with the CT values.
5
Whole-Genome Sequencing of Aspergillus sp.
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Genomic DNA from all fungal strains was prepared using lysis buffer (Supporting Information Table S6 ). Whole genome scanning of Aspergillus sp. SCSIO SX7S7 was finished using the 2nd generation Illumina sequencing platforms and 3rd generation PacBio RS (Shanghai Biozeron Biotechnology). The engineered yeast Saccharomyces cerevisiae JHY686-YH27 (link) was used to construct all of the A. nidulans A1154 ΔEM expression recombination plasmids.
Aspergillus sp. SCSIO SX7S7 was statically grown on PDB medium at 28 °C for 5 days and the mycelia were then collected. The total RNA was extracted from the mycelia following the manufacturer of the Coolaber® Fungal RNA Extraction Kit (Coolaber Technology, China) protocol. One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, China) was used to prepare the cDNA of SCSIO SX7S7.
Aspergillus sp. SCSIO SX7S7 was statically grown on PDB medium at 28 °C for 5 days and the mycelia were then collected. The total RNA was extracted from the mycelia following the manufacturer of the Coolaber® Fungal RNA Extraction Kit (Coolaber Technology, China) protocol. One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen, China) was used to prepare the cDNA of SCSIO SX7S7.
6
Transcriptome Analysis of Rice Plants
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For mRNA‐seq, total RNAs were purified from 2‐wk‐old seedlings and 60‐d plants (including inflorescence tissues) after germination using the RNeasy Plant Mini Kit (Qiagen, 74904). Library construction and Illumina sequencing were performed by the Beijing Genome Institute (Wuhan, China). Oligo(dT)‐attached magnetic beads were used to purify mRNA. Sequencing reads were mapped to the rice genome (RGAP7.0) using the Tophat program. Clean reads were mapped to the assembled unique genes by Bowtie 2 (v.2.2.5) and the expression levels of genes were calculated using rsem (v.1.2.8) and normalised to fragments per kilobase of transcript per million mapped reads (FPKM).
For gene expression analysis, total RNAs were isolated with TRIzol reagent (Sangon Biotech, Shanghai, China), and first‐strand cDNA was reverse transcribed from 5 μg of total RNA using the One‐Step gDNA Removal and cDNA Synthesis SuperMix Kit (Transgen, Beijing, China). RT‐qPCR analysis was performed using the PerfectStart Green qPCR SuperMix (Transgen). Primers for qRT–PCR are listed in Supporting Information TableS1 .
For gene expression analysis, total RNAs were isolated with TRIzol reagent (Sangon Biotech, Shanghai, China), and first‐strand cDNA was reverse transcribed from 5 μg of total RNA using the One‐Step gDNA Removal and cDNA Synthesis SuperMix Kit (Transgen, Beijing, China). RT‐qPCR analysis was performed using the PerfectStart Green qPCR SuperMix (Transgen). Primers for qRT–PCR are listed in Supporting Information Table
7
Tilapia Liver Total RNA Extraction
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Total RNA derived from Tilapia liver tissues were extracted using TRIzol0020 reagent (Transgen, China) according to the manufacturer's protocol. The quality of total RNA was measured spectrophotometrically (NanoDrop 2000, Thermo Scientific) and by electrophoresis on 1% agarose gel. First-strand cDNA from RNA of the best quality (absorbance 260/280 > 1.8 and 260/230 > 1.8) was synthesised using One-Step gDNA removal and cDNA Synthesis SuperMix kit (Transgen, China).
8
Watermelon ClHSP70 Gene Expression Analysis
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Total RNA was extracted from samples by Total RNA kit (TIANGEN, Beijing, China), and reverse-transcribed by One-Step gDNA Removal and cDNA Synthesis Super Mix kit (TransGen, Beijing, China) according to the manufacturer’s instructions. The final cDNA was used as the template for subsequent quantitative real-time PCR (qRT-PCR).
Specific primers for detection of ClHSP70 gene expression (Supplementary Table S3 ) were designed using Primer 5 software. The yellow-leaf-specific proein8 (ClYLS8) and β-actin (ClACT) genes from watermelon were used as the reference genes (Kong et al., 2014 (link)). qRT-PCR was conducted using ChamQ universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) with a 20 μL system on the qTOWER³ Series-Real-Time Thermal Cyclers (Analytik Jena, Thüringen, Germany). The experimental procedure of qRT-PCR carried out in accordance with the instruction manual of SYBR qPCR Master Mix, including an initial activation of 95°C for 30 s, then 40 cycles of 95°C for 10 s, 60°C for 30 s. The 2−△△CT method was used to calculate the relative transcript levels of ClHSP70s (Schmittgen and Livek, 2008 (link)).
Specific primers for detection of ClHSP70 gene expression (
9
Validation of RNA-seq by qRT-PCR
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Validation of RNA-seq results was performed for 15 genes selected from DEGs that are involved in photosynthesis and other pathways using quantitative RT-PCR (qRT-PCR). The sequences of the gene primers are provided in Supplementary data S3 . Total RNA was extracted as mentioned above for RNA-Seq and first-strand cDNA was synthesized using a TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit utilizing oligodT primers (TransGen Biotech, Beijing, China). qRT-PCR was carried out in the ABI Step One Plus Real-Time PCR System (Applied Biosystems, USA) utilizing the SYBR Premix Ex Taq RT-PCR kit (Takara Bio, Japan). qRT-PCR programs were as follows: (i) 95 °C for 30 s, (ii) 40 cycles of denaturation at 95 °C for 5 s, annealing at 60 °C for 10s and extension at 72 °C for 10s. Rice ATP synthase subunit beta (OsAtpB, Os10g21266) gene was employed as negative controls in this study. The two rice reference gene Osactin1 (Os03g50885) and Osubiquitin (Os03g03920) were used as internal control genes for expression normalization.
10
RNA Extraction and RT-qPCR Analysis
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Total RNA was isolated from three biological replicates of each plant sample using the plant RNA extraction kit (Biofit, Chengdu, China) following the manufacturer’s protocol. Genomic DNA was eliminated, and cDNA was synthesized according to the instructions of TransScript (One-Step gDNA Removal and cDNA Synthesis Supermix kit, Transgen Biotech, Beijing, China). RT-qPCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazyme, Nanjing, China) kit to prepare the reaction mixture, which was analyzed with the ABI-QuantStudio6 Flex real-time fluorescence quantitative PCR instrument. The PgActin gene served as an internal reference for normalizing variations in gene expression. The relative expression levels of the target genes were quantified using the ΔΔCt method as described by Livak and Schmittgen [51 (link)]. Each treatment was subjected to three biological replicates. The primers used for RT-qPCR are listed in Table S2 .
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