Total RNA was extracted by TransZol Plant (Transgen) and it was reverse-transcribed with PrimerScriptTM RT regent Kit with gDNA Eraser (Takara) according to the manufacturer’s instruction. A 5 μl aliquot of 1:50 diluted cDNA was used as the template in a 10 μl PCR system. The qRT-PCR was performed using TransStar Green qPCR Supermix (Transgen) after a pre-incubation at 95°C for 30s, followed by 40 cycles of denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s in an Applied Biosystems 7500 Fast Real-Time PCR Systems. The gene-specific primers of qPCR were listed in S6 Table and wheat actin was used as an internal standard to normalize the gene expression. The quantification of gene expression was determined in three independent samples.
Transzol plant
Manufactured by Transgene
Sourced in China
TransZol Plant is a laboratory reagent used for the extraction and purification of total RNA from plant tissues. It is a monophasic solution containing guanidine thiocyanate and phenol that efficiently lyses plant cells and deactivates RNase enzymes to preserve RNA integrity during the extraction process. The product enables researchers to obtain high-quality, intact RNA samples from a variety of plant sources for downstream applications such as gene expression analysis and RT-PCR.
Automatically generated - may contain errors
Lab products found in correlation
Cfx96 real time system, by Bio-Rad (2 mentions)
Reverse transcription kit, by Vazyme (2 mentions)
Primerscript tm rt regent kit with gdna eraser, by Takara Bio (1 mentions)
Transstar green qpcr supermix, by Transgene (1 mentions)
7500 fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Iq5 system, by Bio-Rad (1 mentions)
Sybr green 1 kit, by Vazyme (1 mentions)
Hiscript q rt supermix for qpcr gdna wiper, by Vazyme (1 mentions)
Transtaq hifi pcr super mix, by Transgene (1 mentions)
All in one first strand cdna synthesis supermix for pcr, by Transgene (1 mentions)
Sybr green mix, by Vazyme (1 mentions)
Perfectstart green qpcr supermix dye 2, by Transgene (1 mentions)
Hiscript 2 1st strand cdna synthesis kit gdna wiper, by Vazyme (1 mentions)
Sybr green pcr master mix, by Vazyme (1 mentions)
M mlv reverse transcriptase kit, by Promega (1 mentions)
Quantstudio 5 real time system, by Thermo Fisher Scientific (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Iq5 instrument, by Bio-Rad (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Taq pro universal sybr qpcr master mix, by Vazyme (1 mentions)
11 protocols using transzol plant
1
Quantitative real-time PCR analysis of gene expression
2
Quantitative PCR Analysis of Gene Expression
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Total RNA was extracted from immature endosperms, embryos and other tissues using TransZol Plant (Transgen). Five hundred nanogram of total RNA was used for first‐strand cDNA synthesis using HiScript® QRT SuperMix for qPCR (+gDNA wiper) (Vazyme). All qPCR analyses were carried out in a Bio‐Rad iQ5 system (Bio‐Rad iQ5 Real Time PCR, ABI 7500) using the SYBR Green I kit (Vazyme). The 2−ΔΔCT method was used to calculate the relative transcript level of the target gene with ZmActin (Zm00001d010159) as the endogenous control. The PCR program was conducted as follows: (1) 5 min at 94 °C; (2) 40 cycles of 10 s at 95 °C, 30 s at 58 °C. The 20‐μL reaction volumes contained 2 μL cDNA, 0.4 μL L/R primers (10 μm ), 10 μL 2 × qPCR SYBR Green I mix and 7.2 μL double‐distilled water. Statistically significant differences in gene expression levels were analysed by Student's t‐test. All primers used for qPCR are listed in Table S3 .
3
Transcriptional Analysis of Tobacco Stress Response
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Healthy tobacco leaves and tobacco samples treated with the pGR-107 effector recombinant vector, Bax, and pGR-107 empty vector for 5 days were collected and ground after flash freezing in liquid nitrogen. Total RNA of tobacco was extracted with TransZol Plant (TransGen, Beijing, China), and cDNA was obtained by reverse transcription with All-in-One First-Strand cDNA Synthesis SuperMix for PCR (TransGen Biotech, Beijing, China). The tobacco Actin gene was used as an internal reference gene, and 2×TransTaq HiFi PCR SuperMix (TransGen Biotech, Beijing, China) was used for PCR amplification to detect the expression of effectors in tobacco.
4
Transcriptome Analysis of Wheat Samples
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Total RNA was extracted from samples using TransZol Plant (TransGen Biotech, ET121-01), and first-strand cDNAs (20 μL) were synthesized from 1 μg starting total RNA using a reverse transcription kit (Vazyme Biotech, R223-01) according to the manufacturer’s instructions. The first-strand cDNAs was diluted with H2O to 80 μL. For RT-qPCR, the reaction mixture was composed of the 1 μL cDNA template, 0.2 mΜ primers, and 5 μL SYBR Green Mix (Vazyme Biotech, Q121-02/03) in a final volume of 10 μL. Amplification was performed using a CFX96 real-time system (BioRad). Differences in relative transcript levels were calculated using the 2−ΔCT method (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/cms_040980.pdf ) relative to wheat TaACTIN (TraesCS5B02G124100). RT-qPCR was performed as technical triplicates per sample. Three biological replicates were performed, with similar results; the results from one replicate are shown in the figures.
5
Quantitative Real-Time PCR Analysis of Transgenic Tobacco
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The total RNA was extracted from the plants using TransZol Plant (Transgen, Beijing, China) according to the manufacturer’s protocol. First−strand cDNA was reverse transcribed using the HiScript®® II 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, Nanjing, China). Quantitative real−time PCR (qRT−PCR) was performed using the PerfectStart®® Green qPCR SuperMix(+Dye II) (Transgen, Beijing, China) in a Light Cycler 480 II fluorescent PCR instrument (Roche Diagnostics Corporation, Indianapolis, Indiana, USA). The qRT−PCR protocol was as follows: 95 °C for 5 min and 40 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, followed by a final extension at 72 °C for 7 min. Using the GAPDH gene as an internal control, the relative expression levels in the transgenic tobacco lines and non−transgenic tobacco were detected. The 2−∆∆Ct method was used for quantification [30 (link)], and the variation in expression was analyzed based on three biological replicates. The primers used for the qRT−PCR are shown in Table S2 .
6
Temporal Dynamics of Seed Transcripts
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Total RNAs was extracted from seeds at the 1‐ to 6‐DAP, 8‐DAP, 10‐DAP, and from seeds without embryo of 15‐DAP, 20‐DAP and 25‐DAP using the TransZol Plant (TransGen Biotech, ET121‐01). First‐strand cDNAs were generated using a reverse transcription kit (Vazyme Biotech, R223‐01) according to the manufacturer's instructions. RT‐qPCR assays were performed using the SYBR Green PCR Master Mix (Vazyme Biotech, Q121‐02/03) with a CFX96 real‐time system (BioRad). The wheat TaACTIN (TraesCS5B02G124100) was used for standardization. The comparative CT (2−ΔCT) method (Schmittgen and Livak, 2008 (link)) was used to quantify the different mRNA levels. Three biological replicates and three technical replicates for each biological replicate were performed per gene. The results from one replicate are shown in the figures. Statistical test was performed by applying Student's t‐test at P < 0.05. The RT‐qPCR primers used in this study are listed in Data Set S3 .
7
Quantitative RNA Expression Analysis of Sweet Potato
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Total RNA was isolated from sweet potato leaves in September using TransZol Plant (Transgen Biotech Inc., Beijing, China) following the manufacturer’s instructions. First-strand cDNA were reverse transcribed using a Reverse Transcriptase M-MLV Kit (Promega, Madison, WI, USA). The qRT-PCR assay was performed on an ABI QuantStudio5 Real-time system (ABI, Foster City, CA, USA), and in 10 μL of reaction containing 2× TransStart Top Green qPCR SuperMix (Transgen Biotech Inc., Beijing, China), 10 μM of solution from each primer (Supplementary Table S1 ), and 100 ng of cDNA. Thermocycling conditions were: initial denaturation at 95 °C for 2 min, followed by 40 cycles for 15 s at 95 °C, and 1 min at 60 °C. The relative expression of anthocyanin biosynthetic genes was assessed by the comparative threshold cycle (Ct) method [20 (link)]. The sweet potato actin gene (NCBI accession: EU250003) served as an internal control for signal normalization. Expression levels were evaluated as technical duplicates of biological triplicates from separate plant samples.
8
Quantitative Analysis of miR167 Expression
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Total RNA was isolated from various plant organs or grains using TRIzol reagent (Invitrogen) or TransZol Plant (Transgen, ET121-01) according to the manufacturer’s instructions. First-strand cDNA was synthesized from 1 μg total RNA using the Quantscript RT Kit (Tiangen, KR103). Stem–loop qPCR was used to detect the expression of miR167. For qRT–PCR, Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Q712-02) was added to the reaction system, and the reaction was run on a Bio-Rad iQ5 instrument (BioRad, Hercules, CA, USA); the β-actin gene was used as an internal control. Relative expression level was calculated by the 2−ΔΔCt method. The primers used are listed in Supplemental Table 5 . The PCR procedure was as follows: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 15 s, and extension at 72°C for 30 s.
9
Quantitative Analysis of Plant Transcripts
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Total RNA was isolated from nodules using the TransZol Plant (TransGen Biotech) with three biological replicates. For quantitative real-time PCR (qRT-PCR), complementary DNA was constructed using 1 lg total RNA as input materials following the instructions of the HiScript II Q RT SuperMix kit (Vazyme, Nanjing, China). qRT-PCR was performed using the TransStart Tip Green qPCR SuperMix (TransGen Biotech) on a Real-Time PCR Detection System (CFX384; Bio-Rad). Ubiquitin (Ubq1, Lj5g3v2060710) and ATPase (Lj5g3v2169420) were used as reference genes. Primers used for qRT-PCR are provided in Table S1.
10
Mapping Defective Kernel Locus via BSR-seq
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The cns1-ref allele was crossed into B73, and selfed F 2 populations were used for BSR-seq. WT and mutant kernels were collected from the same ear on 12 DAP. The pericarps were removed and 60 kernels of either WT or mutant phenotype were pooled for BSR analysis. Total RNA was extracted from each pool using TransZol Plant (TransGen, Beijing, China) and subjected to sequencing by Anoroad Gene Technology (Beijing, China). The data were analyzed by Allwegene Technologies (Beijing, China). In brief, whole chromosomes were scanned using the mean value of SNP-index derived from a 1-Mb window and scanning was performed with a step size of 1 kb. For digestion-ligation-amplification analysis, genomic DNA was isolated from the leaves of WT and heterozygous plants, obtained through self-pollination to identify their phenotypes. Mu-tagged sequences were obtained using the Genome Walker Kit (Clontech, Mountain View, CA, USA), and the Muflanking sequences linked with defective kernel phenotype were sequenced and analyzed.
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