Another independent experiment was conducted in which the freshly detached shoot sample of 15-day-old Se-2-treated B. rapa plants was immediately stored in liquid nitrogen. For the extraction of total RNA, the TransZol Up reagent (TransGen Biotech, Beijing, China) was used. RNA quality and quantity were analyzed using the Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For complementary DNA (cDNA) production, 2 µg RNA was transferred into the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit according to the manufacturer’s instructions (TransGen Biotech, Beijing, China). Subsequently, a 1:10 dilution of cDNA was used for quantitative real-time PCR analysis with the TransStart Tip Green qPCR SuperMix (TransGen Biotech, Beijing, China) attached to a Roche LightCycler 480 thermal cycler instrument (384-well; Roche, Basel, Switzerland). The primers listed below were used to assess the expression levels of the APX, SOD, POD, and CAT genes (Zhang et al., 2020 (link)).
Transscript one step gdna removal and cdna synthesis supermix kit
Manufactured by Transgene
Sourced in China
The TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit is a laboratory product designed to remove genomic DNA and synthesize cDNA in a single-step reaction.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (42 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (21 mentions)
Transstart tip green qpcr supermix, by Transgene (12 mentions)
Trizol, by Thermo Fisher Scientific (10 mentions)
Transstart top green qpcr supermix kit, by Transgene (10 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (10 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (9 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (7 mentions)
Transstart top green qpcr supermix, by Transgene (6 mentions)
Lightcycler 480 2, by Roche (5 mentions)
Ultrasybr mixture, by CWBIO (5 mentions)
Smart race cdna amplification kit, by Takara Bio (5 mentions)
Transzol up reagent, by Transgene (4 mentions)
Rneasy mini kit, by Qiagen (4 mentions)
Sybr green pcr supermix, by Bio-Rad (4 mentions)
Rneasy plant mini kit, by Qiagen (4 mentions)
Transzol up plus rna kit, by Transgene (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (4 mentions)
Cfx96 real time system, by Bio-Rad (4 mentions)
166 protocols using transscript one step gdna removal and cdna synthesis supermix kit
1
RNA extraction and qRT-PCR analysis
2
Quantitative Transcriptomic Analysis of Plant Tissues
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RNA samples were isolated from the roots and flowers tissues. Reverse transcription was performed using the TransScript® One-Step gDNA Removal and cDNA Synthesis Super Mix kit (TransGene, Beijing, China) following manufacturer’s instructions. The reaction was carried out at 42 °C for 15 min and 80 °C for 5 s. UltraSYBR Mixture (CWBIO, Beijing, China) and LightCycler480 II (Roche, Switzerland) Real-Time PCR System were used to conduct real-time quantification. The reaction mixture (20 μL) contained 10 μL of 2 × UltraSYBR Mixture, 0.5 μL of each forward and reverse primers, and 1 μL (150 ng/ul) of template cDNA. The PCR amplification procedure was as follow: 95 °C for 10 min and 40 cycles of 95 °C for 15 s, 60 °C for 1 min. The gene-specific primers were designed using Primer 5.0 software and were listed in Additional file 3 : Table S3. The GAPDH gene was used as an internal standard, Ct values were determined based on three biological replicates of each sample and calculated using the 2–△△Ct relative quantitative method [38 (link)].
3
Quantitative Analysis of mRNA and miRNA
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The total RNA of tissues and cells was extracted with TRIzol reagent (ComWin Biotech, China). Reverse transcription of mRNA was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, China) based on the manufacturer’s protocols. miRNAs were reverse transcribed with a specific stem-loop RT-PCR. Then, qRT-PCR was conducted with SYBR Green (TransGen Biotech, China) on an Applied Biosystems 7500 Sequence Detection System. Peptidylprolyl isomerase A (PPIA) was used to normalize the relative mRNA expression, and small nucleolar RNA U6 was used to normalize the relative miRNA expression. The relative levels of mRNA and miRNA were calculated using the power formula: 2−ΔCt (ΔCt = Cttarget gene–Ctnormalizer). The primer sequences are listed in Additional file 1 : Table S1.
4
Quantifying miRNA Expression by RT-qPCR
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Total RNA was extracted from samples and cells by applying TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.). The TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit was purchased from Beijing Transgen Biotech Co., Ltd. (Beijing, China). Then total RNA was converted into cDNA through RT according to the manufacturer's protocol. Following RT, qPCR was performed using the TransScript® Tip Green qPCR SuperMix kit according to the manufacturer's protocol (Transgen Biotech Co., Ltd.) with an ABI 7900HT Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.). The following forward and reverse primers were used for RT-qPCR: miR-20a, forward 5′-TAAAGTGCTTATAGTGCAG-3′ and reverse 5′-TGCGTGTCGTGGAGTC-3′ (LNA); U6, forward 5′-CTCGCTTCGGCAGCACATATACT-3′ and reverse 5′-ACGCTTCACGAATTTGCGTGTC-3′. The PCR reaction mixtures contained 2× TransScript® Tip Green qPCR SuperMix (10 µl), 4 µM primers (2 µl), 1 µl cDNA and 7 µl ddH2O in a total volume of 20 µl. The PCR thermocycling conditions were as follows: 94°C for 30 sec, and 40 cycles of 5 sec at 94°C and 30 sec at 60°C. The comparative 2−ΔΔCq method (26 (link)) was used for relative quantification and statistical analysis.
5
RT-PCR Analysis of Smooth Muscle Genes
6
Transcriptional Analysis of Pneumococcal Mutants
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When wild-type S. pneumoniae, 1609- mutant and 1609 complement strains were grown to logarithmic growth phase (OD600 = 0.3), total RNA from each strain was extracted with TRIzol reagent (Invitrogen, United States) following the manufacturer’s protocol. The purity and concentration of the isolated RNA were determined using a NanoDrop 2000 UV-VIS Spectrophotometer (Thermo Scientific, United States). cDNA was generated from 1 μg RNA without DNA contamination using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (TransGen Biotech, China) according to the kit’s specifications. RT-qPCR was carried out using EvaGreen Dye (Bio-Rad, United States) in a Miniopticon Real-Time PCR System (Bio-Rad, United States). The cycle threshold (Ct) value was recorded, and the relative quantification of specific gene expression was calculated using the 2–ΔΔCt method (Livak and Schmittgen, 2001 (link)), with gyrB as an internal control. The results are shown as the 1609- mutant or 1609 complement against the wild-type (WT) strain. The primer sequences are shown in Supplementary Table S1 . All data were evaluated with three independent biological experiments.
7
Total RNA Extraction and cDNA Synthesis
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Total RNA was extracted from the collected samples using the Trizol Reagent (Invitrogen, Shanghai, China) and the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. The RNA purity was measured with a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Rockford, USA) and the integrity was checked by agarose gel electrophoresis. One microgram total RNA was reverse-transcribed to cDNA by using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China).
8
Quantitative Analysis of Fatty Acid Metabolism
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The expression levels of the genes related to fatty acid biosynthesis and degradation metabolism were detected using quantitative real-time PCR (qRT-PCR) following a method described previously [53 (link)]. In brief, the bacterial cells were harvested and the total RNA of each sample was isolated with a Biospin Total RNA Extraction Kit (Dobiothec, Fuzhou, China). An amount of 1 mg of total RNA was used for reverse transcription with the TransScript One-Step gDNA Removal and cDNA Synthesis Supermix Kit (TransGen Biotech, Beijing, China). qRT-PCR was performed in 96-well plates with PerfectStart Green qPCR SuperMix (TransGen Biotech, Beijing, China). The primers for each gene were listed in Table S1 . Each primer pair was specific, and the relative expression of each gene was determined by the comparative threshold cycle method (2-ΔΔCT method).
9
RNA Extraction and RT-PCR Analysis
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Total RNA was prepared from leaves using a Quick RNA Isolation kit (HUAYUEYANG, Beijing). For RT‐PCR, the first‐strand cDNA was synthesized from 1.5 mg total RNA using the TransScript One‐Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, China). Semi‐quantitative PCR was performed for gene expression analysis using gene‐specific (DMp008Os‐F and DMp008Os‐R) and rice ACTIN (OsrActin‐F and OsrActin‐R) primers. Real‐time PCR was performed on an optical 96‐well plate in a BIO‐RAD CFX96 Real‐Time system using TransStart Tip Green qPCR SuperMix (TransGen, China). Actin was used as an endogenous control. Primers used in this study were listed in Table S9 .
10
RNA Extraction and RT-qPCR Analysis
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Total RNA was extracted using the EZNA Plant RNA Kit (R6827, OMEGA, America), and cDNA was synthesized for quantitative real-time PCR (RT‒qPCR) by the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Kit (AT311, TransGen Biotech, Beijing, China) according to the manufacturer’s method. RT‒qPCR was carried out using SYBR® Green real-time PCR Master Mix (QPK-201, TOYOBO, OSAKA, Japan) in a CFX96 TouchTM real-time PCR detection system (Bio-Rad, Hercules, CA, USA). The housekeeping genes PdbEF and Pdbactin were selected as internal controls, and the primers used for RT‒qPCR analysis are listed in Supplementary Table S1 . Relative gene expression levels were measured using the 2−ΔΔCt method (Livaka and Schmittgen, 2001 (link)). Genes with changes in the expression level greater than 2-fold were considered to be significantly regulated.
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