Transcript analysis of HlGST and HlGST2 genes was performed through real-time PCR in a Mx3005P qPCR system (Agilent Technologies, Santa Clara, USA) using TransStart® Top Green qPCR SuperMix (TransGen Biotech) according to the manufacturer's directions. The qPCR assays were carried out in 96-well polypropylene plates (Axygen, San Jose, USA) in a 20 μL reaction volume containing 1 μL of cDNA template, 10 μL of 2× TransStart® Top Green qPCR SuperMix, 0.4 μL of each 10 μM primer, 0.4 μL of Passive Reference Dye (TransGen Biotech) and 7.8 μL of H 2 O. The thermal cycling program used is as follows: 94 ºC for 30 s, then 40 cycles of 94 ºC for 5 s and 60 ºC for 30 s. Veri cation of primers with high ampli cation speci city were done by the observation of different peaks in corresponding melting curves. Triplicate reaction for each DNA sample were contained in each plate. After each assay, melting curves were traced to con rm that the uorescence signal was retrieved from speci c PCR products and also ensure that there are no primer dimers. Ct represents the relative gene expression levels for each gene in each sample and it is transformed into relative values or it is relatively quanti ed (RQ) by the 2 -ΔΔCT method, where ΔΔC T = (C T, Target -C T, Actin ) sample -(C T, Target -C T, Actin ) control [25] (link).
Transstart top green qpcr supermix
Manufactured by Transgene
Sourced in China, United States, Germany, Switzerland, Finland, Canada
The TransStart Top Green qPCR SuperMix is a ready-to-use master mix for quantitative PCR (qPCR) applications. It contains all the necessary components for efficient and sensitive qPCR, including a DNA polymerase, buffers, dNTPs, and a green fluorescent dye for real-time detection.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (82 mentions)
Transscript one step gdna removal and cdna synthesis supermix, by Transgene (34 mentions)
Trizol, by Thermo Fisher Scientific (22 mentions)
Revertaid first strand cdna synthesis kit, by Thermo Fisher Scientific (18 mentions)
Primescript rt reagent kit, by Takara Bio (17 mentions)
Easyscript one step gdna removal and cdna synthesis supermix, by Transgene (16 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (15 mentions)
Rnaiso plus, by Takara Bio (15 mentions)
Transscript first strand cdna synthesis supermix, by Transgene (15 mentions)
Primescript rt reagent kit with gdna eraser, by Takara Bio (14 mentions)
Rnaprep pure plant kit, by Tiangen Biotech (12 mentions)
Rneasy mini kit, by Qiagen (12 mentions)
Transscript all in one first strand cdna synthesis supermix, by Transgene (11 mentions)
Easyscript first strand cdna synthesis supermix, by Transgene (11 mentions)
Trizol reagent, by Takara Bio (10 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (10 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (9 mentions)
Lightcycler 480 system, by Roche (9 mentions)
Cfx96 real time system, by Bio-Rad (8 mentions)
Abi 7500 real time pcr system, by Thermo Fisher Scientific (8 mentions)
446 protocols using transstart top green qpcr supermix
1
Real-time PCR analysis of HlGST genes
2
Quantitative RT-PCR Analysis of Arabidopsis Genes
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Total RNA of various Arabidopsis tissues were extracted using Trizol reagent (Sigma) and then reverse-transcribed into cDNA with a Reverse Transcription System (TOYOBO). The cDNAs of rosette leaves of the cpnb1-4 homozygous mutants were used as the templates for PCR analysis with the gene-specific primers. qRT-PCR of CPNA2 was performed using TransStart Top Green qPCR SuperMix (TransGen, China) with a Rotor-Gene 6000 machine (Corbett Research) and the relative expression levels normalized to GAPDH were analyzed by the double standard curves method as described previously [56 (link)]. qRT-PCR of KASI was performed using TransStart Top Green qPCR SuperMix (TransGen, China) with a Bio-rad CFX Connect machine (BIO-RAD) and the relative expression levels normalized to GAPDH were analyzed by the comparative CT method as described previously [57 (link),58 (link)]. Three biological and three technical replicates of each sample were made for qRT-PCR analysis. Primers used in the experiments were listed in S4 Table .
3
Quantitative Gene Expression Analysis via qPCR
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Gene amplification was performed by qPCR in a Mx3005P qPCR system (Agilent Technologies, Santa Clara, USA) using TransStart® Top Green qPCR SuperMix (TransGen Biotech) following the manufacturer’s instructions. The qPCR assays were conducted in 96-well polypropylene plates (Axygen, San Jose, USA) in a 20 μl reaction volume comprising 1 μl of cDNA template, 0.4 μl of each 10 μM primer (Table 1 ), 10 μl of 2× TransStart® Top Green qPCR SuperMix, 0.4 μl of Passive Reference Dye (TransGen Biotech) and 7.8 μl of H2O. The thermal cycling program was as follows: 94 °C for 30 s, followed by 40 cycles of 94 °C for 5 s and 60 °C for 30 s. The primers with high amplification specificity were verified by different peaks observed in corresponding melting curves. Each plate contained triplicate reactions for each DNA sample. Melting curves were also traced after each assay to confirm that the fluorescence signal was retrieved from specific PCR products and to ensure the absence of primer dimers. The relative gene expression levels for each gene in each sample were calculated as Ct and transformed into relative values (RQ) by the 2−ΔΔCT method, where ΔΔCT = (CT, Target − CT, Actin) sample − (CT, Target − CT, Actin) control [30 (link)].
4
Quantifying Fungal Stress Response with qPCR
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Zea mays leaves injected with purified EG1 expressed by P. pastoris GS115 were harvested at different time points and ground in liquid nitrogen for RNA extraction with TRIzol. The first strand of cDNA was synthesized from 1 μg total RNA using TransScript All-in-One First Strand cDNA Synthesis SuperMix (Transgen, China). qRT-PCR was assayed by TransStart Top Green qPCR SuperMix (Transgen, China). Each PCR tube contained 10 μL of Top Green qPCR SuperMix, 25 ng of cDNA, and 0.2 μM of each primer. The thermal cycling conditions were 30 s at 94°C, followed by 40 cycles of 5 s at 94°C and 15 s at 55°C by Roche LightCycler 96.
Rhizoctonia solani was cultured in PDA with different GABA concentrations for 3 days and was collected in liquid nitrogen for RNA extraction and qRT-PCR as described above.
For biomass measurement, total DNA was extracted by the Plant Genomic DNA Kit (TIANGEN, China), and qPCR was assayed by the TransStart Top Green qPCR SuperMix (Transgen, China).
Rhizoctonia solani was cultured in PDA with different GABA concentrations for 3 days and was collected in liquid nitrogen for RNA extraction and qRT-PCR as described above.
For biomass measurement, total DNA was extracted by the Plant Genomic DNA Kit (TIANGEN, China), and qPCR was assayed by the TransStart Top Green qPCR SuperMix (Transgen, China).
5
Quantitative Analysis of Drug Resistance Genes
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Total RNA was isolated with trizol reagent (Ambition, life technologies, USA) from Pumc-91/ADM and T24/DDP cells which were treated with TMP (0, 2 mM and 4 mM) according to manufacturer’s instructions. cDNA was generated using Oligo d (T) (Dingguo Biotech), dNTP (Genview) under the action of M-MLV reverse transcriptase (Promega). Quantitative real-time PCR was done by using TransStart Top Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd) with LightCycler 480 Real Time PCR System. The PCR primer sequences used were as follows: GAPDH forward: 5’-TTTGGTATCGTGGAAGGACT-3’ and reverse: 5’-AGTAGAGGCAGGGATGATGT-3’ ; MRP1 forward: 5’-TTGCCGTCTACGTGACCATT-3’ and reverse: 5’-AGGCGTTTGAGGGAGACACT-3’ ; LRP forward: 5’-TATGTGCCATCTGCCAAA GT-3’ and reverse: 5’-CATGTAGGTGCTTCCAATCA-3’ ; GST forward: 5’-TTCCTGTGGCATAATGTGAT-3’ and reverse: 5’-CTGATTCAA AGGCAAATCTC-3’ ; TOPO-II forward: 5’-AGGCATCGCATCTTGTTTAG-3’ and reverse: 5’ -CTGTCTCCGGTCTTCCATAA-3’ ; BCL-2 forward: 5’-GACAACATCGCCCTGTGGAT-3’ and reverse: 5’- AGGGCCAAACTGAGCAGAGT-3’ . Quantitative real-time PCR were done by using TransStart Top Green qPCR SuperMix (Beijing TransGen Biotech Co., Ltd) with LightCycler 480 Real Time PCR System. GAPDH was used as endogenous reference. Relative gene expression was calculated using the 2-ΔΔCt method [25 (link)]. All of the experiments were repeated three times.
6
Quantifying Developmental Stage-Specific RNA Expression
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Total RNA was extracted from eggs in different developmental stages (80 mg each) using an AxyPrep™ Multisource Total RNA Miniprep Kit (Axygen). Then, total RNA from each sample was reverse transcribed to cDNA using TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix (TransGen Biotech) following the manufacturer’s protocol. qPCR was performed in an Mx3005P system (Agilent Technologies) using TransStart® Top Green qPCR SuperMix (TransGen Biotech) following the manufacturer’s instructions. The reaction system was as follows: 10 μl of 2× TransStart® Top Green qPCR SuperMix, 7.8 μl of H2O, 1 μl of cDNA template, 0.4 μl of Passive Reference Dye (TransGen Biotech) and 0.4 μl of each primer at 10 μM (Table 1 ). The parameters of the machine were set as described previously [34 (link)]. The results were normalized to β-actin and analyses of gene expression were performed using the 2−ΔΔCq method [36 (link)].
7
Quantitative RT-PCR Workflow for Gene Expression
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Highly purified RNA was extracted using the CTAB method [30] (link). The TransScript ® First-Strand cDNA Synthesis SuperMix (TransGen) was used to obtain the cDNA, with the following reaction system: 500 ng of total RNA, 1 µL of anchored oligo(dT) 18 , 10 µL of 2 × ES reaction mix, 1 µL of EasyScript ® RT/RI enzyme mix and appropriate ddH 2 O to make the total volume to 20 µL. The cDNA was diluted 10-fold (cDNA:nuclease free water = 1:10) for further qRT-PCR analysis. Gene-specific primers were designed using Primer Premier 6.25, and NCBI primer-BLAST was used to check their specificity. TransStart ® Top Green qPCR Super Mix (TransGen Biotech, Beijing, China) was employed to carry out the qRT-PCR, with the following reaction system: 10 µL of 2 × TransStart ® Top Green qPCR Super Mix, 7 µL of double-distilled H 2 O, 1 µL of diluted template, and 2 µL of forward primer and reverse primer. qTOWER 3G Cycler and qPCR software (Analytik Jena, Germany) were used as a work program and the 2 -∆∆CT method [31] (link) was used to perform the relative gene expression level analysis. The PtrActin was used as the internal reference gene. Triplicate independent technical and biological replications were used in the analysis. The primers for qRT-PCR are listed in Table S1.
8
Quantitative RT-PCR Analysis of Gene Expression
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We amplified the PCR products for qRT-PCR analysis in triplicate using 2×TransStart™ TOP Green qPCR SuperMix (TransGen Biotech Inc., Beijing, China) in 10μl qRT-PCR assays. PCR was performed using the QuantStudio 7 Flex Real-time PCR System (Applied Biosystems, Foster City, CA, United States). The cycling conditions consisted of denaturation at 95°C for 30s, followed by 40cycles of denaturation at 95°C for 5s, annealing at 58°C for 15s, and extension at 72°C for 10s. The reference genes, CmCAC and NbACTIN, were used as the internal controls (Obrero et al., 2011 (link); Nie et al., 2020 (link)). The gene-specific primers for CmRCC1 and the NbPIN gene family are listed in Supplementary Table S1 . Relative gene expression was determined as previously described by Livak and Schmittgen (2001) (link).
9
Quantitative gene expression analysis
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The transcript levels of the identified genes were analyzed by qRT-PCR using the above-synthesized cDNAs as templates, 2×TransStart® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China), and gene-specific primers (Supplementary Materials Table S2 ) in a 20 μL reaction mix. The reaction was run on a Step-Two Plus real-time PCR system (Applied Biosystems, San Francisco, CA, USA). PbACTIN was used as the reference gene. At least three replicates were included in each experiment, and experiments were repeated three times.
10
Quantifying Gene Expression by qRT-PCR
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First-strand cDNA was synthesized from 1.0 μg of RNA using oligo (dT) primers by using a PrimeScript® RT Reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) in accordance with the manufacturer's protocol. The cDNA was diluted to a final concentration of 300 ng·μL−1 and used as the template for qRT-PCR. qRT-PCR was performed on the LightCyler® 480 real-time detection system (Roche Diagnostics, Switzerland). The intercalation dye SYBR Green (TransStart®) was used as a fluorescent reporter. Translation elongation factor 2 was used as a reference gene to normalize gene expression in according with a previously published report (Sherif et al., 2012 (link)). In brief, 15 μL of the PCR system contained 300 ng of cDNA, 10 mmol of each primer, and 7.5 μL of 2 × TransStart® Top Green qPCR SuperMix (TransGen, Beijing, China). The reaction was performed at 95°C for 30 s, followed by 45 cycles of 95°C for 5 s, 60°C for 30 s, 72°C for 30 s. Relative gene expression was calculated using the comparative 2−ΔΔCT method (Livak and Schmittgen, 2001 (link)). The qRT-PCR results are shown as the means ± SD of three independent biological replicates.
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