Expression levels of E1 and GmFT2a/5a in wild type plants and T2 homozygous mutants were analyzed under LD and SD conditions, respectively. Every 5-day interval after 10 days after emergence (DAE), at 10 am (4 h after light), the trifoliate leaves were sampled from plants with different genotypes under LD and SD conditions. Total RNAs were extracted using TransZol Up Plus RNA Kit (TransGen Biotech). For reverse transcription, the first-strand cDNA synthesis was performed using the TransScript First-strand cDNA Synthesis SuperMix Kit (TransGen, China). For qRT-PCR, gene expressions were examined using cDNA templates on an Applied Biosystems 7300 Real-Time PCR System. The relative gene expression levels followed the method (Pfaffl, 2001 (link)). The mRNA level of GmActin (Glyma18g52780) was used as a reference for normalization. Specific primers we used in this study were list in Supplementary Table 2
Transscript first strand cdna synthesis supermix kit
Manufactured by Transgene
Sourced in China
The TransScript First-Strand cDNA Synthesis SuperMix Kit is a laboratory equipment product that is used for the reverse transcription of RNA into complementary DNA (cDNA). It contains a premixed solution with all the necessary components to perform this process efficiently.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (29 mentions)
Trizol, by Thermo Fisher Scientific (6 mentions)
Opticon 2 real time pcr detection system, by Bio-Rad (5 mentions)
Cfxtm real time thermal cycler, by Bio-Rad (4 mentions)
Transstart top green qpcr suppermix kit, by Transgene (4 mentions)
Trizol reagent, by CoWin Biotech (4 mentions)
Nanodrop 2000c, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Transzol up plus rna kit, by Transgene (3 mentions)
Sybr green, by Roche (3 mentions)
Sybr green pcr master mix, by Roche (3 mentions)
Sybr green qpcr supermix, by Transgene (3 mentions)
7300 real time pcr system, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taq, by Takara Bio (2 mentions)
Easypure plant rna kit, by Transgene (2 mentions)
Cfx manager system, by Bio-Rad (2 mentions)
Dnase 1, by Promega (2 mentions)
Stepone real time pcr system, by Thermo Fisher Scientific (2 mentions)
Transstart top green qpcr supermix kit, by Transgene (2 mentions)
Transstart green qpcr supermix, by Transgene (2 mentions)
67 protocols using transscript first strand cdna synthesis supermix kit
1
Expression Profiling of E1 and GmFT2a/5a
2
Circadian Clock Gene Expression
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Total RNA was extracted from individual SCN tissues using Trizol reagent (Invitrogen) according to manufacturer’s instruction. RNA concentration and purity were determined using the NanoDrop 2000 (Thermo Fisher, USA), and RNA integrity was confirmed by agarose gel electrophoresis. cDNA synthesis was performed using the TransScript First-Strand cDNA Synthesis Super Mix kit (TransGen Biotech Co., China) and 20 ng of total RNA. qPCR reactions were prepared with SYBR Premix Ex Taq (TaKaRa) and run on the CFX96 Real-Time qPCR System (Bio-Rad, USA). Primer sequences were as follows: Clock forward, 5′-TTAGATCACAGGGCACCACC-3′; Clock reverse, 5′-GTGCTCGTGACATTTTGCCA-3′; Bmal1 forward, 5′-GGCCTTCATTGCACCTTCCTT-3′; Bmal1 reverse, 5′-GAACCGGAGAGTAGGTCGGT-3′; Per1 forward, 5′-TCGAAACCAGGACACCTTCTCT-3′; Per1 reverse, 5′-GGGCACCCCGAAACACA-3′; Per2 forward, 5′-CATTGAACTTGAGACTGAGGT-3′; Per2 forward, 5′-AAGGGAACACACTGAGAGGAT-3′; GAPDH forward, 5′-CAAGGTCATCCATGACAACTTTG-3′; GAPDH reverse, 5′-GTCCACCACCCTGTTGCTGTAG-3′.
3
Quantifying ZFP580 Expression in H9c2 Cells
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Total RNA from H9c2 cells treated with 600 µM CoCl2 at various time intervals (0, 4, 8, 12, 16, 20 or 24 h) was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. First-strand cDNA samples were synthesized using a TransScript First-Strand cDNA Synthesis Supermix kit according to the manufacturer's protocol (Beijing Transgen Biotech, Co., Ltd., Beijing, China). GAPDH RNA levels were quantified in all of the samples as an internal control, and mRNA levels were calculated relative to GAPDH mRNA. qPCR was performed in a 25 µl volume with SYBR® Green PCR Master Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.). Each gene analysis was repeated at least 3 times, and all RT-qPCR experiments were performed in triplicate using the ABI 7500 Real-Time PCR platform (Applied Biosystems; Thermo Fisher Scientific, Inc.) The specific primers used were as follows: Forward, 5′-ACATCATTTCGTCTTTTCTTCTG-3′ and reverse, 5′-GGTGCTTTTGTCATTTCTTCCAC-3′ for ZFP580. The PCR conditions for ZFP580 were 15 sec at 95°C, 34 sec at 63°C and 45 sec at 72°C for 40 cycles. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a housekeeping gene, served as an internal control. The fold-change in expression of the gene of interest between the two samples was calculated using the ΔΔCq method (19 (link)).
4
Validating Fungal Gene Expression through qPCR
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Real‐time quantitative PCR was used to verify the gene expression level calculated from transcriptomic data. DEGs that may regulate fungal growth and aflatoxin production were verified and selected for further investigation. Crude RNA was used to synthesize cDNA using a transScript® first‐strand cDNA synthesis superMix kit (Transgen), where the 20 μL reaction system consisted of 10 μl SYBR® Fast qPCR Mix (2x), 0.5 μl of each primer (10 μmol/l) and 1 μl cDNA. The real‐time quantitative PCR program was set to the following sequence: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and finally, 60°C for 10 s. The β‐tubulin gene was used as an endogenous control, with three biological replicates assessed for each sample. Relative expression levels were calculated using the 2−ΔΔCT method.
5
SARS-CoV-2 Pseudovirus Detection Comparison
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SARS-CoV-2 pseudoviruses containing RdRP and E target sequences were obtained from Maccura Biotechnology (Sichuan, China). SARS-CoV-2 RNA was extracted using the MagicPure® Simple Viral DNA/RNA Kit (TransGen Biotech, Beijing, China). Next, the RNA samples were diluted into five different concentrations (105, 104, 103, 102, 101 copies/μL) and reverse transcribed using the TransScript® First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech, Beijing, China). Also, these samples were tested simultaneously to compare the performances of the GO-multiplex and multiplex qPCR techniques.
6
Earthworm Protein Effects on Cell Signaling
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Cells
were incubated with the proteins from tail-amputated earthworm at
0.8, 1, and 1.2 μg/mL or with the proteins from un-amputated
earthworm at 1 μg/mL for 48 h. bFGF (0.1 μg/mL) was used
as the positive control. Total RNA was extracted from 1 million cells
by RNeasyTM Plus Animal RNA Spin Column Extraction Kit
(Beyotime, China). The RNA solution was measured for absorbance at
260 and 280 nm. RNA is considered to be of high purity when the A260/A280
ratio is between 1.7 and 2.1. We used 0.05–5 μg of RNA
to obtain a cDNA library through the reverse transcription process
by TransScript First-Strand cDNA Synthesis SuperMix Kit (TransGen,
China). To amplify the cDNA of K-Ras, Raf-1, B-Raf, Erk1, and Erk2. The primer sequences are as follows with GAPDH as the internal control:
K-Ras, 5′-GCGCTGACCTAGGAATGTTG-3′,
and 5′-AGGAGTAGTACAGTTCATGAC-3′;
Raf-1, 5′-CATCAATGGAGCACATACAG-3′,
and 5′-AGGCAGTCATGTAAGCTCAT-3′;
B-Raf, 5′-ATTGTTACCCAGTGGTGTGAG-3′,
and 5′-TCTTGAGGTCTCTGTGGATG-3′;
Erk1, 5′-CCAAGTCAGACTCCAAAGCC-3′,
and 5′-GGTCATAGTACTGCTCCAGG-3′;
Erk2, 5′-GAAGCACCATTCAAGTTCGAC-3′,
and 5′-AAGATCTGTATCCTGGCTGG-3′;
GADPH, 5′-CTTTGGTATCGTGGAAGGACTC-3′,
and 5′-GTAGAGGCAGGGATGATGTTCT-3′.
were incubated with the proteins from tail-amputated earthworm at
0.8, 1, and 1.2 μg/mL or with the proteins from un-amputated
earthworm at 1 μg/mL for 48 h. bFGF (0.1 μg/mL) was used
as the positive control. Total RNA was extracted from 1 million cells
by RNeasyTM Plus Animal RNA Spin Column Extraction Kit
(Beyotime, China). The RNA solution was measured for absorbance at
260 and 280 nm. RNA is considered to be of high purity when the A260/A280
ratio is between 1.7 and 2.1. We used 0.05–5 μg of RNA
to obtain a cDNA library through the reverse transcription process
by TransScript First-Strand cDNA Synthesis SuperMix Kit (TransGen,
China). To amplify the cDNA of K-Ras, Raf-1, B-Raf, Erk1, and Erk2. The primer sequences are as follows with GAPDH as the internal control:
K-Ras, 5′-GCGCTGACCTAGGAATGTTG-3′,
and 5′-AGGAGTAGTACAGTTCATGAC-3′;
Raf-1, 5′-CATCAATGGAGCACATACAG-3′,
and 5′-AGGCAGTCATGTAAGCTCAT-3′;
B-Raf, 5′-ATTGTTACCCAGTGGTGTGAG-3′,
and 5′-TCTTGAGGTCTCTGTGGATG-3′;
Erk1, 5′-CCAAGTCAGACTCCAAAGCC-3′,
and 5′-GGTCATAGTACTGCTCCAGG-3′;
Erk2, 5′-GAAGCACCATTCAAGTTCGAC-3′,
and 5′-AAGATCTGTATCCTGGCTGG-3′;
GADPH, 5′-CTTTGGTATCGTGGAAGGACTC-3′,
and 5′-GTAGAGGCAGGGATGATGTTCT-3′.
7
Plant RNA Extraction and qRT-PCR Analysis
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Total RNA was extracted from samples using the EasyPure Plant RNA Kit (Transgen Biotech, China) and then reverse transcribed to produce cDNA using a TransScript First-Strand cDNA Synthesis SuperMix Kit (TransGen Biotech). qRT-PCR assays were performed as reported previously (Zhou et al., 2018 (link)). All of the primers used for qRT-PCR are listed in Supplementary Table S1 .
8
Quantitative Real-Time PCR Analysis
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High quality total RNA (1 μg) was used to prepare first-strand cDNA using the TransScript First-Strand cDNA Synthesis SuperMix kit (TransGen Biotech, Beijing, China) following the manufacturer's protocol.
Quantitative real-time PCR (qRT-PCR) was performed according to the manufacturer's instructions using a TP8000 Real-time PCR detection system and the SYBR premix Ex Taq kit (TAKARA, Japan) with the following PCR program: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 53°C for 10 s, and 72°C for 20 s. All PCR reactions consisted of three technical replicates. Transcript abundance of each gene was normalized to ubiquitin with the comparative Ct method (Livak and Schmittgen, 2001 (link); Udvardi et al., 2008 (link)). Oligonucleotides used in this study are given in TableS1 . Three independent biological replicates for each sample and three technical replicates for each biological replicate were analyzed.
Quantitative real-time PCR (qRT-PCR) was performed according to the manufacturer's instructions using a TP8000 Real-time PCR detection system and the SYBR premix Ex Taq kit (TAKARA, Japan) with the following PCR program: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 53°C for 10 s, and 72°C for 20 s. All PCR reactions consisted of three technical replicates. Transcript abundance of each gene was normalized to ubiquitin with the comparative Ct method (Livak and Schmittgen, 2001 (link); Udvardi et al., 2008 (link)). Oligonucleotides used in this study are given in Table
9
RNA Extraction and qPCR Gene Expression Analysis
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Total RNA was extracted using TRIZOL (Invitrogen). cDNA was synthesized with a TransScript First-strand cDNA Synthesis SuperMix kit from TransGen. The reaction mix included total RNA (500 ng), 1 μl anchored oligo (dT), 10 μl 2 × TS Reaction Mix, and 1 μl TransScript RT/RI Enzyme Mix (the anchored oligo (dT) was replaced with the RT primer corresponding to the target gene when the miRNA cDNA first-strand was synthesized). The reaction conditions were 30 min at 42°C then 85°C for 5 min. Quantitative PCR (qPCR) was performed with the StepOnePlus System (ABI) using the TransStartTM EcoGreen qPCR SuperMix (TransGen). The amplification conditions were 94°C for 30 s, followed by 40 cycles of 94°C for 5 s, and 60°C for 30 s. U6 RNA or GAPDH were used as endogenous controls for miRNA or other mRNA levels. The relative gene expression was analyzed using the 2ŻΔΔCT method.
10
Evaluating Gene Expression Profiles in Stomach Tissue
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Each stomach tissue sample including control, model, omeprazole, and triterpenes middle groups was used for RNA extraction with TRIZOL (Invitrogen, Carlsbad, CA, USA). cDNA was synthesized with 1 mg of total RNA, using Trans Script first-strand cDNA synthesis Super Mix kit (Beijing Trans Gen Biotech, China). Differential gene expression was evaluated by real time polymerase chain reaction (RT-PCR), using TransStart™ Top Green RT-PCR Super Mix kit (Beijing Trans Gen Biotech, China). Primers used to amplify H+-K+-ATPase, PLA2 were from invitrogen and expression of these transcripts was quantified against the housekeeping gene β-actin, which was amplified using the forward 5’-ATCATTGGACGCATCGCCTCTCTGG-3’, 5’-TGACAGCAGGAAGCGAACGA-3’ and the reverse 5’-GTCTTCTGTGGTGTC CGCCGTGTGG-3’, 5’-GACTCATACAGTGCCTT-3’. Expression levels of target genes were analyzed using the CFX manager system (BIO-RAD, USA).
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