RNA was extracted and isolated from rat myocardial tissue by homogenization, centrifugation and dissolution. A PCR tube was taken, a solution containing more than 100 ng of RNA was added as a template for reverse transcription, and 1 µl of reverse transcription primer was added for reverse transcription. This process was performed according to the instructions of RevertAid First Strand cDNA Synthesis Kit from TransGen Biotech Co. (Beijing, China). qRT-PCR analysis was then performed using the ABI Prism 7000 system (Abcam, Fremont, CA, USA) to detect mRNA expression. The relative mRNA expression levels of each molecule were normalized by subtracting the corresponding GAPDH threshold cycle (CT), which was performed using the ΔΔCT comparison method. A list of real-time PCR primer sequences is presented in Table 1 .
Revertaid first strand cdna synthesis kit
Manufactured by Transgene
Sourced in China
The RevertAid First Strand cDNA Synthesis Kit is a laboratory product designed for the synthesis of first-strand cDNA from RNA templates. The kit contains the necessary components, including a reverse transcriptase enzyme, to facilitate the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Rnaprep pure plant kit, by Tiangen Biotech (3 mentions)
Sybr green pcr master mix, by Transgene (3 mentions)
Abi 7500 real time system, by Thermo Fisher Scientific (2 mentions)
Faststart universal sybr green master mix, by Roche (2 mentions)
Trizol reagent, by Transgene (2 mentions)
Trizol reagent, by Takara Bio (2 mentions)
Cfx96 system, by Bio-Rad (2 mentions)
Lightcycler 480 sybr green master system, by Roche (2 mentions)
Rna isolation kit, by Promega (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Transzol up reagent, by Thermo Fisher Scientific (2 mentions)
Easypure plant rna kit, by Transgene (2 mentions)
Sybr premix ex taq kit, by Transgene (2 mentions)
Abi quantitative pcr q6 detection system, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Fungal rna kit, by Omega Bio-Tek (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Perfectstart uni rt qpcr kit, by Transgene (1 mentions)
Transzol up reagent, by Transgene (1 mentions)
30 protocols using revertaid first strand cdna synthesis kit
1
Rat Myocardial RNA Extraction and qRT-PCR
2
Quantitative Real-Time PCR Analysis
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The cDNA was synthesized using total RNA by the RevertAid First-Strand cDNA Synthesis kit (TransGen Biotech, Beijing, China). The qRT-PCR reactions were analyzed with PerfectStart Uni RT-qPCR Kit (TransGen Biotech, Beijing, China). The housekeeping gene of Glyceraldehyde-3-dehydrogenase (gapdh) and the internal control gene of Ras-related small GTPase (ras) were used as an internal reference [40 (link)], and relative gene expression levels were calculated using the 2−ΔΔCt method [41 (link)]. The qRT-PCR analysis of each sample was performed in three biological replicates.
3
Quantitative Real-Time PCR Analysis of Epithelial-Mesenchymal Transition Markers
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Total RNA was extracted from cells with TransZol UP reagent (Transgene, Strasbourg, France) and reverse transcribed with the Revert Aid First Strand cDNA Synthesis Kit (Transgene). The following q-PCR primer sequences were used: LHPP forward: 5-CAAACTGTGTGGTAATTGCAGA-3, reverse: 5-CCAGAGGTCTCCTTGTAGTAAC-3; snail forward: 5-CCTTCGTCCTTCTCCTCTACTT-3, reverse: 5-GCTTCGGATGTGCATCTTGA-3; laminin-5 forward: 5-CCATGAATTTCTCCTACTCGCC-3, reverse: 5-CTCCGATGCTGATATCCTTGAT-3; L1CAM forward: 5-GCTGGTTCATCGGCTTTGTG-3, reverse: 5-CTGTACTCGCCGAAGGTCTC-3; N-cadherin forward: 5-CGATAAGGATCAACCCCATACA-3, reverse: 5-TTCAAAGTCGATTGGTTTGACC-3; vimentin forward: 5-AGTCCACTGAGTACCGGAGAC-3, reverse: 5-CATTTCACGCATCTGGCGTTC-3; E-cadherin forward: 5-AGTCACTGACACCAACGATAAT-3, reverse: 5-ATCGTTGTTCACTGGATTTGTG-3;GAPDH forward: 5-CACCCACTCCTCCACCTTTGA-3, reverse: 5-TCTCTCTTCCTCTTGTGCTCTTGC-3. PCR was performed with Fast Start Universal SYBR Green Master Mix (Roche, Basel, Switzerland), and the fluorescence was measured using an ABI 7500 Real Time System (Applied Biosystems, Life Technologies, Foster City, CA, U.S.A.) by following the manufacturer’s instructions. Data were analyzed using the 2−ΔΔCt method, and GAPDH was regarded as an internal control.
4
Total RNA Isolation and cDNA Synthesis
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Total RNA from frozen HMTs and CMMTs was isolated with a TRIzol reagent (TransGen, Beijing, China). The concentration and purity of RNA were determined at A260, 280, and A230 using NanoVue Plus, and RNA integrity was evaluated by separating 1 μg total RNA on 1.5% agarose gel containing 1% formaldehyde. Equal amounts of RNA samples (250 ng) were reverse transcribed to cDNA following instructions of the Revert Aid First Strand cDNA Synthesis Kit (TransGen, Beijing, China) and then stored at −20 °C until use.
5
Quantitative RT-PCR Analysis of Fibroblast Markers
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Total RNA was extracted with Trizol reagent (Takara, Carlsbad, CA, USA) according to the manufacturer's protocol. The cDNA was synthesized using the RevertAid First-Strand cDNA Synthesis Kit (TransGene, Beijing, China). For qRT-PCR, each sample was analyzed in triplicate with amplification conditions of 1 cycle at 95°C for 10 min, then 45 cycles of 95°C for 15 s and 60°C for 60 s. The relative expression of mRNA was calculated by using the formula 2-∆∆CT. Primer sequences were as follows: Kindlin-2: sense, 5′-AACCAAGGATGGCTTGATTCC-3′; antisense, 5′-CAGGGCTGCAAACATCATCAT-3′; α-SMA: sense, 5′-GGCAAGTGATCACCATCGGA-3′; antisense, 5′-GTGGTTTCATGGATGCCAGC-3′; vimentin: sense, 5′-TGGACCAGCTAACCAACGAC-3′; antisense, 5′-GCC AGAGACGCATTGTCAAC-3′; fibronectin (FN): sense, 5′-CAGTAGACCTGTGGAGCATTAC-3′; antisense, 5′-CGGCTCCACATCCTCAATAA-3′; and fibroblast activation protein (FAP) sense, 5′-GGCTTATCACCTGA TCGGCAA-3′; antisense, 5′-TTTACTCCCAACAGGC GACC-3′; β-actin: sense, 5′-GGCGGCACCACCATGTA CCCT-3′; antisense, 5′-AGGGGCCGGACTCGTCATACT-3′.
6
Quantitative Gene Expression Analysis
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Total RNA was extracted using the RNAprep Pure Plant kit (Tiangen, Beijing, China) and then reverse transcribed to cDNA using the RevertAid™ First Strand cDNA Synthesis Kit (TransGen, Beijing, China). A quantitative real-time polymerase chain reaction (qRT-PCR) analysis was performed using the SYBR® Green PCR Master Mix (TransGen) and the CFX96 system (Bio-Rad, Hercules, CA, United States). Three replicates were analyzed per sample. The MdActin gene was used as an internal control. Relative mRNA levels were calculated according to the 2−ΔΔCt method (Livak and Schmittgen, 2001 (link)). The qRT-PCR primers are listed in Supplementary Table S2 .
7
Quantitative Real-Time PCR Analysis of mRNA
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Total RNA was extracted using TRIzol® reagent (TransGen Biotech, Beijing, China) as described by the manufacturer. After acquiring high-quality total RNA, mRNAs were reverse transcribed using the RevertAid™ First Strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). Quantitative real-time PCR (qRT-PCR) analyses on the mRNAs were performed using SYBR Green PCR Master Mix (ABI, USA, 4304437) in a Bio-rad CFX96 real-time system. Beta actin was used as an internal control for each mRNA. Primer sequences of all mRNAs were designed using primer 5 (Table 1 ). For mRNA quantification, the reaction conditions were: 95°C for 10 min, followed by 40 cycles of 95°C for 10 s and 59°C for 50 s. Relative mRNA expression was evaluated using the 2−△△CT method [37 (link)]. Differences were considered significant when P < 0.05 using one-way analysis of variance. All statistical analyses were performed using general linear models in R. Visualization of graphs was done with the ggplot package [38 (link)] in R.
8
Validating Differentially Expressed Genes via qRT-PCR
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To validate the expression of 19 significant DEGs observed in RNA-Seq data, reaction was carried out using EvaGreen 2X qPCR MasterMix Kit (abm, Vancouver, Canada) in a Quanstudio™ 5 Real-Time PCR Intruments (Thermo Fisher Scientific, Inc.). First-strand cDNA was synthesized using the RevertAid™First strand cDNA Synthesis Kit (TransGen Biotech, Beijing, China). DEGs primers were designed using the Primer-Blast (/" https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ) and synthesized commercially (Shuoqing, Kunming, China). Actin were selected as reference genes [121 (link)]. The primers used in qRT-PCR analyses are listed in Table S1 . Amplification reaction mixtures were made of 10 μL of Eva Green 2X qPCR Master Mix, 0.5 μL of each forward and reverse primer (10 mM), and 1 μL of cDNA template, and ddH2O was added to a final volume of 20 μL. The amplification cycling program was as follows: enzyme activation was operated at 95 °C for 10 mins, moreover, 40 cycles of 95 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s. The results were analyzed using the software accompanying the Quanstudio™ 5 Real-Time PCR instruments. The relative expression values were obtained by using the 2-ΔΔCt method [122 (link)].
9
Quantitative Gene Expression Analysis in Pyrus
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Total RNA was extracted using the Plant Total RNA Isolation Plus kit (Foregene; http://www.foregene.com ). The first-strand cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (TransGen Biotech, China). The primers used for qRT-PCR were designed using the NCBI online tool (https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ). The qRT-PCR reactions were performed using the LightCycler 480 SYBR GREEN Master system (Roche, USA). Each 10 μL reaction contained 150 ng of template cDNA, 0.5 μM of each primer, and 5 μL of LightCycler 480 SYBR GREEN I Master. All reactions were performed in 96-well plates with three replicates for each cDNA sample. The qRT-PCR amplification conditions were as follows: 3 min at 95 °C followed by 45 cycles of 95 °C for 3 s, 60 °C for 10 s, and 72 °C for 30 s. Fluorescence signal data was collected at 60 °C. The Pyrus GAPDH gene was used as the internal control. Gene expression levels were calculated using the 2−ΔΔCt method [91 (link)]. At least three biological replicates were performed for each experiment.
10
Comparative Gene Expression Analysis in L. lactis
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L. lactis N8-1 and L. lactis N8-2 were cultivated in GM17 medium for 6 h. The cultures were then diluted to obtain comparable cell densities. Total RNA was extracted, which was followed by reverse transcription into first-strand cDNA using the RevertAid First Strand cDNA Synthesis Kit (TransGen, Beijing, China). The gene transcription levels of tufA, butA, and butB were evaluated using RT-qPCR. The tufA [32 (link)] gene was selected as the housekeeping gene, and the comparative CT(2−ΔΔCT) method was used for data analysis. Transcriptions with a fold change > 2 were considered statistically significant.
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