Total RNA from VSMCs was isolated using TRIzol® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) following the manufacturer's instructions. RNA (1 µg) was reverse transcribed into first-strand cDNA using the Reverse Transcriptase kit according to the manufacturer's protocol (TransGen Biotech Co., Ltd.). qPCR was performed using SYBR Green Mixture (Takara Bio, Inc.) in the ABI Prism 7300 Sequence Detection System (Applied Biosystems; Thermo Fisher Scientific, Inc.). The thermocycling conditions were: Initial denaturation for 10 min at 94˚C, followed by 40 of cycles of denaturation for 30 sec at 94˚C, annealing for 30 sec at 55˚C and extension for 30 sec at 72˚C The 2-ΔΔCq method (19 (link)) was applied to determine the relative target gene expression. The sequences for the qPCR primers were as follows: ZFAS1 forward, 5'-AGCGTTTGCTTTGTTCCC-3' and reverse, 5'-CTCCCTCGATGCCCTTCT-3'; GAPDH forward, 5'-GGTCTCCTCTGACTTCAACA-3' and reverse, 5'-AGCCAAATTCGTTGTCATAC-3'. GAPDH was employed as an internal control.
Reverse transcriptase kit
Manufactured by Transgene
Sourced in China
The Reverse transcriptase kit is a laboratory tool designed to convert RNA molecules into complementary DNA (cDNA) strands. It contains the necessary enzymes, buffers, and reagents to perform this reverse transcription process, which is a fundamental step in various molecular biology and genetic research applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (7 mentions)
Lightcycler 480, by Roche (2 mentions)
Sybr green containing pcr kit, by Takara Bio (2 mentions)
Rtaq dna polymerase, by Takara Bio (2 mentions)
Sybr green mixture, by Takara Bio (1 mentions)
Abi prism 7300 sequence detection system, by Thermo Fisher Scientific (1 mentions)
Pet32a, by Takara Bio (1 mentions)
Bl21 competent cells, by Tiangen Biotech (1 mentions)
Trizol reagent, by Transgene (1 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Viral rna isolation kit, by Foregene (1 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (1 mentions)
Nanodrop spectrophotometer, by BioTeke (1 mentions)
Biozol reagent, by Thermo Fisher Scientific (1 mentions)
Transstart top green qpcr supermix, by Transgene (1 mentions)
Sybr green realtime pcr master mix, by Accurate Biology (1 mentions)
Pcr supermix, by Transgene (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Perfectstarttm green qpcr supermix kit, by Transgene (1 mentions)
Steponeplus pcr system, by Thermo Fisher Scientific (1 mentions)
17 protocols using reverse transcriptase kit
1
Quantifying ZFAS1 Expression in VSMCs
2
Profiling Sheep Tissue Transcriptomes
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Six sheep were selected at random from all of the samples (n = 1,382) to act as experimental subjects. Following that, these individuals' heart, liver, spleen, lung, kidney, muscle, tail fat, lymph, rumen, and duodenum were all collected. TRIzol reagent (Invitrogen, Waltham, MA, USA) was used to extract total RNA from the samples, which was then reverse transcribed into cDNA using a reverse transcriptase kit (TransGen Biotech, Beijing, China).
3
Cloning and Sequencing of RIPK3 Protein
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RNA was harvested with TRIzol reagent (TransGen Biotech, Beijing, China) from Hy-Line brown chicks according to the manufacturer’s instructions and reverse transcription was executed with Reverse Transcriptase Kit (TransGen Biotech, Beijing, China). PCR was then performed to amplify the RIPK3 protein gene using the following primers: (Fwd) 5′-CCGGAATTC GAAGTAGATATTTGGAGCAG-3′, (Rev) 5′-CCCAAGCTT TGATGAGGTAAGGGATGT-3′ (The italics represent EcoRI, HindIII restriction sites). The pET-32a (+) (Takara Bio Inc, Shiga, Japan) plasmid and amplified PCR products were subsequently digested by double endonuclease (EcoRI, HindIII: Takara Bio Inc, Shiga, Japan) followed by purification and then ligated together by T4-DNA ligase (Takara Bio Inc, Shiga, Japan) at 4 °C overnight. Subsequently, BL21-competent cells (TianGen Biotech, Beijing, China) following the transformation of the recombinant plasmid were cultured on LB/Amp solid plate. A single positive colony from the plate was inoculated into the liquid medium. Finally, PCR-positive samples were sent for sequencing.
4
Quantification of Enterovirus D68 RNA
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Total RNA was extracted using a viral RNA isolation kit (Foregene, Chengdu, Sichuan, China) according to the manufacturer’s protocol. cDNA was generated using a reverse transcriptase kit (TransGen, Beijing, China). The viral RNA was quantified using qRT-PCR with the SYBR Green reaction mix (GenStar, Beijing, China) in a Roche LightCycler 480 (Roche, Basel, Switzerland). The following primer was used: GAPDH forward primer 5'-GCAAATTCCATGGCACCGT-3'; GAPDH reverse primer 5'-TCGCCCCACTTGATTTTGG-3'; EV-D68 forward primer 5'-TGTTCCCACGGTTGAAAACAA-3'; and EV-D68 reverse primer 5'-TGTCTAGCGTCTCATGGTTTTCAC-3'. The relative levels of EV-D68 RNA in different samples were determined using a comparative 2−△△CT method and normalized to the GAPDH gene (Wei et al., 2016 (link)).
5
Quantitative RT-PCR analysis of Sirt6 cKO in brain
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Total RNA from control and Sirt6 cKO brains was extracted using TRIzol reagent (Invitrogen, 15596026) according to the manufacturer’s instructions. Complementary DNA synthesis was performed with a Reverse Transcriptase Kit (TransGene, AH341-01) following the manufacturer’s instructions. Quantitative RT–PCR was performed using a SYBR Green-containing PCR kit (TaKaRa, RR820A), and signals were detected with a Bio-Rad CFX96 Touch Real-Time PCR detection system. Relative mRNA amounts were assayed by comparing PCR cycles to GAPDH using normalization to control samples. The sequences of primers used in this study are given in Table S2 .
6
Liver Glycolipid and Inflammation Profiling
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Total RNA was extracted from the liver tissues using a Biozol reagent (Invitrogen, Carlsbad, CA, USA), as described previously [32 (link)]. The concentration of extracted RNA was determined using a NanoDrop spectrophotometer (BioTeke, Beijing, China). The RNA was reverse-transcribed into cDNA using a reverse transcriptase kit (TransGen Biotech, Beijing, China) following the manufacturer’s instructions. The relative expression levels of genes involved in glycolipid metabolism as well as inflammatory factors (IL-6, IL-β, and TNF-a) and MCP-1 in the liver were measured. The PCR amplification was performed with Trans Start® Top Green qPCR SuperMix (TransGen Biotech, Beijing, China) using the ABI 7500 Fast Dx PCR instrument (Applied Biosystems, Foster City, CA, USA). The relative expression levels of the quantitative genes were identified using the 2−ΔΔCt method, with the β-actin set as the reference gene and the NC group as the control. The primer sequences are listed in Supplementary Table S1 .
7
Quantitative Analysis of m6A-modified RNAs
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M6A-modified RNAs from the IP sample and RNAs from the Input sample were reverse–transcribed into cDNA using a reverse transcriptase kit (TransGen Biotech, Beijing, China). Quantitative real-time polymerase chain reaction (qPCR) was performed using the cDNA as the template on a LightCycler 480 instrument (Roche Applied Science, Mannheim, Germany) with the SYBR Green Realtime PCR Master Mix (Accurate Biotechnology, Hunan, China). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The primer information is presented in Supplementary Table 1
8
Robust RNA Isolation and qRT-PCR Analysis
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RNA isolation and qRT-PCR analysis were performed as previously presented [70 (link)]. Briefly, total RNA was extracted with Trizol reagent (Sigma). Two µg of total RNA was reverse transcribed into cDNA according to the standard procedures of the reverse transcriptase kit (TransGen, AE341-02). PCR reaction was performed in technical triplicates using PCR SuperMix (TransGen, AS111-01). The expression values were analyzed with ABI 7300 detection system (Thermo Scientific). The used PCR primers are listed in Supplementary Table 2 .
9
Quantifying mRNA Expression via qPCR
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After treatment with either solvent control PBS/DMSO or SeO2/GSK-J4 for different time periods, total RNA was extracted from cells using RNAiso Plus Reagent (Takara, Japan) in accordance with the manufacturer's instructions. Next, 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcriptase Kit (TransGen, Beijing, China). The mRNA expression was determined using real-time PCR with the PerfectStartTM Green qPCR SuperMix Kit (TransGen, Beijing, China) on a StepOne Plus PCR system (Applied Biosystems, Waltham, MA, USA). The 2−ΔΔCT formula was used to calculate the expression of the genes, with the human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene serving as the internal reference. The primer information is listed in Additional file 1 : Table S1.
10
RNA Extraction and Quantitative RT-PCR Analysis
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RNA extraction utilized Transzol reagent (TransGen Biotech, China), and RNA purity was determined by absorbance measurements at 260 and 280 nm. Reverse transcription was carried out with a Reverse Transcriptase kit (TransGen Biotech), and transcript abundance was quantitated on a 7500 Real-Time PCR System (Applied Biosystems, USA) using the SYBR Green detection system (TransGen Biotech).
Target gene expression was normalized to Gapdh mRNA amounts. The 2−ΔΔCt method was utilized for data analysis. The primers used are listed in Table S1.
Target gene expression was normalized to Gapdh mRNA amounts. The 2−ΔΔCt method was utilized for data analysis. The primers used are listed in Table S1.
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