Total RNA was extracted using TRIzol reagent (Ambion). mRNA was reverse transcribed into cDNA using a RevertAidH Minus Kit (Thermo Scientific), and qPCR was performed using SYBR Green master using the set of primers described in Supplementary Table 1 . For quantitative analysis of miRNA, cDNA was synthesised using a miScript II RT Kit (Qiagen). The expression of miRNAs was evaluated using miScript Primer Assays (Qiagen) together with the miScript SYBR Green PCR Kit (Qiagen). The relative miRNA and mRNA expression levels were normalised to those of the internal controls, U6 for miRNA and GAPDH for mRNA. The primer sequences are provided in Supplementary Table 1 .
Revertaid h minus kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
The RevertAid H Minus kit is a reverse transcription kit designed for the synthesis of first-strand cDNA from RNA templates. It contains the RevertAid H Minus M-MuLV Reverse Transcriptase enzyme, necessary buffers, and reagents for the reverse transcription process.
Automatically generated - may contain errors
Lab products found in correlation
Viia7 real time pcr detection system, by Thermo Fisher Scientific (3 mentions)
Absolute blue sybr green mastermix reagent, by Thermo Fisher Scientific (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Miscript primer assay, by Qiagen (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Miscript sybr green pcr kit, by Qiagen (1 mentions)
Miscript 2 rt kit, by Qiagen (1 mentions)
Sds 2, by Thermo Fisher Scientific (1 mentions)
Abi prism 7900ht sequence detection system, by Thermo Fisher Scientific (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Stepone thermocycler, by Thermo Fisher Scientific (1 mentions)
Nanodrop system, by Thermo Fisher Scientific (1 mentions)
Sybr green, by Bio-Rad (1 mentions)
Trizol reagent, by Merck Group (1 mentions)
Multiplex pcr kit, by Qiagen (1 mentions)
Ampure xp kit, by Beckman Coulter (1 mentions)
Rnaeasy kit, by Qiagen (1 mentions)
Trizol ls reagent, by Thermo Fisher Scientific (1 mentions)
Rnase free dnase 1, by Thermo Fisher Scientific (1 mentions)
17 protocols using revertaid h minus kit
1
Quantitative analysis of RNA and miRNA
2
RNA Expression Analysis in Monocytes
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RNA was isolated from monocytes incubated with MPO-ANCA or control IgG ± LPS for 18 hours using a Qiagen RNeasy mini kit according to the manufacturer’s instructions. RNA (200 ng) was converted to cDNA using a RevertAid H Minus kit (Thermo Fisher Scientific) and stored at –80°C. Hydrolysis probes (TaqMan) were used (Thermo Fisher Scientific). The first strand cDNA obtained was amplified using the recommended TaqMan primer/probe mastermix in 384-well plates using the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) in duplicate. The sequence detector SDS 2.4 software (Applied Biosystems) was used to export the Ct values (threshold cycle). The reference gene bGus was used as an endogenous control. The comparative Ct method was used (2–ΔΔCt) for quantification.
3
Quantification of GPCR and Inflammatory Genes
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The total RNA was isolated from THP-1 cells using TRIzol reagent, according to the manufacturer’s protocol. First-strand cDNA was synthesized from 2 µg of RNA using oligo(dT) primers with RevertAid H Minus kit (Thermo Fisher Scientific, USA). The qRT-PCR procedure was performed using ABsolute Blue QPCR SYBR Green Master Mix reagent (Thermo Fisher Scientific, USA) with a ViiA7 real-time PCR detection system (Applied Biosystems, USA). The primer pairs that were employed for HCA2/GPR109A were forward 5′-TGATGCTCTTCATGTTGGCT-3′ and reverse 5′-GCTGAAGCTGCTGCACAA-3′; for FFA2/GPR43 were forward 5′-GCCTGGGTTATGTCCTTTGGTC-3′ and reverse 5′-CGGTGAAGTTCTCGTAGCAGGT-3′; for FFA3/GPR41 were forward 5′-TGCTGTTCCTGCCTTTCCGCAT-3′ and reverse 5′-GGAAGCGTTCAATGCTCACAGC-3′; for IL-6 were forward 5′-AGACAGCCACTCACCTCTTCAG-3′ and reverse 5′-TTCTGCCAGTGCCTCTTTGCTG-3′; for MCP-1 were forward 5′-AGAATCACCAGCAGCAAGTGTCC-3′ and reverse 5′-TCCTGAACCCACTTCTGCTTGG-3′; for TNF-α were forward 5′-CCCAGGGACCTCTCTCTAATCA-3′ and reverse 5′-AGCTGCCCCTCAGCTTGAG-3′; for RPS29 were forward 5′-CAAGATGGGTCACCAGCAG-3′ and reverse 5′- ATATTTCCGGATCAGACCGT-3′. The mRNA levels were quantified and normalized to the levels of the reference gene RPS29 using the 2−ΔΔCt method and presented as relative expression compared to the values for untreated cells.
4
RNA extraction and gene expression analysis
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Total RNA was extracted with the RNeasy kit (Qiagen) according to the manufacturer's instructions; quality and quantity were measured on a Nanodrop system (Thermo Fisher Scientific). cDNA synthesis was performed with the Revertaid H minus kit (Thermo Fisher Scientific). cDNA expression analysis was performed using Sybr Green (Bio-Rad) qRT-PCR on a StepOne thermocycler (Thermo Fisher Scientific). CDA forward: GGGG ACA AGT TTG TAC AAA AAA GCA GGC TAT GGC TAT GGC CCA GAA GCG T. CDA reverse: GGGG AC CAC TTT GTA CAA GAA AGC TGG GTT CAC TGA GTC TTC TG.
5
Profiling Purinergic Receptor Expression
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Total RNA was isolated and purified from cultured mouse TPCs (passage 1) with Trizol followed by complementary DNA synthesis with RevertAid H Minus kit (#K1632 Thermo Fisher) according to the manufacturer’s instructions. Controls in which the reverse transcriptase was omitted were routinely performed. PCR amplification was performed during 30 thermal cycles (95°C, 20 s; 58°C, 20 s; 72°C, 20 s). The following specific primer pairs were used for PCR amplification:
| Target | Forward primer 5´−3´ | Reverse primer 5´−3´ |
|---|---|---|
| P2X1 | ||
| P2X2 | ||
| P2X3 | ||
| P2X4 | ||
| P2X5 | ||
| P2X6 | ||
| P2X7 | ||
| P2Y1 | ||
| P2Y2 | ||
| P2Y4 | ||
| P2Y6 | ||
| P2Y12 | ||
| GAPDH |
6
Quantitative Analysis of Gene Expression
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Total RNA was isolated from GH3 cells usingTRIzol reagent, according to the manufacturer’s protocol. First-strand cDNA was synthesized from 1.6 µg RNA with RevertAidH Minus kit (ThermoFisherScientific). qRT-PCR was performed using Absolute Blue SYBR green MasterMix reagent (ThermoFisherScientific) with a ViiA7 real-time PCR detection system (AppliedBiosystems). The primer pairs that were employed for MUC16 were forward 5′-GCCTAGGAAGAACCAAAACTCA-3′ and reverse 5′-TCCAATGTGTAGTTCCCCAGT-3′; for GRHL2 were forward 5′-CCTCTGCCTGAGTCAAGACC-3′ and reverse 5′-TAGGAGCTGTGGCTGGCTAT-3′; for MACC1 were forward 5′- CCTGGATGCCTTAGGTGGTA-3′ and reverse 5′-CCCACCCAGGACTCTGATTA-3′; for GUSB were forward 5′-GACTGATCCTTCCATGTATCCCA-3′ and reverse 5′-CCCGCATAGTTGAAGAAGTCG-3′. mRNA levels were quantified and normalized to levels of reference gene GUSB using the 2−ΔΔCt method and presented as relative expression compared with values of untreated cells.
7
RNA Extraction and qRT-PCR Analysis
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Total RNA was isolated from cultured cells using TRIzol reagent (Sigma, USA), according to the manufacturer's protocol. First-strand cDNA was synthesized from 2 µg RNA using oligo (dT) primers with Revert Aid H Minus kit (Thermo Fisher Scientific, USA). qRT-PCR was performed using Absolute Blue SYBR green Master Mix reagent (Thermo Fisher Scientific, USA) with a ViiA7 real-time PCR detection system (Applied Biosystems, USA). The primer pairs used for the gene amplifications are listed in Table 3 mRNA levels were quantified and normalized to levels of reference gene RPS29 using the 2−ΔΔCt method and presented as relative expression compared with values of untreated cells.
8
Amplification of BCR H-CDR3 Regions
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Total RNA was reverse transcribed into cDNA using the RevertAid H Minus Kit (Thermo Scientific, USA). Based on the gene composition of BCR H-CDR3 regions, 19 upstream primers and 5 downstream primers of the mice were designed and synthesis [21 (link)]. Then cDNA was used as a template to amplify the BCR H-CDR3 regions by multiplex PCR with the specifically designed primers using the Qiagen Multiplex-PCR Kit (Qiagen, Germany). PCR cycling conditions were 95 °C for 15 min, 25 cycles of 94 °C for the 15 s and 60 °C for 3 min, and 70 °C for 10 min, followed by the final step at 4 °C as a holding temperature. Subsequently, the multiplex PCR products were purified using Beckman Agencourt AMPure XP Kit (Beckman, USA). The purified product was then subjected to a second round of multiplex PCR reaction. PCR cycling conditions were 98 °C for 1 min, 25 cycles of 98 °C for the 20 s and 65 °C for 30 s, and 72 °C for 5 min, followed by the final step at 4 °C as a holding temperature. After end-repair, dA-tailing, adapter ligation and PCR amplification, the specific DNA fragments were purified again and subjected to library construction.
9
Schneiders S2 Cells RT-qPCR Protocol
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RT-qPCR was employed as described previously.29 1 million Schneiders S2 cells were seeded in 1 ml medium containing 28 μg of total dsRNA on 12-well tissue culture plates. RNA was extracted using the RNAeasy kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). RNA was transcribed into cDNA using the RevertAid-H minus kit (ThermoFisher, Darmstadt, Germany). qPCR was performed in a Roche Lightcycler using the Steinbrenner Cybergreen 5x qPCR-mastermix (Steinbrenner, Wiesbaden, Germany). Primers used in this study are listed in Table S6 .
10
RNA Isolation and cDNA Synthesis from Rice Leaves
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Total RNA was isolated from 0.2 g fresh rice leaves using TRIzol™ LS reagent (Invitrogen; http://www.invitrogen.com ). Three µg of the total RNA was digested with RNase-free DNase I (Thermo Scientific) to remove genomic DNA contamination. The poly(A)-RNA concentration was determined using NanoDrop 2000 spectrophotometer (Thermo Scientific). Samples with a 260/280 ratio of 1.9–2.1 and a 260/230 ratio ≥ 2.0 were chosen to determine the quality and purity of the RNA preparations. The integrity of the purified RNA was checked on 2% formamide denaturing gel. Subsequently, first-strand cDNA was synthesized in a 20 μL reaction mixture by using RevertAid H minus kit (Fermentas) following the manufacturer’s instructions and stored at −20 °C until use.
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