Total RNA was isolated using RNA isolator (#R401‐01‐AA, Vazyme Biotech) following the manufacturer's protocol. Then, the cDNA library was constructed using HiScript II Q RT SuperMix for qPCR Reverse Transcription Kit (#R223‐01, Vazyme Biotech) according to the manufacturer's protocol. Quantitative real‐time PCR (qRT‐PCR) was performed using AceQ® Universal SYBR® qPCR Master Mix (#Q511, Vazyme Biotech). Primers were synthesized by Integrated DNA Technologies. The relative expression levels of genes were calculated via the 2−ΔΔCt method. The geometric mean of GAPDH was used as normalizer for studies. The primers used in qRT‐PCR were listed in Supplementary Table S5 .
Rna isolator
Manufactured by Vazyme
Sourced in China
The RNA Isolator is a lab equipment designed for the extraction and purification of RNA from various biological samples. It utilizes a specialized protocol to efficiently isolate high-quality RNA for downstream applications, such as reverse transcription and gene expression analysis.
Automatically generated - may contain errors
Lab products found in correlation
Quantstudio 5 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (3 mentions)
Hiscript 2 1st strand cdna synthesis kit, by Vazyme (3 mentions)
Hieff qpcr sybr green master mix, by Yeasen (3 mentions)
Hiscript 2 first strand cdna synthesis kit, by Vazyme (2 mentions)
Chamq sybr color qpcr master mix, by Vazyme (2 mentions)
Hiscript q rt supermix for qpcr, by Vazyme (2 mentions)
Lightcycler 480 2, by Roche (2 mentions)
Nano 100 micro spectrophotometer, by Allsheng (2 mentions)
Cfx system, by Bio-Rad (2 mentions)
Aceq qpcr sybr green master mix kit, by Vazyme (2 mentions)
Chamq sybr qpcr master mix, by Vazyme (2 mentions)
Sybr green master mix, by Vazyme (2 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (1 mentions)
Hiscript 2 qrt supermix for qpcr reverse transcription kit, by Vazyme (1 mentions)
Sybr green qpcr master mix kit, by Vazyme (1 mentions)
Hifair 2 1st strand cdna synthesis supermix, by Yeasen (1 mentions)
7500 fast real time pcr instrument, by Thermo Fisher Scientific (1 mentions)
Abi 7500, by Thermo Fisher Scientific (1 mentions)
Hiscript q rt supermix with gdna eraser, by Vazyme (1 mentions)
26 protocols using rna isolator
1
Quantitative Real-Time PCR for Gene Expression
2
Quantifying Target mRNA Expression
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Total RNA was extracted from frozen organs using an RNA isolator (Vazyme, Nanjing, China), and then 2 μg of RNA was used to reverse-transcribe into cDNA using the HiScript® III RT SuperMix for qRNA (Vazyme, Nanjing, China). qRT-PCR was performed in duplicate using AceQ® qPCR (Vazyme, Nanjing, China) with specific primers for target sequences. The 2−ΔΔCt relative quantification method, using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) for normalization, was used to estimate the amount of target mRNA in samples, and fold ratios were calculated relative to mRNA expression levels from control samples. The primer sequences used, including forward and reverse sequences for each gene, are shown in the Supplementary Material (Table S2 ).
3
Quantifying TLR4 mRNA Expression using RT-qPCR
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Following transfection for 48 h, cells were collected and washed with PBS twice. Next, based on the one-step method (20 (link)), total RNA was extracted by adding 1 ml RNA isolator (Vazyme Biotech Co., Ltd.). As the template for PCR, cDNA was synthesized using the HiScript II First Strand cDNA Synthesis kit according to the manufacturer's protocol (Vazyme Biotech Co., Ltd.). The internal reference was human GAPDH. The fluorescent signal was obtained using SYBR-Green I. Based on the manufacturer's protocol of the qPCR SYBR-Green Master Mix kit (Vazyme Biotech Co., Ltd.), qPCR was performed. The thermocycling conditions were as follows: 95°C for 5 min (pre-denaturation), followed by 40 cycles at 95°C for 10 sec and 60°C for 30 sec, and dissociation at 95°C for 15 sec, 60°C for 1 min and 95°C for 15 sec. For each sample, each experiment was repeated three times. The forward primer for GAPDH was 3′-CACTACCGTACCTGACACCA-5′ and the reverse primer was 3′-ATGTCGTTGTCCCACCACCT-5′. The TLR4 sequence was synthesized by GeneCopoeia, Inc. (cat. no. HQP054754). The relative expression levels of TLR4 mRNA (ΔCq=CqTLR4-CqGAPDH) were calculated using the 2−ΔΔCq method (21 (link)). RT-qPCR was used to evaluate the silencing effect of shRNA TLR4 expression levels and to select the interference plasmid with the greatest silencing effect.
4
Quantifying Gene Expression in bEnd.3 Cells
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Total RNA from bEnd.3 cells were isolated with an RNA isolator (Vazyme, Nanjing, China). The concentration of RNA was measured by a NANO-100 micro-spectrophotometer (ALLSHENG, China), and the absorbance at 260/280 nm was considered to detect the purity of RNA. HiScript® Q RT SuperMix for qPCR (Vazyme, Nanjing, China) was used to synthesize cDNA from RNA. Subsequently, qRT-PCR was performed with ChamQ SYBR Color qPCR Master Mix (Vazyme, Nanjing, China) on LightCycler 480 II (Roche, Basel, Switzerland). mRNAs in the tested samples were standardized with Actb, and the 2−ΔΔCt method was used for relative quantification. Primer sequences are listed in Table S1 .
5
Gene Expression Analysis by RT-qPCR
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Total RNA was extracted from the cells using Trizol (RNA Isolator (Vazyme Biotech Co., Ltd, Nanjing)). Reverse transcription (RT) and qPCR were performed in accordance with the manufacturer’s instructions (Vazyme Biotech Co., Ltd, Nanjing). RT-qPCR for each gene was repeated three times. Quantification amounts were normalized to GAPDH levels. Primers are listed in Table A2
6
Gene Expression Analysis by qPCR
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Total RNA was extracted and purified with an RNA isolator (Vazyme, R401-01) according to the standard protocol. Hifair II 1ST Strand cDNA Synthesis SuperMix (Yeasen, 11123ES60) was used to perform reverse transcription. Hieff UNICON qPCR SYBR Green Master Mix (NO Rox) (Yeasen, 11198ES08) was used to perform real-time quantitative PCR. The relative mRNA level of specific genes was normalized to the internal reference level. The primers used are listed in Table 1 .
7
Gene Expression Analysis in Colorectal Cells
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NCM460 cells and HT29 cells were seeded in 6-well plates (1-2 × 105 cells per well). HT-29 cells were treated with WZB117 (0 and 300 μmol/L) for 24 h. After 24 h, the culture medium was removed, and cells were washed 2-3 times with PBS at 4°C, harvested, and lysed. Total RNA from NCM460 and HT-29 cells was extracted using the RNA isolator (Vazyme, Nanjing, China). Complementary DNA (cDNA) was synthesized from 1 ng of total RNA using the NovoScript Plus All-in-one 1st Strand cDNA Synthesis SuperMix Kit, which contains genomic DNA (gDNA) removal (Jiangsu Novogene Bioinformatics Technology Co., Ltd.). Real-time quantitative polymerase chain reaction (qPCR) was performed using the QuantStudio 5 Real-Time PCR System (Applied Biosystems, United States) with the Hieff qPCR SYBR Green Master Mix (Shanghai Yisen Biological Technology Co., Ltd.). The primers listed in Table 1 were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.
8
Quantifying Gene Expression in Kidney Cells
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1 μg of total RNA were extracted from HK2 cells or rat kidney tissues with the RNA isolator (Vazyme, China) and were reversed into cDNA with the HiScript II RT SupeMix (Vazyme, China). The qRT-PCR (40 cycles) was performed with the ChamQ Universal SYBR qPCR Master Mix (Vazyme, China) by using the Applied Biosystem 7500 fast Real-Time PCR instrument. The human GADPH and the rat Actin were used to normalize the mRNA levels of ATP1A1, DNMT1, DNMT3A and DNMT3B. The following primers were used:
human ATP1A1 forward: 5′-GACGTGATAAGTATGAGCCTG-3′, human ATP1A1 reverse: 5′-AATCCCCGGCTCAAGTCTGT-3’; human DNMT1 forward: 5′-GGTTCTTCCTCCTGGAGAATGTC-3′, human DNMT1 reverse: 5′-GTCTGGGCCACGCCGTACTG-3’; human DNMT3A forward: 5′-AGTTAGCAGCAGGGAGACGA-3′, human DNMT3A reverse: 5′-AAGAGGTAACAGCGGCTTCTA-3’; human DNMT3B forward: 5′-AGGGAAGACTCGATCCTCGTC-3′, human DNMT3B reverse: 5′-GTGTGTAGCTTAGCAGACTGG-3’; human GAPDH forward: 5′-CTTTGGTATCGTGGAAGGAC-3′, human GAPDH reverse: 5′-GAAATGAGCTTGACAAAGTG-3’; rat Atp1a1 forward: 5′-TATCTGCAAATGGCTGCAAG-3′, rat Atp1a1 reverse: 5′-CCCAGTGTACACAACGATGC-3’; rat Actin forward: 5′-AGCCATGTACGTAGCCATCC-3′, rat Actin reverse: 5′-TCTCAGCTGTGGTGGTGAAG-3’.
human ATP1A1 forward: 5′-GACGTGATAAGTATGAGCCTG-3′, human ATP1A1 reverse: 5′-AATCCCCGGCTCAAGTCTGT-3’; human DNMT1 forward: 5′-GGTTCTTCCTCCTGGAGAATGTC-3′, human DNMT1 reverse: 5′-GTCTGGGCCACGCCGTACTG-3’; human DNMT3A forward: 5′-AGTTAGCAGCAGGGAGACGA-3′, human DNMT3A reverse: 5′-AAGAGGTAACAGCGGCTTCTA-3’; human DNMT3B forward: 5′-AGGGAAGACTCGATCCTCGTC-3′, human DNMT3B reverse: 5′-GTGTGTAGCTTAGCAGACTGG-3’; human GAPDH forward: 5′-CTTTGGTATCGTGGAAGGAC-3′, human GAPDH reverse: 5′-GAAATGAGCTTGACAAAGTG-3’; rat Atp1a1 forward: 5′-TATCTGCAAATGGCTGCAAG-3′, rat Atp1a1 reverse: 5′-CCCAGTGTACACAACGATGC-3’; rat Actin forward: 5′-AGCCATGTACGTAGCCATCC-3′, rat Actin reverse: 5′-TCTCAGCTGTGGTGGTGAAG-3’.
9
Liver RNA Extraction and qPCR Analysis
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Total RNA was extracted from liver tissues using an RNA isolator (Vazyme Biotech Co., Ltd., Nanjing, Jiangsu, China) in accordance with the manufacturer’s instructions. Reverse transcription was performed using a HiScript® II 1st Strand cDNA Synthesis kit (Vazyme Biotech Co., Ltd., Nanjing, China). Real-time PCR was performed using a Bio-Rad CFX System and AceQ qPCR SYBR Green Master Mix kit (Vazyme Biotech Co., Ltd., Nanjing, China) following the method described previously [28 (link),29 (link)]. The primer sequences listed in Table 1 are designed for mice genes.
10
Real-Time qPCR Gene Expression Analysis
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Total RNA was extracted using RNA isolator (R401-01, Vazyme, Nanjing, China) and reverse-transcribed into cDNA using a HiScript qRT SuperMix with gDNA Eraser (R122-01, Vazyme) according to the manufacturer’s instructions. RT-qPCR was performed on an ABI 7500 (Applied Biosystems, Shanghai, China) using the ChamQ SYBR qPCR Master Mix (Q311-02, Vazyme, Nanjing, China). Primers for RT-qPCR are listed in Supplemental Table S1 . Gene expression levels were calculated using the 2−ΔΔCt method and normalized to β-actin mRNA expression.
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