Total RNA was extracted from renal tissues using RNA isolater Total RNA Extraction Reagent (Vazyme Biotech) according to the manufacturer’s instructions. The concentration and the purity of RNA were measured spectrophotometrically using Nano-100 (Allsheng, Hangzhou, Zhejiang, China). mRNA transcript levels of α-SMA, collagen I, and vimentin were amplified with ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Vazyme Biotech). GAPDH was used as the endogenous control to normalize the amount of cDNA added to each reaction (ΔCT), and the mean ΔCT of control samples was used as the calibrator to calculate the ΔΔCT. Quantitation of each transcript was by the comparative CT method. In this method, the relative quantity of target mRNA, normalized to the endogenous control and relative to the calibrator, is equal to 2−ΔΔCT. Each experiment was carried out in triplicate at least twice; the results are expressed as means±SEM of representative triplicates. Primer sequences were as follows: α-SMA, forward 5′-GTGATCACCATCGGGAATGA-3′, reverse 5′-CAGCAATGCCTGGGTACATG-3′; vimentin, forward 5′-GATCGATGTGGACGTTTCCAA-3′, reverse 5′-ATACTGCTGGCGCACATCAC-3′; collagen, forward 5′-AACCCCAAGGAGAAGAAGCA-3′, reverse 5′-AGCGTGCTGTAGGTGAATCG-3′, GAPDH, forward 5′- CAGGGCTGCCTTCTCTTGTG-3′, reverse 5′-GATGGTGATGGGTTTCCCGT-3′.
Rna isolater total rna extraction reagent
Manufactured by Vazyme
Sourced in China
The RNA Isolater Total RNA Extraction Reagent is a laboratory product designed for the isolation and purification of total RNA from various biological samples. It is a complete solution for efficient RNA extraction, enabling the recovery of high-quality RNA for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Aceq qpcr sybr green master mix, by Vazyme (11 mentions)
Chamq universal sybr qpcr master mix, by Vazyme (8 mentions)
Hiscript 3 rt supermix for qpcr gdna wiper, by Vazyme (6 mentions)
Hiscript 2 q rt supermix, by Vazyme (6 mentions)
Hiscript 3 1st strand cdna synthesis kit gdna wiper, by Vazyme (5 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (5 mentions)
Chamq sybr qpcr master mix, by Vazyme (4 mentions)
Hiscript 2 q rt supermix for qpcr gdna wiper, by Vazyme (3 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (3 mentions)
Primescript rt reagent kit, by Takara Bio (3 mentions)
7500 real time pcr system, by Thermo Fisher Scientific (3 mentions)
Trypsin edta, by Thermo Fisher Scientific (3 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (3 mentions)
Chamq sybr qpcr master mix low rox premixed, by Vazyme (2 mentions)
Iq5 quantitative pcr detection system, by Bio-Rad (2 mentions)
Hiscript 2 q select rt supermix, by Vazyme (2 mentions)
Take3 plate, by Agilent Technologies (2 mentions)
Chamqtm sybr qpcr master mix, by Vazyme (2 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (2 mentions)
Hiscript 3 rt supermix, by Vazyme (2 mentions)
59 protocols using rna isolater total rna extraction reagent
1
Renal Tissue RNA Extraction and qRT-PCR Analysis
2
Biochemical Assays for Kidney Injury
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serum creatinine (Scr) and blood urea nitrogen (BUN) assay kits were purchased from Nanjing Jiancheng Biotech Co., Ltd. (Nanjing, Jiangsu, China). Enzyme-linked immunosorbent assay (ELISA) kit to determine the concentration of urinary albumin (UAlb) was purchased from JinYiBai Biological Technology Co., Ltd. (Nanjing, Jiangsu, China). Enhanced BCA Protein Assay Kit was obtained from Beyotime Institute of Biotechnology (Nanjing, Jiangsu, China). Anti-α-smooth muscle actin (α-SMA) antibody (ab32575) was purchased from Abcam (Cambridge, UK). Anti-vimentin (5741) antibody and anti-eNOS antibody (32,027) were obtained from Cell Signaling Technology (Beverly, MA, USA). Anti-collagen I (AF7001) was obtained from Affbiotech (Cincinnati, OH, USA). Anti-GAPDH (MB001) was purchased from Bioworld Technology (St. Louis Park, MN, USA). The IRDye (680RD or 800CW)-labeled secondary antibodies were purchased from LI-COR Biotechnology (Lincoln, NE, USA). ChamQ SYBR qPCR Master Mix (Low ROX Premixed) (Q331-02) and RNA isolater Total RNA Extraction Reagent (R401-01) were purchased from Vazyme Biotech Co., Ltd. (Nanjing, Jiangsu, China). All the inorganic chemicals were from Sigma-Aldrich (St. Louis, MO, USA).
3
Validating RNA-seq Data with RT-qPCR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To validate the RNA-seq data, three miRNAs were randomly selected for RT-qPCR confirmation with ChamQ™ universal SYBR® RT-qPCR master mix (Vazyme, China). Total RNAs were extracted using RNA Isolater Total RNA Extraction Reagent (Vazyme, China). For miRNA determination, 1 µg of total RNA was reversed using the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, China) with miRNA-specific stem-loop primers. For gene quantification, the cDNA was prepared using HiScript® III All-in-one RT SuperMix Perfect for RT-qPCR (Vazyme, China) with the gDNA wiper. The PCR was conducted with a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The specificity of each primer pair was ensured by analyzing the melting curve. Experiments were performed with three independent biological replicates and technical replicates. The relative quantity of miRNAs and mRNAs was analyzed by RT-qPCR and β-actin was used as the reference gene. All primers were synthesized by the Sangon Biotech Co., Ltd. (Shanghai, China) (Table S1 ). Data were analyzed by the 2−ΔΔCT method.
4
Extraction and Analysis of miRNA from Tissues and EVs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from tissues and EVs as described previously. In brief, total RNA was extracted from all samples by RNA isolater Total RNA Extraction Reagent (Vazyme, China), and cDNA was synthesized using a reverse transcriptase kit (Vazyme, China). For tissue samples from the DSS group, the isolated RNA was purified using LiCl. In brief, a 0.1 volume of 8 M LiCl (Sigma-Aldrich) solution was added to a 1 volume of RNA solution and incubated on ice for 2 h before being centrifuged (14000 × g, 30 min, 4°C), after which the pellets were collected, and the process was repeated twice. Subsequently, 0.1 volume of 3 M sodium acetate (Thermo Scientific) and 2 volumes of ethanol were added to 1 volume of RNA solution to reprecipitate RNA without DSS. For EV samples, a settling agent (Takara #9094, Japan) was added with ethanol to extract the miRNAs. Subsequently, before reverse transcription, the RNA was processed with Poly(A) polymerase (NEBio, China) for the tailing reaction. The primers for miRNA reverse transcription were designed by miRprimer.64 (link) Quantitative real-time PCR (RT-PCR) was performed as described previously.65 (link) The primer sequences are shown in Table S1.
5
Quantitative Analysis of Gene and miRNA Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Tissue samples were subjected to total RNA isolation using the RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China) and reverse-transcribed into cDNA using the HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme) according to the manufacturer’s instructions. qPCR was then performed using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) in the iQ5 quantitative PCR detection system (Bio-Rad, Richmond, CA,USA). The extraction, reverse and qPCR of miRNA were using a miRcute kit (TIANGEN BIOTECH, Beijing) in accordance with the manufacturer’s introductions. We used 2−ΔΔCt method to analysis the relative expression of genes. The primer sequence of the genes were listed below: SOX1, Forward (F): 5′-AGACCTAGATGCCAACAATTGG-3′, Reverse (R): 5′-GCACCACTACGACTTAGTCCG-3′; GAPDH, F: 5′-GGAGCGAGATCCCTCCAAAAT-3′, R: 5′-GGCTGTTGTCATACTTCTCATGG-3′; U6, F: 5′-CTCGCTTCGGCAGCACA-3′, R: 5′-ATCCAGTGCAGGGTCCGAGG-3′; hsa-miR-155-5p, F: 5′-CGCGTTAATGCTAATCGTGATAGGGGTT-3′, R: 5′-ATCCAGTGCAGGGTCCGAGG-3′.
6
Hippocampal RNA Extraction and qPCR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA of the hippocampus samples (N = 3 for each group) was isolated using RNA isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China) in accordance with the manufacturer's instructions. Complementary DNA (cDNA) was synthesized with 1 µg total RNA using HiScript II Q Select RT SuperMix (Vazyme, Nanjing, China). Real-time PCR was performed with routine activating (95°C, 5 min), denaturing (95°C, 15 s) and annealing (72°C, 1 min) programs to evaluate the expression of mRNA. Speci c gene primers were designed by Generay and are presented in Table S11.
7
Quantitative PCR analysis of PDAC
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from fresh PDAC cell line samples and human tissues samples with RNA Isolater Total RNA Extraction Reagent (Vazyme, China) and reverse-transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNA wiper) (Vazyme) according to the manufacturer's instructions. Then, quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme) in an iQ5™ quantitative PCR detection system (Bio-Rad, USA). The primers used in the study were as follows: KRT8, 5′-CAGAAGTCCTACAAGGTGTCCA-3′ and 5′-CTCTGGTTGACCGTAACTGCG-3′, KRT18, 5′-TCGCAAATACTGTGGACAATGC-3′ and 5′-GCAGTCGTGTGATATTGGTGT-3′, YWHAZ, 5′-TGATCCCCAATGCTTCACAAG-3′ and 5′-GCCAAGTAACGGTAGTAATCTCC-3′, LAD1, 5′-GATACCACACGGCCATACGG-3′ and 5′-GAGCCACGAATAACTCAGTGC-3′, and GAPDH, 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTGTTGTCATACTTCTCATGG-3′. The data was analysed using the 2−∆∆Ct method.
8
Quantification of Circular RNAs Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from samples using RNA Isolater Total RNA Extraction Reagent (Vazyme, Nanjing, China). cDNA was synthesized by the GoScript Reverse transcription (RT) System (Promega, WI, USA). The expression levels of circRNA_25775, circRNA_33798, circRNA_25487, circRNA_18052 and circRNA_3684 were analyzed using the CFX96 Q-PCR Detection System (Bio-Rad, CA, USA). The reaction system with 14 μL volume consisted of 3 μL of cDNA, 1.4 μL of primers, 7 μL of AceQ qPCR SYBR Green Master Mix (Vazyme Biotech Co., NJ, USA), and 0.4 μL of ultrapure water. The relative expression levels were calculated by relative quantification (2−ΔΔCt) method.
9
Quantifying CLN7 mRNA in MEFs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from WT and CLN7-KO MEFs using RNA Isolater Total RNA Extraction Reagent (Vazyme) according to the manufacturer’s instructions. Purified RNA was quantified using NanoDrop 2000 (Thermo Fisher Scientific). The cDNA was synthesized by reverse transcription using HiScript II Q RT SuperMix (Vazyme). RT-qPCR was performed on LightCycler 96 (Roche) using SYBR Green Master (Vazyme). The following primers were used: 5′-TGTTGCCGTTGTCCGATCAT-3′ (forward) and 5′-GATGTCCCACGTCACACCTT-3′ (reverse) for CLN7 and 5′-TGATGGGTGTGAACCACGAG-3′ (forward) and 5′-GCCCTTCCACAATGCCAAAG-3′ (reverse) for Gapdh. The 2-ΔΔCt method was used to calculate the relative mRNA level of CLN7.
10
Comprehensive RT-PCR and RNA-seq Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For RT-PCR, total RNA was isolated from cells with RNA Isolater Total RNA Extraction Reagent (Vazyme Biotech) according to the manufacturer’s instructions. The isolated RNA was reverse transcribed into complementary DNA using HiScript II (Vazyme Biotech). Quantitative real-time PCR was performed with a CFX96 Real-Time system (Bio-Rad) and SYBR Green PCR master mix (Yeasen). The fold change in the gene expression was calculated using the comparative Ct method, and three replicates were tested for each complementary DNA sample. ACTB or Actb were used as an internal reference. Primers used in this study were as follows:
hCTGF forward: 5′-AAAAGTGCATCCGTACTCCCA-3′,
hCTGF reverse: 5′-CCGTCGGTACATACTCCACAG-3′;
hCYR61 forward: 5′-GGTCAAAGTTACCGGGCAGT-3′,
hCYR61 reverse: 5′-GGAGGCATCGAATCCCAGC-3′;
hACTB forward: 5′-ATCATGAAGTGTGACGTGGA-3′;
hACTB reverse: 5′-CTCAGGAGGAGCAATGATCT-3′.
For RNA-seq, HGC-27 cells in 6-well plates were treated with DSF, TH, or CX. Total RNA was extracted. RNA quality was assessed using a 2100 Expert Bioanalyzer (Agilent) and sent for library preparation and sequencing using the Illumina Hiseq2000 platform of Majorbio Biotech. The data were analyzed on the free online Majorbio I-Sanger Cloud Platform (www.majorbio.com ). The GEO accession numbers for the high-throughput sequencing reported in this paper is GSE168618 .
hCTGF forward: 5′-AAAAGTGCATCCGTACTCCCA-3′,
hCTGF reverse: 5′-CCGTCGGTACATACTCCACAG-3′;
hCYR61 forward: 5′-GGTCAAAGTTACCGGGCAGT-3′,
hCYR61 reverse: 5′-GGAGGCATCGAATCCCAGC-3′;
hACTB forward: 5′-ATCATGAAGTGTGACGTGGA-3′;
hACTB reverse: 5′-CTCAGGAGGAGCAATGATCT-3′.
For RNA-seq, HGC-27 cells in 6-well plates were treated with DSF, TH, or CX. Total RNA was extracted. RNA quality was assessed using a 2100 Expert Bioanalyzer (Agilent) and sent for library preparation and sequencing using the Illumina Hiseq2000 platform of Majorbio Biotech. The data were analyzed on the free online Majorbio I-Sanger Cloud Platform (
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!