Cells were seeded in 12-well plastic bottom cell culture plates and treated as described above. Three parallel experiments were performed in every treatment. Total RNA was isolated with the TRIZOL RNA isolation kit (Fisher), and mRNA was reversely transcribed with the RNAscript II kit (ABI). The stem-loop method was used for microRNA reverse transcription. The qPCR system was prepared with the SYBR green qPCR kit with designed primers (Supplementary table 1 ) and performed on StepOnePlus real-time PCR (ABI).
Trizol rna isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States, France
The TRIzol RNA Isolation Kit is a reagent designed for the extraction and purification of total RNA from a variety of biological samples, including cells, tissues, and body fluids. The kit utilizes a monophasic solution of phenol and guanidine isothiocyanate to effectively lyse cells and denature proteins, allowing for the isolation of high-quality, intact RNA.
Automatically generated - may contain errors
Lab products found in correlation
Iscript cdna synthesis kit, by Bio-Rad (6 mentions)
Iq sybr green supermix, by Bio-Rad (4 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (4 mentions)
Agilent rna 6000 nano kit, by Agilent Technologies (4 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
Picopure rna isolation kit, by Thermo Fisher Scientific (3 mentions)
Sybr green master mix, by Bio-Rad (3 mentions)
Cfx96 system, by Bio-Rad (3 mentions)
Transscript first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Quantitect sybr green pcr kit, by Qiagen (2 mentions)
Hiseq 2500 platform, by Illumina (2 mentions)
Quadromacs starting kit, by Miltenyi Biotec (2 mentions)
Qubit dsdna br assay kit, by Thermo Fisher Scientific (2 mentions)
Dneasy blood tissue kit, by Qiagen (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Dna free dna removal kit, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Sybr green mix, by Thermo Fisher Scientific (1 mentions)
First strand cdna synthesis kit, by Thermo Fisher Scientific (1 mentions)
Cck 8, by Merck Group (1 mentions)
45 protocols using trizol rna isolation kit
1
Quantitative Gene Expression Analysis
2
Transcriptome Analysis of Sorted BM CD34+ Cells
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RNA from sorted BM CD34 + cells was isolated using the TRIzol RNA Isolation Kit (Fisher Scientific, Waltham, MA) followed by RNA-Seq library construction. FASTQ files were processed in TopHat2 using the default options [11] . The t-statistics from the differential expression analysis were used to rank the genes for this analysis. Twenty thousand gene permutations were used to calculate statistical significance, and an FDR <0.25 was considered for statistical significance of a gene set.
3
RNA-seq of Plant Tissue Samples
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For each sample, total RNA was extracted from c. 200 mg tissue, using the TRIzol RNA Isolation Kit (Thermo Fisher Scientific). After DNA removal (DNA‐free™ DNA Removal Kit, Ambion, Austin, TX, USA), RNA was purified and the quality checked using an Agilent 2100 bioanalyser. Only samples with a RIN > 8.6 were used for Illumina sequencing. Three biological replicates were performed for each sampling stage. Paired‐end RNA‐sequencing (2 × 125 nt) was carried out at the GeT‐PlaGe core facility (INRA Toulouse) using a TruSeq Illumina SBS Kit V4 and a HiSeq 2500 platform.
4
Quantifying Gene Expression in HepG2 Cells
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Total RNA was isolated from HepG2 cells using a TRIzol RNA isolation kit (Thermo Fisher Scientific, Inc.) and the purity and concentration of the RNA samples were determined by a DN2000 ultramicro nucleic acid analyzer (Thermo Fisher Scientific, Inc.). The total RNA was reverse transcribed into cDNA using a First Strand cDNA Synthesis kit (Thermo Fisher Scientific, Inc), according to the manufacturer's instructions. RT-qPCR reactions were performed as follows: 25°C for 10 min, 48°C for 60 min and 95°C for 5 min. The qPCR was performed using SYBR green mix from Thermo Fisher Scientific, Inc. The reaction conditions included an initial pre-denaturation step at 95°C for 30 sec, followed by 40 cycles of thermal steps consisting of 95°C for 5 sec and 60°C for 30 sec. β-Actin was used as an internal control. The fold-change was calculated by the 2−∆∆Cq method (23 (link)). The primer sequences used for each gene are shown in Table I .
5
RNA Extraction and qPCR Analysis
6
Quantitative Analysis of miRNA and mRNA
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The total RNAs of GL261 cells were purified and enriched with a TRIzol RNA Isolation Kit (Thermo Fisher Scientific). miRNAs were extended by the stem-loop method, and total RNA reverse transcription was performed with the TransScript First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). After reverse transcription, qPCR was performed to analyze the expression levels of miRNA and mRNA transcripts using specific primers and the QuantiTect SYBR-Green PCR kit (Qiagen, Dusseldorf, Germany). The β-actin and U6 small RNA levels were measured to normalize the mRNA and miRNA levels, respectively. Expression levels were calculated according to the 2−ΔΔCt method. The primers are listed in Table S1.
7
qPCR Analysis of Gene Expression
8
CCK-8 Assay for Cell Viability
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CCK-8 (Cat # 96992) was purchased from Sigma (St. Louis, MS, USA). Fetal bovine serum (FBS) was purchased from Ausbian (Sydney, Australia). DMEM (10-013–CVR) was purchased from Corning Inc. (Corning, NY, USA). Trypsin (Cat # T4665) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). D-Hanks Hanks was obtained from Shanghai GeneChem Co. Ltd. (Shanghai, China). 4-hydroytamoxifen (4-OH-TAM) was purchased from Sigma (St. Louis, MS, USA). TRIZOL RNA Isolation Kit (Catalog #: 12183555) was purchased from Thermo Fisher Scientific (Waltham, MA, USA). M-MLV (M1705), dNTPs (U1240) and RNase inhibitor (N2115) were obtained from Promega (Madison, WI, USA). Oligo dT (B0205) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). Bulge-LoopTM miRNA qPCR Primer Sets were synthesized by Ibibio (Guangzhou, Guangdong, China). Reverse and forward primers were synthesized by Shanghai Genechem Co., LTD (Shanghai, China). SYBR Master Mixture (DRR041B) was obtained from TAKARA (Daliang, Liaoning, China). Reagents for reverse transcription were purchased from Axygen (Union City, CA, USA).
9
Quantitative Gene Expression Analysis
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RNA was extracted from cells, and a quantitative reverse transcription PCR (qPCR) assay was performed to evaluate gene expression. RNA extraction and purification used the TRIzol RNA Isolation Kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. Total RNA was then treated with reverse transcription using the iScript cDNA synthesis Kit (Bio-rad, Hercules, CA, USA). Primer sequences of the genes used for qPCR are listed in Table A1 . qPCR was performed using the Bio-Rad IQ SYBR green supermix. Gene expression was normalized to human GAPDH. Three biological replicates were used for each analysis.
10
Gene Expression Analysis of Vascular Cells
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