Trizol extraction method

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TRIzol extraction method is a widely used technique for the isolation and purification of RNA from a variety of biological samples. It utilizes a mixture of guanidinium thiocyanate, phenol, and chloroform to effectively separate the RNA from DNA, proteins, and other cellular components. The resulting RNA can then be used for various downstream applications, such as gene expression analysis, reverse transcription, and cloning.

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TRIzol (38370 citations) TRIzol method (692 citations) TRIzol extraction (213 citations) TRIzol reagent extraction method (9 citations) Trizol RNA extraction method (8 citations) TRIzol one‑step extraction method (2 citations)

48 protocols using trizol extraction method

Genomic and Transcriptomic DNA/RNA Extraction

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Genomic DNA was isolated from GCT tissue and paired normal adjacent tissue (when available) using either the TRIzol® extraction method (Invitrogen Life Technologies, California) or a QIAamp DNA Mini Kit (Qiagen Sciences, Maryland) according to the manufacturer’s recommended protocol. DNA yield was quantified using 1 μl DNA on a NanoDrop™ spectrophotometer (Thermo Scientific, Maryland). Extracted DNA was stored at −80 °C until further analysis.
Total RNA was extracted from fresh frozen tissue using the TRIzol® extraction method (Invitrogen Life Technologies, California) according to the manufacturer's protocol. Following extraction, RNA was cleaned using the RNeasy Mini Kit (Qiagen, Maryland) according to the manufacturer's recommended protocol. RNA yield was then quantified using 2 μl on a NanoDrop™ spectrophotometer (Thermo Scientific, Maryland). Extracted RNA was stored at -80^O C until further analysis.

Poynter J.N., Bestrashniy J.R., Silverstein K.A., Hooten A.J., Lees C., Ross J.A, & Tolar J. (2015). Cross platform analysis of methylation, miRNA and stem cell gene expression data in germ cell tumors highlights characteristic differences by tumor histology. BMC Cancer, 15, 769.

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Transcriptome Profiling of RNA-Seq Data

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Total cellular RNA was isolated using the Trizol extraction method (Thermo Fisher, Carlsbad, United States of America). Subsequently, 10 ng–100 ng of RNA was utilized for preparing sequencing libraries with the TruSeq RNA Sample Preparation Kit v2 (Illumina). All samples were sequenced on the Illumina platform Hiseq X Ten with paired-end 101bp reads. To analyze the data, the sequencing reads were mapped to the human reference genome (GRCh37) using STAR (2.7.1a). HTSeq count was employed to calculate read counts for each gene, and fragments per kilobase of an exon model per million (FPKM) were calculated to quantify gene expression levels. Differential gene expression analysis was performed using the DESeq2 package in R. Genes with |log2 (fold change)| > 2 were considered differentially expressed between the two conditions for each group.

Chen Y., Fang H., Sun H., Wu X., Xu Y., Zhou B.B, & Li H. (2024). Up-regulation of ABCG1 is associated with methotrexate resistance in acute lymphoblastic leukemia cells. Frontiers in Pharmacology, 14, 1331687.

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Gene Expression Analysis in Rgs2/5 dbKO Mice

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Total RNA was extracted from heart apexes of male and female WT and Rgs2/5 dbKO mice via a Trizol extraction method (ThermoFisher Scientific) and utilizing an Omni Bead Ruptor 24 homogenizer (Omni International) with tissue homogenizer tubes. RNA was purified using the Purelink RNA Mini Kit (ThermoFisher) and then reverse-transcribed using a Maxima First Strand cDNA Synthesis Kit (ThermoFisher Scientific), all according to the manufacturer’s instructions. The primer probes listed in Table S4 were used in the real-time PCR assays with TaqMan gene expression master mix (ThermoFisher Scientific): The ΔC_t method (where C_t is threshold cycle) was used to calculate mRNA expression after normalization to Gapdh expression.

Dahlen S.A., Bernadyn T.F., Dixon A.J., Sun B., Xia J., Owens E.A, & Osei-Owusu P. (2022). Dual loss of regulator of G protein signaling 2 and 5 exacerbates ventricular myocyte arrhythmias and disrupts the fine-tuning of Gi/o signaling. Journal of molecular and cellular cardiology, 170, 34-46.

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Gene Expression Analysis in Rgs2/5 dbKO Mice

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Quantitative Analysis of RGS Genes

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Expression levels of RGS2, RGS4, and RGS5 were determined by real‐time PCR using RNA extracted from uterine arteries of NP, D10, D15, D18, and PPD3 WT, Rgs2^−/−, and Rgs5^−/− mice and from mesenteric arteries and aorta of NP, D15, and PPD3 WT, Rgs2^−/−, and Rgs5^−/− mice. Total RNA was isolated using the TRIzol extraction method (Thermo Fisher Scientific, Waltham, MA), with tissue homogenizer tubes and an Omni Bead Ruptor 24 homogenizer (Omni International, Kennesaw, GA). RNA was purified using the Purelink RNA Mini Kit (Thermo Fisher Scientific). RNA was then reverse‐transcribed into cDNA using a Maxima First Strand cDNA Synthesis Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. The following primer probes were used in the real‐time PCR assays with TaqMan gene expression master mix (Thermo Fisher Scientific), as directed by the manufacturer: Rgs2, Mm01292909_g1; Rgs5, Mm00654112_m1; Rgs4, Mm00501389_m1; and Gapdh, Mm99999915_g1. The ∆C_t method (where C_t is threshold cycle) was used to calculate RGS2, RGS4, and RGS5 mRNA expression after normalization to Gapdh expression.

Koch J.N., Dahlen S.A., Owens E.A, & Osei‐Owusu P. (2019). Regulator of G Protein Signaling 2 Facilitates Uterine Artery Adaptation During Pregnancy in Mice. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 8(9), e010917.

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Molecular Characterization of Neural Cells

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Genomic DNA was extracted from neurons and astrocytes by DNeasy Blood and Tissue Kit (Qiagen). Semiquantitative polymerase chain reaction (PCR)-based amplification of sex-determining region protein gene on Y chromosome (Sry) was done as previously reported [17 (link)] to identify and confirm male cells. We used the interleukin 3 (Il3) gene as an autosomal gene and an internal control for both sexes. PCR products were identified based on size (Sry, 402 base pairs (bp); Il3, 544 bp). RNA was extracted from male and female cells by Trizol extraction method (Life Technologies Inc., 15596-026) and mirVana RNA extraction kit (Thermo Fisher Scientific, AM1560), respectively. Quantitative reverse transcription PCR (qRT-PCR) for X-inactive specific transcripts (Xist) was done for female cells as previously described [17 (link),52 (link)].

Liyanage V.R., Olson C.O., Zachariah R.M., Davie J.R, & Rastegar M. (2019). DNA Methylation Contributes to the Differential Expression Levels of Mecp2 in Male Mice Neurons and Astrocytes. International Journal of Molecular Sciences, 20(8), 1845.

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Transcriptomic Analysis of INS-1 Cells

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Expression of genes related to insulin regulation, oxidative stress response, and DNA damage were analyzed using quantitative real-time PCR. Total RNA was collected from INS-1 cells using a standard Trizol extraction method (Life Technologies) as previously published (Beaver et al. 2017 (link); Wong et al. 2013 (link); Wong et al. 2015 (link)). cDNA was synthesized using 1 μg of total RNA and SuperScript III First-Strand Synthesis SuperMix (Life Technologies). Real time PCR was accomplished using primers that amplify all known transcript isoforms of each gene as a single product of expected size. Rat-specific primer sequences were as indicated (Table 1). Reactions were performed using Fast SYBR Green Mastermix (Life Technologies) on 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). PCR conditions were as follows: 95°C for 20 s, followed by 40 cycles of denaturing at 95°C for 1 s, annealing and extension at 58°C for 20 s, followed by a standard dissociation curve. A dilution series of 10³, 10⁴, 10⁵, 10⁶, and 10⁷ copies of template DNA served as internal standard for quantification. Data represent the copy number of the gene of interest normalized to the copy number of the housekeeping gene, 18S ribosomal RNA (Rn18s also called 18s, and then expressed relative to the mean levels found in ZA cells with no arsenic exposure.

Cao A.L., Beaver L.M., Wong C.P., Hudson L.G, & Ho E. (2019). Zinc deficiency alters the susceptibility of pancreatic beta cells (INS-1) to arsenic exposure. Biometals : an international journal on the role of metal ions in biology, biochemistry, and medicine, 32(6), 845-859.

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Ovine liver tissue RNA isolation and characterization

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Sheep liver tissue (1g) was collected from a slaughter house located under Kolkata Municipality Corporation. Adult males (n=6) of about 1-1.5 years of age were selected for collection of samples. Liver tissue was immersed in Trizol in vial and transported in ice to the laboratory for RNA isolation. Total RNA was isolated using TRIzol extraction method (Life Technologies, USA), as per the standard procedure and was further utilized for cDNA synthesis (56 ). Since the samples were collected from the slaughter house, ethical approval was not necessary. cDNA concentration was estimated and samples above 1200 microgram per ml were considered for further study. cDNA was employed for the characterization of ovine CD14 gene.

Rawat K., Pal A., Banerjee S., Pal A., Mandal S.C, & Batabyal S. (2021). Ovine CD14- an Immune Response Gene Has a Role Against Gastrointestinal Nematode Haemonchus contortus—A Novel Report. Frontiers in Immunology, 12, 664877.

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RNA-seq of B Cell Subpopulations

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Total RNA from all B cell subpopulations was isolated using Trizol extraction method (Life Technologies), purified by RNeasy MinElute spin column (Qiagen) and treated with DNase I (Thermo Fisher) following the manufacturer’s instructions. Total RNA was quantified using NanoDrop Specthophotometer (NanoDrop Technologies). High-quality purified total RNA (300–600 ng) from each sample was used for library preparation according to the user's manual of Truseq Stranded Total ribo-depleted RNA sample preparation kit (Illumina). Library quality was assessed using Agilent 2100 Bioanalyzer (Agilent Technologies) and the quantity was determined using Qubit (Life Technologies). Strand-specific RNA libraries were multiplexed (four samples per lane) and the sequencing was done with Illumina HiSeq 2500 (Illumina) as 50 base paired-end runs.

Agirre X., Meydan C., Jiang Y., Garate L., Doane A.S., Li Z., Verma A., Paiva B., Martín-Subero J.I., Elemento O., Mason C.E., Prosper F, & Melnick A. (2019). Long non-coding RNAs discriminate the stages and gene regulatory states of human humoral immune response. Nature Communications, 10, 821.

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Quantitative RT-PCR Analysis of Mouse Tissues

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Total RNA was extracted from both the spleens and livers of experimental mice by TRIzol extraction method (purchased from Life technologies (Carlsbad, CA)), making sure to minimize any contaminating genomic DNA material. cDNA was prepared by iScript reverse transcriptase and RT PCR reactions were done by using IQ SYBR green super mix and a CFX 96 RT-PCR cycler (purchased from Bio-Rad, Hercules, CA, USA). Pre-validated primers were selected from the Primer bank website (http://pga.mgh.harvard.edu/primerbank) and are listed in Supplementary Table 1. Samples were run in technical triplicates, and appropriate positive and negative controls were included for each RT-PCR experiment. Data obtained were normalized using the housekeeping gene β-actin and presented as the fold induction over uninfected WT mice.

Terrazas C., Varikuti S., Oghumu S., Steinkamp H.M., Ardic N., Kimble J., Nakhasi H, & Satoskar A.R. (2017). Ly6Chi inflammatory monocytes promote susceptibility to Leishmania donovani infection. Scientific Reports, 7, 14693.

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