Trizol reagent protocol

Manufactured by Merck Group

Trizol reagent is a guanidinium-based solution used for the extraction and isolation of total RNA from various biological samples. It is a mono-phasic solution containing phenol and guanidine isothiocyanate that effectively lyses cells and denatures cellular components, allowing for the selective isolation of intact RNA.

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Lab products found in correlation

M mlv reverse transcriptase, by Promega (2 mentions) Nanodrop, by Thermo Fisher Scientific (1 mentions) Improm 2 reverse transcription kit, by Promega (1 mentions) Cfx96 real time pcr system, by Bio-Rad (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

TRIzol reagent (2090 citations) Trizol (1168 citations) Trizol protocol (5 citations)

4 protocols using trizol reagent protocol

Quantitative PCR Analysis of MSC Gene Expression

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Using Trizol reagent protocol (Sigma), total RNA was isolated from naïve MSCs according to the manufacturer's instructions. The quality and quantity of RNA were quantified using a Nanodrop (Thermo-Scientific, Rockford, IL). First-strand cDNA synthesis was performed following the manufacturer's instructions (Improm II reverse transcription kit; Promega, Madison, WI) using oligo dT primers. Real-time qPCR was performed in triplicate using SYBR Green Master Mix and a Real-time PCR system (CFX96; BioRad Laboratories, Hercules, CA). The following primers were used:

AT2R-F: 5′-GTTCCCCTTGTTTGGTGTAT-3′.

AT2R-R: 5′-CATCTTCAGGACTTGGTCAC-3′.

GAPDH-F: 5′-CCCATCACCATCTTCCAGGAG-3′.

GAPDH-R: 5′-GTTGTCATGGATGACCTTGGCC-3′.

IL-6-R-F: 5′-GCCGTGTTACTGGTGAGGAA-3′.

IL-6-R-R: 5′-AACTGGCAGAAAAACCGCTGC-3′.

TNFα-R-F 5′-CCCGAGTCTCAACCCTCAAC-3′.

TNFα-R-R 5′-GTTCCTTCAAGCTCCCCCTC-3′.

VEGFR-2-F 5′-CTGGATTCGTGGAGGAGAAATC-3′.

VEGFR-2-R 5′-GAGATGCTCCAAGGTCAGAAAG-3′.

Ikhapoh I.A., Pelham C.J, & Agrawal D.K. (2015). Atherogenic Cytokines Regulate VEGF-A-Induced Differentiation of Bone Marrow-Derived Mesenchymal Stem Cells into Endothelial Cells. Stem Cells International, 2015, 498328.

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RNA Extraction and Real-Time PCR Analysis

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Total RNA was extracted by a single-step phenol–chloroform–isoamyl alcohol extraction procedure modified from the protocol previously described^{3 (link)}. Total RNA was isolated from cells by using the Trizol reagent protocol (Sigma). cDNA was synthesized using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). For real-time PCR analysis, reverse transcription was performed using 1 μg of total RNA and oligo (dT) primer in a 20 μl reaction according to the manufacturer’s protocol (Applied Biosystems, Foster City, California, USA). Real-time PCR was performed using the Mx3005 qPCR system (Stratagene, La Jolla, CA, USA) with SYBR green (Applied Biosystems, Foster City, CA, USA). The relative abundance of each mRNA was calculated using the ΔΔCt method and normalized to GAPDH. Primers are listed in Supplementary Table S3.

Yen J.H., Huang S.T., Huang H.S., Fong Y.C., Wu Y.Y., Chiang J.H, & Su Y.C. (2018). HGK-sestrin 2 signaling-mediated autophagy contributes to antitumor efficacy of Tanshinone IIA in human osteosarcoma cells. Cell Death & Disease, 9(10), 1003.

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RNA Extraction, cDNA Synthesis, and qPCR Analysis

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Total RNA was isolated from cells by using the Trizol reagent protocol (Sigma). cDNA was synthesised using M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). PCR was performed using TaKaRa Taq™ Polymerase. Each sample was tested in triplicate and normalized with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as an endogenous control. Sequences for the PCR primers are listed in Additional file 1.

Yen J.H., Lin L.C., Chen M.C., Sarang Z., Leong P.Y., Chang I.C., Hsu J.D., Chen J.H., Hsieh Y.F., Pallai A., Köröskényi K., Szondy Z, & Tsay G.J. (2015). The metastatic tumor antigen 1-transglutaminase-2 pathway is involved in self-limitation of monosodium urate crystal-induced inflammation by upregulating TGF-β1. Arthritis Research & Therapy, 17(1), 65.

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Transcriptomic Analysis of Drosophila Tissues

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Whole heads, including brain and mouthparts, and abdomens, containing both gonads and gut, were severed from 3-day old adult females and males of either WT or F3 generation GFR flies using fine tweezers. No attempt was made to keep flies virgins. A total of approximately 20 heads and 10 abdomens were pooled per head and abdomen sample, respectively. Three biological samples were collected per condition and RNA was subsequently extracted using a TRIzol™ Reagent protocol (Sigma). In brief, samples were homogenized in TRIzol™ reagent with sterile pestles, incubated at room temperature, and phases were separated using 1–bromo–3–chloropropane (BCP) phase separation reagent. RNA was collected from the aqueous phase, transferred into new tubes and precipitated with isopropanol. Pellets were washed, dried and rehydrated in RNase free water.

Baião G.C., Schneider D.I., Miller W.J, & Klasson L. (2019). The effect of Wolbachia on gene expression in Drosophila paulistorum and its implications for symbiont-induced host speciation. BMC Genomics, 20, 465.

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