Trizol rna extraction protocol

Manufactured by Thermo Fisher Scientific

The TRIzol RNA extraction protocol is a method for the isolation and purification of total RNA from biological samples. It utilizes a monophasic solution of phenol and guanidine isothiocyanate to effectively lyse cells and separate RNA from DNA and proteins. The protocol provides a simple and efficient way to extract high-quality RNA for various downstream applications.

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TRIzol (38370 citations) TRIzol extraction (213 citations) TRIzol protocol (131 citations) RNA Trizol (35 citations) TRIzol extraction protocol (24 citations) TRIzol® RNA extraction (18 citations) RNA extraction protocol (3 citations) TRIzol protocol for RNA extraction (2 citations)

6 protocols using trizol rna extraction protocol

Quantitative Detection of Turkey Reovirus

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Duodenum, jejunum, cecum, bursa of Fabricius, cloacal swab, heart, liver, spleen, kidney and gastrocnemius, and digital flexor tendons were collected and 100 mg of each tissue sample was homogenized in Hanks’ balanced salt solution (HBSS) containing 2% donor horse serum. The homogenate was then centrifuged at 1500 g for 20 min and the supernatant was subjected to RNA extraction using QIAamp Viral RNA mini kit (Qiagen, Valencia, CA). Swabs were treated as tissue samples. RNA was extracted using TRIZOL RNA extraction protocol (Life Technologies, Carlsbad, CA) from 200μl sample of whole blood (with anticoagulant). Copy numbers of the S4 gene were then determined by a previously developed quantitative RT-PCR method specific for turkey reovirus S4 gene [18 (link)]. It has been reported that one TCID₅₀ of TARV-MN4 was equivalent to11.6± 0.2RNA copies of the S4 gene [18 (link)].

Sharafeldin T.A., Mor S.K., Sobhy N.M., Xing Z., Reed K.M., Goyal S.M, & Porter R.E. (2015). A Newly Emergent Turkey Arthritis Reovirus Shows Dominant Enteric Tropism and Induces Significantly Elevated Innate Antiviral and T Helper-1 Cytokine Responses. PLoS ONE, 10(12), e0144085.

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Quantitative Analysis of Intestinal Gene Expression

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Total RNA was extracted from small intestinal tissues using the TRIzol RNA extraction protocol (Life Technologies Inc.). 1 μg of RNA was extracted and reverse transcribed to obtain the cDNA template. The real-time fluorescent quantitative PCR reaction system was set up, and 40 cycles of real-time detection were performed on the computer to obtain CT values. Subsequently, the relative expression levels were calculated. The primers for the target gene ODC and the internal reference (β-actin) are listed in Table 1.

Xu X., Huang X., Xiao L., Wang J., Yang X, & Wu Y. (2024). Mechanism of electro-acupuncture in alleviating intestinal injury in septic mice via polyamine-related M2-macrophage polarization. Frontiers in Immunology, 15, 1373876.

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RNA Extraction from CCA Samples

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RNA was extracted from CCA samples using a modified TRIzol® RNA extraction protocol from Invitrogen. CCA samples were removed from RNAlater and then homogenised in Trizol (1 mL) at room temperature for 6 mins at 30 Hz using a QIAgen TissueLyser. After the initial 3 mins of homogenisation, samples were removed, placed on ice for 5 mins, and then homogenised for the remaining 3 mins. Further processing followed the manufacturer’s protocol, using bromochloropropane for phase separation, and high-salt solution for precipitation. RNA pellets were resuspended in DNase/RNase-free distilled water (20 µl). Total RNA quantity was determined spectrophotometrically using Invitrogen Qubit® Broad Range RNA kit. RNA yield ranged from 5.36 ng RNA/µl to 800 ng RNA/µl. Presence of contaminating DNA was checked randomly in samples using an Invitrogen Qubit® DNA High Sensitivity kit and returned readings “too low for detection”.

Page T.M., McDougall C, & Diaz-Pulido G. (2019). De novo transcriptome assembly for four species of crustose coralline algae and analysis of unique orthologous genes. Scientific Reports, 9, 12611.

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Nascent RNA Sequencing of Mouse ES Cells

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Mouse ES cells were pulse labeled with 1 mM EU in prewarmed ES culture
medium for 45 min at 37°C, 5% CO₂. The media was removed, and
the cells were collected with TRIzol Reagent (Invitrogen). Total RNA was
isolated using the standard TRIzol RNA extraction protocol (Invitrogen).
Biotin-azide was then conjugated to EU with the Click-iT Nascent RNA Capture Kit
(Thermo Fisher Scientific, C10365). Both custom biotinylated, spike-in control
RNAs were then added to 1.7 μg of each biotinylated sample (5e-5
μg of Chr16 and 5e-4 μg of ChrX). Biotin-EU-RNA and spike-in
controls, where applicable, were pulled down from total RNA using
streptavidin-coated magnetic beads (Invitrogen) and purified by TRIzol. The
sequencing library was prepared by using KAPA RNA HyperPrep Kit with RiboErase
(HMR) (KK8561) according to the manufacturer’s protocols. Sequencing was
performed at the Genome Sequencing Facility at St. Jude Children’s
Research Hospital on the Illumina HiSeq platform in single-end mode with 75 bp
per read.

Zhu Z., Chen X., Guo A., Manzano T., Walsh P.J., Wills K.M., Halliburton R., Radko-Juettner S., Carter R.D., Partridge J.F., Green D.R., Zhang J, & Roberts C.W. (2023). Mitotic bookmarking by SWI/SNF subunits. Nature, 618(7963), 180-187.

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RNA Extraction and cDNA Synthesis

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Total RNA was extracted from RAW cells using the TRIzol RNA extraction protocol (Invitrogen, Life Technologies) and treated with DNase (Qiagen). DNA-free RNA (500 ng) was mixed with 50 ng of random hexamers and 50 μM oligo (dT) (Invitrogen), and cDNA was synthesized by Superscript III reverse transcriptase (Invitrogen) following the manufacturer’s recommendations.

Sarno A., Leite A., Augusto C., Muller I., de Ângelis L., Pimentel L., Queiroz A, & Arruda S. (2024). Impaired macrophage and memory T-cell responses to Bacillus Calmette-Guerin nonpolar lipid extract. Frontiers in Immunology, 14, 1263352.

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Gene Expression Analysis by qRT-PCR

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Total RNA was extracted using the standard TRIZOL RNA extraction protocol (15596026, Invitrogen). After cDNA synthetization by high-capacity cDNA reverse transcription kit (4368813, Applied Biosystems™), mixed with Maxima® SYBR Green/ROX q-PCR Master Mix (K0221, Thermo Scientific™) and primers. The sequences of these primers are shown in Table 1. The real-time PCR reaction was performed and analyzed using a Q7 real-time PCR system (Applied Biosystems™), and relative expression was determined as follows. 1 Primer sequence of target gene. MMP1:matrix metalloproteinase 1; MMP3: matrix metalloproteinase 3; COL1A1: type I collagen alpha1 chains; COL1A2: type I collagen alpha2 chains; COL3A1: type III collagen alpha1 chains; SDHA: Recombinant Succinate Dehydrogenase Complex Subunit A; GAPDH: glyceraldehyde-3-phosphate dehydrogenase.

Ge G., Wang Y., Xu Y., Pu W., Tan Y., Liu P., Ding H., Lu Y.M., Wang J., Liu W, & Ma Y. (2023). Induced skin aging by blue-light irradiation in human skin fibroblasts via TGF-β, JNK and EGFR pathways. Journal of dermatological science, 111(2).

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