Trizol total rna isolation reagent

Manufactured by Thermo Fisher Scientific

Sourced in United States, China

Trizol total RNA isolation reagent is a phenol-based reagent used for the extraction and purification of total RNA from various biological samples, including cells, tissues, and microorganisms. It is designed to effectively isolate high-quality RNA for downstream applications, such as gene expression analysis, RT-PCR, and Northern blotting.

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60 protocols using trizol total rna isolation reagent

Quantification of miRNA-19a Expression

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Total RNA was extracted from the cells at a density of 75% confluence using TRIzol total RNA isolation reagent (Gibco, Waltham, MA, USA) as per the manufacturers instructions. Total RNA (2 µg) was transcribed with stem-loop RT primers. The primer sequences used were: miR-19a (5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCGGAAC-3); U6 (5-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACAAAATATGGAAC-3).
The produced cDNA was quantified by SYBR Premix Ex Taq (Takara Bio, Inc., Otsu, Japan). The primer sequences used were: miR-19a, 5-TGCGGTTCACAGTGGCTAAG-3; U6 forward, 5-TGCGGGTGCTCGCTTCGGCAGC-3 and reverse, 5-CCAGTGCAGGGTCCGAGGT-3. The reactions were incubated at 95°C for 5 min (95°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec) for 40 cycles and maintained at 10°C. The expression of mRNA was quantified by 2^−ΔΔCT.

Hu W., Jin P., Ding C, & Liu W. (2016). miR-19a/b modulates lung cancer cells metastasis through suppression of MXD1 expression. Oncology Letters, 12(3), 1901-1905.

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Quantitative RT-PCR analysis of erbB receptors

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Total RNA was isolated from cells, using Trizol^® total RNA isolation reagent (Gibco, Gaitherburg, MD, USA), according to the manufacture's recommendations. cDNA for qRT-PCR analyses was synthesized with the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). For erbB receptors qRT-PCR, cDNA was analyzed using the appropriate Taqman probes by an 7900 HT Sequence Detection System (Applied Biosystems, Foster City, CA, USA). Gene expression modulation was meseaured by the −2^ΔΔCT method [55 (link)] and normalized to β-actin levels as endogenous control.

Ciardiello C., Roca M.S., Noto A., Bruzzese F., Moccia T., Vitagliano C., Gennaro E.D., Ciliberto G., Roscilli G., Aurisicchio L., Marra E., Mancini R., Budillon A, & Leone A. (2016). Synergistic antitumor activity of histone deacetylase inhibitors and anti-ErbB3 antibody in NSCLC primary cultures via modulation of ErbB receptors expression. Oncotarget, 7(15), 19559-19574.

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RNA Isolation and qRT-PCR Analysis

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Total RNA was isolated from cells, using Trizol® total RNA isolation reagent (Gibco, Gaitherburg, MD, USA), according to the manufacture’s recommendations. cDNA for qRT-PCR analyses was synthesized with the QuantiTect Reverse Transcription Kit (Qiagen, Valencia, CA, USA). mRNA expression levels were quantified by the fluorescent dye SYBR-green method (Qiagen, Valencia, CA, USA). Gene expression modulation was meseaured by the 2^−ΔΔCT method and normalized to β-actin levels as endogenous control.

Iannelli F., Roca M.S., Lombardi R., Ciardiello C., Grumetti L., De Rienzo S., Moccia T., Vitagliano C., Sorice A., Costantini S., Milone M.R., Pucci B., Leone A., Di Gennaro E., Mancini R., Ciliberto G., Bruzzese F, & Budillon A. (2020). Synergistic antitumor interaction of valproic acid and simvastatin sensitizes prostate cancer to docetaxel by targeting CSCs compartment via YAP inhibition. Journal of Experimental & Clinical Cancer Research : CR, 39, 213.

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Quantifying Renal Cancer Stem Cell Gene Expression

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To investigate the mRNA levels of renal cancer stem cell related genes, total RNA was extracted using Trizol® total RNA isolation reagent (Gibco Life Technologies, Grand Island, NY) following the manufacturer’s protocol. One μg of RNA was reverse transcribed with M-MLV reverse transcriptase (Invitrogen Life Technologies Inc., Grand Island, NY) with random primers. cDNA was diluted 1:10 in the PCR reactions. SOX2 expression levels were measured by quantitative real-time PCR using the SYBR Green assay (Applied Biosystems, Carlsbad, CA, USA) on a StepOne instrument (Applied Biosystems). RRN18S was used as reference endogenous gene (Applied Biosystems, Carlsbad, CA, USA).
SOX-2 amplification was performed using the following primers:
SOX2- FW TACAGCATGTCCTACTCGCAG.
SOX2 -RW GAGGAAGAGGTAACCACAGGG.
Values are expressed in terms of 2-ΔΔCT where ΔΔCT = ΔCTsample- ΔCTcalibrator.; ΔCT is the difference in threshold cycles between the mRNA and RRN18S amplicons, and CT is a parameter given by ABI PRISM 7700 Sequence Detector software by negative correlation with an internal reference (Applied Biosystem Life Technologies Inc., Grand Island, NY).

di Martino S., De Luca G., Grassi L., Federici G., Alfonsi R., Signore M., Addario A., De Salvo L., Francescangeli F., Sanchez M., Tirelli V., Muto G., Sperduti I., Sentinelli S., Costantini M., Pasquini L., Milella M., Haoui M., Simone G., Gallucci M., De Maria R, & Bonci D. (2018). Renal cancer: new models and approach for personalizing therapy. Journal of Experimental & Clinical Cancer Research : CR, 37, 217.

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Quantifying gene expression by RT-qPCR

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Total RNA was extracted using TRIzol total RNA isolation reagent (Gibco, Thermo Fisher Scientific, Inc.). cDNA was synthesized using the RT-PCR kit. Primer sequences were as follows: p53, forward 5′-GGCCATCTACAAGCAGTCA-3′ and reverse 5′-GGGCAGTGCTCGCTTAG-3′; MCP-1, forward 5′-TGTCTGGACCCATTCCTTCT-3′ and reverse 5′-ACCAGCAAGATGATCCCAAT-3′; ICAM-1, forward 5′-CGACTGGACGAGAGG GATTG-3′ and reverse 5′-TTATGACTGCGGCTGCTACC-3′; and β-actin, forward 5′-TCGTGCGTGACATTAAGGAG-3′ and reverse 5′-ATGCCAGGGTACATGGTGGT-3′. The Q-PCR system contained: Sense primer 0.5 μl (10 μM), antisense primer 0.5 μl (10 μM), cDNA (1 μl), double distilled H₂O (8 μl) and SYBR Supermix from the iTaq™ Universal Syber green One Step kit (10 μl). Expression levels of mRNA were quantified by the 2^−ΔΔCt method.

Gao J., Chen Q.Q., Huang Y., Li K.H., Geng X.J., Wang T., Lin Q.S, & Yao R.S. (2021). Design, Synthesis and Pharmacological Evaluation of Naphthofuran Derivatives as Potent SIRT1 Activators. Frontiers in Pharmacology, 12, 653233.

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Quantifying Gene Expression via RT-qPCR

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Total RNA was extracted using Trizol^® total RNA isolation reagent (Gibco®, Life Technologies), followed by the RNeasy Qiaprep (Qiagen) per manufacturer’s protocol. RNA concentration was quantified using a NanoDrop Spectrophotometer (NanoDrop Technologies). cDNA was synthesized from total RNA using random hexamers according to the GeneAmp® RNA PCR Core Kit (Life Technologies) and the High-Capacity cDNA RT kit (Applied Biosystems®, Life Technologies). Real-time PCR was performed using an Applied Biosystems StepOne™ Detection System. Comparative analysis of gene expression levels (ΔΔCt) was carried out using GAPDH as the reference gene. The sequences of the primers are indicated in Additional file 1.

López de Maturana R., Lang V., Zubiarrain A., Sousa A., Vázquez N., Gorostidi A., Águila J., López de Munain A., Rodríguez M, & Sánchez-Pernaute R. (2016). Mutations in LRRK2 impair NF-κB pathway in iPSC-derived neurons. Journal of Neuroinflammation, 13, 295.

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Quantitative Analysis of SPAG6 Expression

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Total bone marrow nucleated cellular RNA was isolated from each sample using TRIzol^® (Total RNA Isolation) reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Total RNA was reverse transcribed into cDNA using a reverse transcription kit (PrimeScript™ RT reagent Kit; Takara Biotechnology Co., Ltd.) according to the manufacturer's instructions. qPCR was subsequently performed using a CFX96 Real Time PCR Detection system (Bio-Rad Laboratories, Inc.). The PCR system and cycle parameters used were as previously described (22 (link)). The total reaction volume was 10 µl and this was prepared as follows: 5 µl TB Green Master Mix (Takara Biotechnology Co., Ltd.), 0.5 µl of each primer, 1 µl cDNA template and 3.0 µl ddH₂O. The thermocycling conditions used for qPCR were as follows: 95°C for 30 sec, followed by 40 cycles at 95°C for 5 sec and 60°C for 30 sec. The relative mRNA expression levels of SPAG6 were calculated using the 2^−ΔΔCq method (23 (link)), and GAPDH acted as the internal control. The following primers sequences were used for qPCR: SPAG6 forward, 5′-AGTGCGACATTCTTCCACAGCTTG-3′ and reverse, 5′-GCGTATCCAGTGCTCCACAATCG-3′; and GAPDH forward, 5′-CTTTGGTATCGTGGAAGGACTC-3′ and reverse, 5′-GTAGAGGCAGGGATGATGTTCT-3′.

Ding L., Luo J., Zhang J.P., Wang J., Li Z.Q., Huang J., Chai L., Mu J., Zhao B., Zhong Y.R., Zhang L.Y, & Liu L. (2021). Aberrant expression of SPAG6 may affect the disease phenotype and serve as a tumor biomarker in BCR/ABL1-negative myeloproliferative neoplasms. Oncology Letters, 23(1), 10.

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Quantifying Gene and MicroRNA Expression

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Total RNA was extracted from frozen tissues or cultured cells using TRIzol total RNA isolation reagent (Invitrogen) according to the manufacturer’s instructions. cDNA was synthesized from total RNA or purified small RNAs using gene-specific primers or random hexamers with the SUPERSCRIPT III Reverse Transcriptase Kit (Invitrogen), according to the manufacturer’s instructions. PCR was then performed using Taq polymerase (TaKaRa) with the specific primers for TGFBR1 (forward: 5′- TCGTCTGCATCTCACTCAT-3′, reverse: 5′-GATAAATCTCTGCCTCACG-3′) and GAPDH as an internal control (forward: 5′-TCTCTGCTCCTCCTGTTC-3′, reverse: 5′-GGTTGAGCACAGGGTACTTTATTGA-3′). MicroRNAs were detected with stem-loop primers purchased from RiboBio as described (Guangzhou RiboBio Co., Ltd). GAPDH and U6 small nucleolar RNA were used for normalization. qPCR was conducted using a QuantiTect SYBR Green PCR Kit (TaKaRa Bio Inc., Japan) on a StepOne Real-Time PCR System (Applied Biosystems, CA, USA). Relative expression levels were calculated using the 2^–ΔΔCt method (Bio-Rad CFX manager software 3.1).

Yang Z., He J., Gao P., Niu Y., Zhang J., Wang L., Liu M., Wei X., Liu C., Zhang C., Wang W., Du J., Li H., Hu W, & Sun G. (2017). miR-769-5p suppressed cell proliferation, migration and invasion by targeting TGFBR1 in non-small cell lung carcinoma. Oncotarget, 8(69), 113558-113570.

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Quantification of miR-373 and ITGA2

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Total RNA was extracted using Trizol total RNA isolation reagent (Invitrogen). The mature form of miR-373 was quantified using Hairpin-it-miRNAs qPCR quantitation kit. In brief, microRNAs were reversely transcribed by stem-loop RT primer, quantitative PCR was then carried out (n = 3). The U6 small nuclear RNA was used as an internal control. The mRNA level of ITGA2 was quantified by the specific primer sets (n = 3). GAPDH was used for normalization.

Ding W., Fan X.L., Xu X., Huang J.Z., Xu S.H., Geng Q., Li R., Chen D, & Yan G.R. (2015). Epigenetic Silencing of ITGA2 by MiR-373 Promotes Cell Migration in Breast Cancer. PLoS ONE, 10(8), e0135128.

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DNA and RNA Isolation from Blood

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Human genomic DNA was isolated from EDTA anti-coagulated peripheral blood using the Puregene DNA isolation kit (Gentra Systems, Minneapolis, MN) by following the vendor’s instruction. Total RNA was purified from peripheral blood leukocytes using TRIzol total RNA isolation reagent (Invitrogen, Carlsbad, CA).

Li Y., Mair D.C., Schuller R.M., Li L, & Wu J. (2015). Genetic Mechanism of Human Neutrophil Antigen 2 Deficiency and Expression Variations. PLoS Genetics, 11(5), e1005255.

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