Taqman 5 nuclease assay

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TaqMan 5' nuclease assay is a real-time PCR (polymerase chain reaction) technique used for the detection and quantification of specific DNA sequences. It utilizes a fluorogenic probe that binds to the target DNA sequence, enabling the monitoring of the amplification process in real-time. The core function of the TaqMan 5' nuclease assay is to provide a sensitive and accurate method for the detection and quantification of DNA targets.

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TaqMan assays (1810 citations) Taqman 5′ nuclease (3 citations)

43 protocols using taqman 5 nuclease assay

Genetic Polymorphisms in IL6 and IL6R

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Genomic DNA was extracted from peripheral blood leukocytes (Miller et al., 1988 (link)). Polymorphisms in the upstream promoter region of the IL6 gene -174 G > C (rs1800795) and in the coding region of IL6R gene (rs2228145) Asp358Ala were analysed by allelic discrimination method (Step One Plus™ Real Time PCR System, Applied Biosystems, FosterCity, CA, USA) with TaqMan 5′ Nuclease Assays using allele specific fluorogenic oligonucleotide probes – C__1839697_20 and C__16170664_10 respectively, following manufacturer's instructions.

Sundaresh A., Oliveira J., Chinnadurai R.K., Rajkumar R.P., Hani L., Krishnamoorthy R., Leboyer M., Negi V.S, & Tamouza R. (2019). IL6/IL6R genetic diversity and plasma IL6 levels in bipolar disorder: An Indo-French study. Heliyon, 5(1), e01124.

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Genetic Variant Determination in Drug Transporters

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Genomic DNA was extracted from peripheral blood leukocytes using proteinase K digestion and standard phenol–chloroform procedures [31 ]. SNPs in ABCB1, ABCC4, SLCO1A2, SLCO1B1, SLCO1B3 and SLCO2B1 genes were determined by allelic discrimination with Taqman 5′-nuclease assays (Supplementary Table 1; real-time PCR, Applied Biosystems, CA, USA) according to the manufacturer's recommended protocol.

Sortica V.A., Lindenau J.D., Cunha M.G., O Ohnishi M.D., R Ventura A.M., Ribeiro-dos-Santos Â.K., Santos S.E., Guimarães L.S, & Hutz M.H. (2017). SLCO1A2, SLCO1B1 and SLCO2B1 polymorphisms influences chloroquine and primaquine treatment in Plasmodium vivax malaria. Pharmacogenomics, 18(15), 1393-1400.

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Genotyping of PTPN2 and IL2RA SNPs

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SNPs in the genes PTPN2 (rs45450798 and rs478582) and IL2RA (rs12722495 and rs2104286) were genotyped using TaqMan 5′ nuclease assays (Applied Biosystems) according to the manufacturer’s protocol.

Yang J.H., Cutler A.J., Ferreira R.C., Reading J.L., Cooper N.J., Wallace C., Clarke P., Smyth D.J., Boyce C.S., Gao G.J., Todd J.A., Wicker L.S, & Tree T.I. (2015). Natural variation in IL-2 sensitivity influences regulatory T cell frequency and function in individuals with long-standing type 1 diabetes. Diabetes, 64(11), 3891-3902.

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Genomic DNA Extraction and SNP Genotyping

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Genomic DNA from all patients was extracted from subject's peripheral blood leukocytes using proteinase K digestion and standard phenol–chloroform procedures [24 ]. Reactions were performed in a total of 8 μl containing 10 ng of genomic DNA. The 13 SNPs in CYP450 and G6PD genes were determined by allelic discrimination assays (7500 Real Time PCR System^®, Applied Biosystems, CA, USA) using Taqman 5′-nuclease assays^® (Applied Biosystems) (Table 1), according to the manufacturer's recommended protocol.

Sortica V.A., Lindenau J.D., Cunha M.G., Ohnishi M.D., Ventura A.M., Ribeiro-dos-Santos Â.K., Santos S.E., Guimarães L.S, & Hutz M.H. (2016). The effect of SNPs in CYP450 in chloroquine/primaquine Plasmodium vivax malaria treatment. Pharmacogenomics, 17(17), 1903-1911.

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Genotyping Variants in Diverse Populations

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All participants from the DHS were previously genotyped for the variants using an Illumina Infinium HumanExome BeadChip^{12 (link),14 (link)}.
Participants from the LBC and from the central European independent replication cohort were genotyped by TaqMan 5′ nuclease assays (Life Technologies). The allelic discrimination probe for PSD3 rs71519934 was not commercially available. A custom assay for this variant has been designed (supplementary material).
Nine individuals denoted as AC homozygotes (n = 3), CT homozygotes (n = 3) and AC/CT heterozygous (n = 3) by the TaqMan assay were Sanger sequenced with consistent results (Supplementary Fig. 2).
UK Biobank participants were genotyped using two highly similar UK BiLEVE or UK Biobank Axiom arrays (>95% overlap). Genotyped data were then imputed based on the 1000 Genomes Phase 3, UK10K haplotype and Haplotype Reference Consortium reference panels^{47 (link)}. Genotype data for the rs71519934 dinucleotide change were not available in the UK Biobank; the rs7003060 (identifying the first nucleotide change of the rs71519934) was among directly genotyped variants and has been used instead.

Mancina R.M., Sasidharan K., Lindblom A., Wei Y., Ciociola E., Jamialahmadi O., Pingitore P., Andréasson A.C., Pellegrini G., Baselli G., Männistö V., Pihlajamäki J., Kärjä V., Grimaudo S., Marini I., Maggioni M., Becattini B., Tavaglione F., Dix C., Castaldo M., Klein S., Perelis M., Pattou F., Thuillier D., Raverdy V., Dongiovanni P., Fracanzani A.L., Stickel F., Hampe J., Buch S., Luukkonen P.K., Prati D., Yki-Järvinen H., Petta S., Xing C., Schafmayer C., Aigner E., Datz C., Lee R.G., Valenti L., Lindén D, & Romeo S. (2022). PSD3 downregulation confers protection against fatty liver disease. Nature Metabolism, 4(1), 60-75.

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Genetic Variant Analysis Protocol

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The rs58542926 C>T (E167K, TM6SF2) and rs738409 C>G (I148M, PNPLA3) genetic variants were assessed in duplicate by TaqMan 5'‐nuclease assays (Life Technologies, Carlsbad, California, USA).

Baselli G.A., Dongiovanni P., Rametta R., Meroni M., Pelusi S., Maggioni M., Badiali S., Pingitore P., Maurotti S., Montalcini T., Taliento A.E., Prati D., Rossi G., Fracanzani A.L., Mancina R.M., Romeo S, & Valenti L. (2020). Liver transcriptomics highlights interleukin-32 as novel NAFLD-related cytokine and candidate biomarker. Gut, 69(10), 1855-1866.

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PNPLA3 and TM6SF2 Genotyping

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The rs738409 C>G (I148M PNPLA3) and the rs58542926 C>T (E167K, TM6SF2) single-nucleotide polymorphisms were assessed in duplicate by TaqMan 5’-nuclease assays (Life Technologies, Carlsbad, CA). The success rate and reproducibility were >99%; random samples were confirmed by direct sequencing. The genotype distributions were in Hardy-Weinberg’s equilibrium and were performed in 134 patients.

Fracanzani A.L., Pisano G., Consonni D., Tiraboschi S., Baragetti A., Bertelli C., Norata G.D., Dongiovanni P., Valenti L., Grigore L., Tonella T., Catapano A, & Fargion S. (2016). Epicardial Adipose Tissue (EAT) Thickness Is Associated with Cardiovascular and Liver Damage in Nonalcoholic Fatty Liver Disease. PLoS ONE, 11(9), e0162473.

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Quantitative RT-PCR Assay for mRNA and miRNA

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The relative quantification of mRNA was performed with a quantitative RT-PCR assay. 400 ng of total RNA was transcribed into cDNA using the cDNA Archive Kit (Life Technologies, Foster City, CA, USA). Each cDNA sample was analyzed by quantitative real-time PCR (qPCR) using the fluorescent TaqMan 5’-nuclease assays (Life Technologies). The analysis was performed with ViiA 7 detection system and software (Life Technologies). Gene expression was standardized to POLR2A expression (2^-ΔCt).
miRNAs were reverse transcribed using TaqMan Micro RNA Reverse Transcription Kit (Life Technologies). Taqman microRNA assays (Life Technologies) were used for miRNA quantification.

Verbanck M., Canouil M., Leloire A., Dhennin V., Coumoul X., Yengo L., Froguel P, & Poulain-Godefroy O. (2017). Low-dose exposure to bisphenols A, F and S of human primary adipocyte impacts coding and non-coding RNA profiles. PLoS ONE, 12(6), e0179583.

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Genetic Variant Screening in LBC Cohort

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The LBC cohort was genotyped for the rs738409 C>G (PNPLA3 I148M), rs58542926 C>T (TM6SF2 E167K), rs1260326 C>T (GCKR P446L), rs641738 C>T MBOAT7, and rs4841132 G>A (LOC157273‐PPP1R3B) variants as described.5, 15 Genotyping of the LBC was performed in duplicate using TaqMan 5′‐nuclease assays (Life Technologies, Carlsbad, CA). Genotype frequencies of the four variants were in agreement with Hardy‐Weinberg proportions (P > 0.1).

Dongiovanni P., Meroni M., Mancina R.M., Baselli G., Rametta R., Pelusi S., Männistö V., Fracanzani A.L., Badiali S., Miele L., Grimaudo S., Petta S., Bugianesi E., Soardo G., Fargion S., Pihlajamäki J., Romeo S, & Valenti L. (2018). Protein phosphatase 1 regulatory subunit 3B gene variation protects against hepatic fat accumulation and fibrosis in individuals at high risk of nonalcoholic fatty liver disease. Hepatology Communications, 2(6), 666-675.

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Evaluating NASH and NAFLD in Severely Obese Individuals

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We evaluated hepatic and adipose tissue samples of 119 unselected severely obese individuals, who underwent percutaneous liver biopsy performed during bariatric surgery, and had no history of at-risk alcohol intake (>30/20 g/day in males/females) or other liver diseases (Bariatric surgery cohort). NASH was diagnosed when steatosis, lobular inflammation and ballooning were concomitantly present. Informed written consent was obtained from each patient and the study protocol was approved by the Ethical Committee of the Fondazione IRCCS Ca’ Granda and conforms to the ethical guidelines of the 1975 Declaration of Helsinki. The clinical characteristics of these patients are listed in Table S1.
Serum free fatty acids (FFAs) were measured in 72 NAFLD patients, of whom samples were available (Hepatology service cohort). Clinical characteristics are listed in Table S2.
Human hepatocytes and hepatic stellate cells (hHSC) were isolated from non-neoplastic tissue obtained from explanted or resected livers of 17 patients, while peripheral blood lymphocytes and monocytes were isolated from 16 healthy subjects. The detailed isolation protocols, and enrolment criteria are described in the Supplementary Methods.
All patients were genotyped for PNPLA3 rs738409 (p.I148M) and MBOAT7 rs641738 by TaqMan 5′-nuclease assays (Life Technologies, Carlsbad, CA) [11 (link),22 (link)].

Meroni M., Dongiovanni P., Longo M., Carli F., Baselli G., Rametta R., Pelusi S., Badiali S., Maggioni M., Gaggini M., Fracanzani A.L., Romeo S., Gatti S., Davidson N.O., Gastaldelli A, & Valenti L. (2020). Mboat7 down-regulation by hyper-insulinemia induces fat accumulation in hepatocytes. EBioMedicine, 52, 102658.

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