Taqman assays

Manufactured by Integrated DNA Technologies

Sourced in United States

TaqMan assays are real-time PCR detection reagents designed for sensitive and specific detection of target DNA sequences. They utilize a fluorogenic probe to enable quantitative measurements of target nucleic acids.

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Lab products found in correlation

Iscript cdna synthesis kit, by Bio-Rad (2 mentions) Quantstudio 3, by Thermo Fisher Scientific (2 mentions) Quick rna miniprep kit, by Zymo Research (2 mentions) Tri reagent, by Merck Group (2 mentions) Steponeplus real time pcr system, by Thermo Fisher Scientific (1 mentions) Iscript kit, by Bio-Rad (1 mentions) Rneasy plus kit, by Qiagen (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) Itaq universal probes one step kit, by Bio-Rad (1 mentions) Superscript 2, by Thermo Fisher Scientific (1 mentions) Steponeplus platform, by Thermo Fisher Scientific (1 mentions) Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) Mirvana kit, by Thermo Fisher Scientific (1 mentions) Sybr green reagent, by Thermo Fisher Scientific (1 mentions) Mirneasy column, by Qiagen (1 mentions) Quantitect rt kit, by Qiagen (1 mentions)

Close spelling variants of this product

TaqMan gene expression assays (8 citations) Real time Taqman qPCR assays (2 citations) TaqMan Gene Expression Assay (Hs00226845_m1) (2 citations) RT-qPCR TaqMan assay (2 citations)

9 protocols using taqman assays

Quantitative RT-PCR for Proteasome Genes

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Total RNA was isolated from 1 × 10⁶ AT2 cells using the Quick-RNA miniprep kit (Zymo Research R1054), and cDNA was synthesized using iScript cDNA Synthesis Kit (BioRad). Quantitative RT-PCR was performed with 25 ng of cDNA per reaction on the StepOne Plus Real Time PCR System (Thermo Fisher Scientific) or QuantStudio 3 (Thermo Fisher Scientific) with Taqman assays (Integrated DNA Technologies) for mouse Psmc4 (RPT3; exons 3–4, Mm.PT.58.7882488 and exons 9–11, Mm.PT.58.12375476), Psma5 (Mm.PT.58.30006634), Psmb5 (Mm.PT.58.6715890), Psmc3 (Mm.PT.58.43458789), Psmd14 (Mm.PT.58.29611235), Sqstm1 (Mm.PT.58.5854953) and Becn1 (Mm.PT.58.31747082). Data were normalized to mouse Actin (Actb, Applied Biosystems, 4352933E).

Sitaraman S., Na C.L., Yang L., Filuta A., Bridges J.P, & Weaver T.E. (2019). Proteasome dysfunction in alveolar type 2 epithelial cells is associated with acute respiratory distress syndrome. Scientific Reports, 9, 12509.

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Quantification of SUPT3H and RUNX2 Expression

Cited 1 time

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Gene expression was quantified using TaqMan chemistry. Pre-designed TaqMan assays (Integrated DNA Technologies) were used to measure the expression of the target genes SUPT3H (Hs.PT.58.3510503) and RUNX2. For RUNX2, two assays were used: one targeting exons 6–7, measuring both the isoforms of the gene (Hs.PT.56a.19568141, termed RUNX2 ALL) and one targeting exons 1–2, therefore only detecting the P1 isoform (Hs.PT.56a.23056352.g). It was not possible to measure the expression of the P2 isoform alone due to the sequence identity between the short isoform and exons 3–8. Expression was assessed relative to the expression of three housekeeping genes: 18S, GAPDH and HPRT1 using the 2^-Δct method as previously described (9 (link)).

Rice S.J., Aubourg G., Sorial A.K., Almarza D., Tselepi M., Deehan D.J., Reynard L.N, & Loughlin J. (2018). Identification of a novel, methylation-dependent, RUNX2 regulatory region associated with osteoarthritis risk. Human Molecular Genetics, 27(19), 3464-3474.

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Quantifying Gene Expression in Tao Drosophila

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Crosses to obtain Tao¹⁶ males and Tao¹⁶/w¹¹¹⁸ females were conducted as described above. Each genotype was dissected in Schneider’s media supplemented with 10% FBS, to obtain 20–50 third instar CNSs. These were homogenized in a 1 mL dounce homogenizer using the RLT-plus buffer from a RNeasy plus kit (Qiagen). Further purification of total RNA followed the manufacturer’s instructions. cDNA was synthesized using the iScript kit (Bio-Rad) and used between 115–250 ng total RNA (varied by experimental repeat but was matched between samples). qRT-PCR was performed using custom-designed Taqman assays (Integrated DNA Technologies) in which the forward primer sequence spans an exon-exon junction. These were utilized in a StepOnePlus Real-Time PCR system (Applied Scientific/Thermo Scientific). Rps17 was used as the reference gene for the delta delta Ct comparison protocol to quantify relative changes in gene expression, all in technical triplicate. No-reverse transcriptase controls were conducted to confirm the purity of each cDNA sample, and lack of gDNA contamination in the qRT-PCR. Four independent biological repeats were performed.

Politano S.F., Salemme R.R., Ashley J., Lopez-Rivera J.A., Bakula T.A., Puhalla K.A., Quinn J.P., Juszczak M.J., Phillip L.K., Carrillo R.A, & Vanderzalm P.J. (2019). Tao negatively regulates BMP signaling during neuromuscular junction development in Drosophila. Developmental neurobiology, 79(4), 335-349.

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RNA Extraction and Real-Time PCR Analysis

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For mRNA expression analysis, total RNA was extracted from cells using an RNeasy mini kit (Qiagen, #74136). Real time PCR was then performed using iTaq Universal Probes One-Step Kit (Biorad, #172-5140) according to manufacturer’s instruction. Expression was normalized to human 18S. The following predesigned labeled primers and probe sets (Taqman assays) from Integrated DNA Technologies (IDT) were used: h18S #HS.PT.39a.22214856 g, hMYC #Hs.PT.58.26770695, hAPEX1 #Hs.PT.56a.3182919, hCDC25A #Hs.PT.58.800341, hCAD #Hs.PT.56a.39091521.

Schukur L., Zimmermann T., Niewoehner O., Kerr G., Gleim S., Bauer-Probst B., Knapp B., Galli G.G., Liang X., Mendiola A., Reece-Hoyes J., Rapti M., Barbosa I., Reschke M., Radimerski T, & Thoma C.R. (2020). Identification of the HECT E3 ligase UBR5 as a regulator of MYC degradation using a CRISPR/Cas9 screen. Scientific Reports, 10, 20044.

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Quantitative Transcriptome Analysis by RT-qPCR

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Total RNA was isolated using TRI Reagent (Sigma-Aldrich, T9424) according to the manufacturer’s instructions. RNA concentration for each sample was determined with NanoDrop™ 2000 Spectrophotometer (Thermo Scientific) by measuring absorbance at 260 nm. RNA (2 µg) was reverse transcribed using Superscript II (Invitrogen, 18064014). For standard RT-PCR (Supplementary Table 2), products were separated on 1% agarose gels and visualized with DDGIT gel scanner (Li-Core). RT-qPCR reactions were performed using Takyon ROX Master Mix (Eurogentec UFRP5XC0501) and the StepOne Plus platform (Applied Biosystems). Target transcript levels (Supplementary Table 3) were normalized to MRPL19 and relative abundance was determined using the ΔΔCt method. Transcript-specific TaqMan assays were purchased from Integrated DNA Technologies.

Almanza A., Mnich K., Blomme A., Robinson C.M., Rodriguez-Blanco G., Kierszniowska S., McGrath E.P., Le Gallo M., Pilalis E., Swinnen J.V., Chatziioannou A., Chevet E., Gorman A.M, & Samali A. (2022). Regulated IRE1α-dependent decay (RIDD)-mediated reprograming of lipid metabolism in cancer. Nature Communications, 13, 2493.

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Quantitative Analysis of Lung Developmental Genes

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Total RNA was isolated from 1 × 10⁶ to 2 × 10⁶ AT2 cells using the Quick-RNA miniprep kit (Zymo Research, R1054), and cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, 1708891). qPCR was performed with 20 ng of cDNA per reaction on QuantStudio 3 (Thermo Fisher Scientific) with Taqman assays (Integrated DNA Technologies) for mouse Sftpc (IDT Mm.PT.58.9922071, exons 1–2), and Bax (IDT Mm.PT.58.14012210, exons 3–4). Multiple housekeeping genes were used to identify stable expression across developmental ages (P4 and P21), and data were normalized to mouse Ppia (IDT Mm.PT.39a.2.gs).

Sitaraman S., Martin E.P., Na C.L., Zhao S., Green J., Deshmukh H., Perl A.K., Bridges J.P., Xu Y, & Weaver T.E. (2021). Surfactant protein C mutation links postnatal type 2 cell dysfunction to adult disease. JCI Insight, 6(14), e142501.

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Transcriptional Profiling of Tendon Repair

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Total RNA was isolated from tendon repair tissues of the control diet group and the high-glucose diet group 2 (control group: n = 5/high-glucose group: n = 5) and 4 weeks (control group: n = 3/high-glucose group: n = 3) after surgery and of intact control tendons (n = 3) using TRI® Reagent (Sigma-Aldrich; Vienna, Austria) according to the manufacturer’s protocol (see also SI methods for details). qRT-PCR was performed as described in Lehner, et al.^{18 (link)}, using TaqMan® assays (Integrated DNA Technologies, Coralville, IA, USA) targeting Sox9 (SRY box 9), Col2a1 (collagen, type II, alpha 1), Acan (aggrecan), Comp (cartilage oligomeric matrix protein), Fabp2 (fatty acid binding protein 2), Pparγ (peroxisome proliferator-activated receptor γ), Runx2 (runt-related transcription factor 2), Col1a1 (collagen type 1 α1), Col3a1 (collagen type 3 α1), Scx (scleraxis), Mkx (mohawk), Tnmd (tenomodulin), Il1b (interleukin 1b), Il6 (interleukin 6), Il10 (interleukin 10) (see also SI methods for details).

Korntner S., Kunkel N., Lehner C., Gehwolf R., Wagner A., Augat P., Stephan D., Heu V., Bauer H.C., Traweger A, & Tempfer H. (2017). A high-glucose diet affects Achilles tendon healing in rats. Scientific Reports, 7, 780.

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Quantitative Analysis of RNA Expression

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RNA was extracted using miRNeasy columns (Qiagen, Valencia, CA) from mouse macrophages and mirVana kit (Ambion, Austin TX) from human monocytes, and cDNA synthesis was performed with 0.5–1.0 μg RNA using QuantiTect RT kit (Qiagen). Expression of genes, including lncRNAs E33 and MIR143HG (human equivalent of E33), was analyzed by quantitative real-time PCR using SYBR green reagents (Life Technologies, Carlsbad, CA) or TaqMan assays (Integrated DNA Technologies, Coralville, IA) on an ABI 7500 instrument using indicated gene primers (Supplementary Table 2). miRNA expression was determined by quantitative reverse transcriptase PCR (RT-QPCR) using miScript (Qiagen) or qScript (Quanta Biosciences, Gaithersburg, MD) kits. RT-QPCR data were analyzed by the 2^{^-ΔΔCt} method, normalized against internal controls (Actb or Ppia for mRNA and lncRNA, and U6 or 18S RNA for miRNA). Results were expressed as fold over control samples.

Reddy M.A., Chen Z., Park J.T., Wang M., Lanting L., Zhang Q., Bhatt K., Leung A., Wu X., Putta S., Sætrom P., Devaraj S, & Natarajan R. (2014). Regulation of Inflammatory Phenotype in Macrophages by a Diabetes-Induced Long Noncoding RNA. Diabetes, 63(12), 4249-4261.

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qPCR Quantification of RUNX2 Isoforms

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cDNA synthesis and quantitative PCR (qPCR) were performed as described previously [11 (link)]. Predesigned TaqMan assays (Integrated DNA Technologies) or assays designed using the Roche probe library system were used to quantify expression of the housekeeping genes HPRT1, 18S and GAPDH and 12 target genes. The relative gene expression was calculated by the 2^−ΔCt method, where ΔCt is the mean Ct value of the three housekeeping genes subtracted from the Ct value of the target gene of interest. For target gene RUNX2, two qPCR assays were used: one targeting exons 6–7, measuring both the main isoforms of the gene (termed RUNX2all), and one targeting exons 1–2, which measures the longer isoform of the gene (termed RUNX2long) [12 (link)].

Shepherd C., Reese A.E., Reynard L.N, & Loughlin J. (2019). Expression analysis of the osteoarthritis genetic susceptibility mapping to the matrix Gla protein gene MGP. Arthritis Research & Therapy, 21, 149.

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