Taqman expression assay

Manufactured by Thermo Fisher Scientific

Sourced in United States, France

TaqMan expression assays are real-time PCR assays designed to quantify the expression of specific genes. They utilize TaqMan probes to detect and measure target gene expression levels in samples.

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TaqMan assays (1810 citations) TaqMan human gene expression assays (13 citations) FAM-labeled TaqMan Gene Expression Assays (11 citations) FAM-tagged TaqMan™ Gene Expression Assays (5 citations) TaqMan Gene Expression Assays (7900 real-time PCR system; (2 citations) TaqMan Gene Expression Assays Hs03063375_ft (2 citations) Taqman 20x gene expression assays for IL-1β (Hs01555410_m1) (2 citations) TaqMan® Gene Expression Assays for ACVR2B and GAPDH (2 citations) Custom Plus TaqMan Gene Expression Assays (2 citations) TaqMan® Gene Expression Assays and Universal PCR Master Mix (2 citations)

54 protocols using taqman expression assay

Reverse Transcription and qPCR Assays

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Reverse transcription of mRNA from 143B and DAN non-transfected and transfected cells was carried out in 100 µl final volume from 400 ng total RNA using High Capacity cDNA Archive kit (Applied Biosystems) according to the manufacturer’s instructions.
Real-time PCR was performed using Viia7 sequence detection system (Applied Biosystems) according to the manufacturer’s protocol. CDKN1A gene was quantified by ΔCt method using TaqMan Expression Assays (Hs00355782_m1 for human and canine cell line) (Applied Biosystems) and normalised to a housekeeping ACTB (Hs99999903_m1 for human cell line and cf 03023880_g1 for canine cell line) (TaqMan Expression Assays; Applied Biosystems).

Leonardi L., Benassi M.S., Pollino S., Locaputo C, & Pazzaglia L. (2020). miR-106B-25 Cluster expression: a comparative human and canine osteosarcoma study. Veterinary Record Open, 7(1), e000379.

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RT-qPCR Analysis of Immune Genes

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Late third-instar larvae were washed and dipped in 70% ethanol followed by rinsing with water. Total RNA extraction was performed as previously described (Arefin et al. 2014 (link)). RNA quality and concentration was determined with NanoDrop 2000 spectrophometer (Thermo Scientific). A total of 1000 ng of total RNA was used to convert cDNA using SuperScript III Reverse Transcriptase (Invitrogen) and oligo(dT) (20-mer). RT-qPCR was performed using KAPA PROBE FAST Universal qPCR Master Mix (Kapa Biosystems) and TaqMan Expression Assays (Applied Biosystems) for Drs and Cecropin A1. Diptericin A1 was synthesized by the custom TaqMan Expression Assays (Applied Biosystems) (Arefin et al. 2015 (link); Dantoft et al. 2013 (link)). A Rotor-gene Q machine (Qiagen) was used to run qPCR. Each sample was run in triplicate. Relative mRNA expression levels were was analyzed by normalizing to the reference gene rpl32 and compared to the control genotype. The data were then transformed to Log2 fold changes and presented as mean ± SDs of the mean of at least three independent biological replicates. In the uninfected wounded situation, two independent biological samples were analyzed and both show a similar pattern.

Arefin B., Kunc M., Krautz R, & Theopold U. (2017). The Immune Phenotype of Three Drosophila Leukemia Models. G3: Genes|Genomes|Genetics, 7(7), 2139-2149.

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Cytokine-Induced SOCS3 Upregulation

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To assay SOCS3 up-regulation in response to cytokine stimulation, naive CD4 T cells were stimulated with or without IL-6 (100 ng/ml; Peprotech), IL-21 (100 ng/ml; Peprotech), IL-23 (100 ng/ml; R&D Systems), or IL-27 (50 ng/ml; R&D Systems) for 18 h. Cells were then washed and pelleted. Total RNA was extracted from PBMCs with the RNeasy Mini Kit (Qiagen). For real-time PCR, 1 µg of total RNA was reverse transcribed (Invitrogen), and the resulting cDNA was amplified by means of PCR with the ABI 7500 Sequencer and TaqMan expression assays (Applied Biosystems). 18S was used as a normalization control. The data were analyzed with the 2^−ΔΔCT method, and results are expressed as mean fold induction.

Spencer S., Köstel Bal S., Egner W., Lango Allen H., Raza S.I., Ma C.A., Gürel M., Zhang Y., Sun G., Sabroe R.A., Greene D., Rae W., Shahin T., Kania K., Ardy R.C., Thian M., Staples E., Pecchia-Bekkum A., Worrall W.P., Stephens J., Brown M., Tuna S., York M., Shackley F., Kerrin D., Sargur R., Condliffe A., Tipu H.N., Kuehn H.S., Rosenzweig S.D., Turro E., Tavaré S., Thrasher A.J., Jodrell D.I., Smith K.G., Boztug K., Milner J.D, & Thaventhiran J.E. (2019). Loss of the interleukin-6 receptor causes immunodeficiency, atopy, and abnormal inflammatory responses. The Journal of Experimental Medicine, 216(9), 1986-1998.

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Validation of MDS Gene Expression

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Real-time quantitative PCR (RT-qPCR) was used to validate the microarray data of the selected genes (LEF1 and DEFA3) in a large cohort of 82 MDS patients (non-overloaded patients: n = 20; iron-overloaded patients: n = 28; and iron-chelated patients: n = 34). RT-qPCR was performed on a StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) using TaqMan Expression assays (Applied Biosystems) according to the manufacturer’s recommendation. The relative gene expression was calculated based on the 2−ΔΔCT method using the B2M gene for normalization.

Votavova H., Urbanova Z., Kundrat D., Dostalova Merkerova M., Vostry M., Hruba M., Cermak J, & Belickova M. (2021). Modulation of the Immune Response by Deferasirox in Myelodysplastic Syndrome Patients. Pharmaceuticals, 14(1), 41.

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Quantitative Analysis of Gastric Cancer Biomarkers

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RNA samples from three GBC cell lines (SNU308, GBD1 and G415) and from four GBC samples (GB82, GB95, GB126 and GB127) were assessed for ESR1, P16, PGP9.5, APC, SSBP2 and GAPDH expression levels using quantitative real-time reverse transcription (qRT-PCR). Reverse transcription was performed with random hexamer primers and Superscript II Reverse Transcriptase (Invitrogen) according to manufacturer’s instructions. qRT-PCR was then carried out on the Applied Biosystems 7900HT Sequence Detection Instrument using TaqMan expression assays (Applied Biosystems). The 2^−ΔΔCt method was used to quantify relative gene expression [38 ].

Kagohara L.T., Schussel J.L., Subbannayya T., Sahasrabuddhe N., Lebron C., Brait M., Maldonado L., Valle B.L., Pirini F., Jahuira M., Lopez J., Letelier P., Brebi-Mieville P., Ili C., Pandey A., Chatterjee A., Sidransky D, & Guerrero-Preston R. (2014). Global and gene-specific DNA methylation pattern discriminates cholecystitis from gallbladder cancer patients in Chile. Future oncology (London, England), 11(2), 233-249.

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Quantitative Real-Time RT-PCR Analysis of Inflammation Markers

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Gene expression levels was measured by quantitative real-time reverse transcriptase polymerase chain reaction (RT-PCR) as previously described [76 (link)]. In short, the acute phase response was assessed by the measurement of Saa3, Saa1 and lipocalin 2 (Lcn2) mRNA expression levels in lung and/or liver tissue. Inflammation was assessed by the measurement of interleukin-6 (Il-6), tumor necrosis factor (Tnf), chemokine ligand 2 (Cxcl2) and chemokine ligand 5 (Cxcl5) mRNA expression levels in lung tissue. Total RNA was isolated using Maxwell® 16 LEV simply RNA Tissue Kit (AS1280, Promega, USA) according to the manufacturer protocol. Complementary DNA (cDNA) was prepared using TaqMan® reverse transcription reagents (Applied Biosystems, USA) according to the manufacturer protocol. Total RNA and cDNA concentrations were measured on NanoDrop 2000c (ThermoFisher, USA). The gene expression levels was determined using predesigned TaqMan expression assays and 18S rRNA as endogenous control (Applied Biosystems, USA). The samples were run in triplicates using ViiA7 Real-Time PCR detector (Applied Biosystems, USA). Negative controls were included in each run of the analysis. The relative expression of the target gene was measured by the comparative C_T method after thoroughly assay optimization and validation.

Danielsen P.H., Bendtsen K.M., Knudsen K.B., Poulsen S.S., Stoeger T, & Vogel U. (2021). Nanomaterial- and shape-dependency of TLR2 and TLR4 mediated signaling following pulmonary exposure to carbonaceous nanomaterials in mice. Particle and Fibre Toxicology, 18, 40.

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Gene Expression Analysis in Cell Lines

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After drug exposure RNA was extracted from cell lines using TRIzol® (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. First strand synthesis was performed using the high-capacity cDNA Reverse Transcription Kit from Applied Biosystems (Foster City, CA, USA). Expression of target genes (Ki67, CDKN1A, PARP1, APC, RARβ2, GSTP1, CCND2, DNMT1, DNMT3a and DNMT3B) was quantified using Taqman expression assays, purchased as pre-developed assays from Applied Biosystems and normalized to the expression of the GUSB housekeeping gene.

Graça I., Sousa E.J., Costa-Pinheiro P., Vieira F.Q., Torres-Ferreira J., Martins M.G., Henrique R, & Jerónimo C. (2014). Anti-neoplastic properties of hydralazine in prostate cancer. Oncotarget, 5(15), 5950-5964.

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Quantitative Real-Time PCR Analysis

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Total RNA was extracted with Trizol and reverse transcribed using the Maxima First Strand cDNA Synthesis Kit kit (Thermo Scientific, Waltham, MA). qRT-PCR was performed on a StepOne Plus apparatus using the Absolute Blue qPCR Rox Mix (Thermo) and TaqMan expression assays (Applied Biosystems, Carlsbad, CA) or the SYBR Green mix (Applied Biosystems) and custom-designed primers (sequences available upon request). Each sample was run in triplicate and 18S was used to control for input RNA. Data analysis was based on the Ct method, and experiments were repeated at least three times using at least two independent organ pools (at least five mice/pool).

Champa D., Russo M.A., Liao X.H., Refetoff S., Ghossein R.A, & Di Cristofano A. (2014). Obatoclax overcomes resistance to cell death in aggressive thyroid carcinomas by countering Bcl2a1 and Mcl1 overexpression. Endocrine-related cancer, 21(5), 755-767.

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Gene Expression and Cytokine Profiling in Activated PBMCs

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U3C transfected cells and frozen stored PBMCs from the patient and healthy donors were used. Gene expression was assayed in cells left unstimulated or stimulated with IFN-γ (1,000 IU/ml) for 6 h by qRT-PCR using Taqman expression assays (Applied Biosystems). GAPDH was used as normalization control. Evaluation of cytokine release was performed in cells stimulated with IFN-γ+LPS (200ng/ml) for 24 h. Culture supernatants were assayed further using a custom bead based cytokine assay (Bio-Plex, BioRad, Hercules, CA).

Sampaio E.P., Ding L., Rose S.R., Cruz P., Hsu A.P., Kashyap A., Rosen L.B., Smelkinson M., Tavella T.A., Ferre E.M., Wierman M.K., Zerbe C.S., Lionakis M.S, & Holland S.M. (2017). NOVEL STAT1 MUTATION DISRUPTS SMALL UBIQUITIN-RELATED MODIFIER (SUMO) CONJUGATION CAUSING GAIN OF FUNCTION. The Journal of allergy and clinical immunology, 141(5), 1844-1853.e2.

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Quantitative Analysis of Transporter Genes

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RNA was isolated from cell lines using Trizol (Invitrogen) chloroform (Sigma-Aldrich) extraction. cDNA was generated from 1 µg of RNA using the SuperScript IV First-Strand Synthesis System (Invitrogen) according to the manufacturer’s instructions. PCR with reverse transcription was performed using TaqMan master mix (Life Technologies) at 2× dilution; TaqMan expression assays (Applied Biosystems) for OCTN1 (Hs00268200_m1; FAM), ENT1 (Hs01085704_g1; FAM), STA5A (Hs00559643_m1; FAM) and GAPDH (Hs02758991_g1; VIC) were used. A QuantStudio 3 system (Life Technologies) was used to perform quantitative PCR. Analyses were performed in technical triplicates using the delta Ct method.

Drenberg C.D., Shelat A., Dang J., Cotton A., Orwick S.J., Li M., Jeon J.Y., Fu Q., Buelow D.R., Pioso M., Hu S., Inaba H., Ribeiro R.C., Rubnitz J.E., Gruber T.A., Guy R.K, & Baker S.D. (2019). A high-throughput screen indicates gemcitabine and JAK inhibitors may be useful for treating pediatric AML. Nature Communications, 10, 2189.

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