Taqman gene expression probe

Manufactured by Thermo Fisher Scientific

Sourced in United States, United Kingdom

TaqMan gene expression probes are molecular biology reagents used to detect and quantify specific nucleic acid sequences in samples. They are designed to provide highly sensitive and specific detection of target genes during real-time PCR experiments.

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TaqMan probes (2822 citations) TaqMan Gene Expression Assays probes (18 citations) TaqMan Gene Expression Assay primers and probes (5 citations) TaqMan gene expression assay primer/probes (5 citations) Inventoried TaqMan Gene expression assay probes (3 citations) Taqman Gene Expression assay FAM/TAMRA probes (2 citations) TaqMan Gene Expression Assays primers and probes (2 citations) TaqMan gene expression probes and reagents (2 citations) Taqman gene expression probes for EHD1-4 (2 citations) Taqman probes gene expression assay (2 citations)

97 protocols using taqman gene expression probe

Profiling Colorectal Cancer Biomarkers

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The China Japan Union Hospital of Jilin University was the source of 30 paired samples (tumor samples from surgical resection and surrounding healthy tissues) collected retrospectively. Informed consent for using tissues for research was obtained from all enrolled patients. The Institutional Review Board of the China Japan Union Hospital approved the study. This study utilized samples from patients who did not display any co-morbid manifestations; tissues were subjected to snap freezing and liquid nitrogen storage. For the independent testing, paired samples were collected from 21 CRC patients undergoing surgical resection who did not have any comorbidities or did not undergo any neoadjuvant chemotherapy. Total RNA from the samples was isolated with TriZol as per instructions of the manufacturer (ThermoFisher Scientific, Shanghai, China). Quantitative RT-PCR was done using TaqMan miRNA or TaqMan gene expression probes (ThermoFisher Scientific) for FOSL2, miR-597-5p (Assay ID: 001551), and miR-143-5p (Assay ID: 002146) as described before (16 (link)). Internal controls were RNU6B (Assay ID: 001093) and ACTB (Assay ID: Hs03023943_g1) for data normalization for miRNA and mRNA expressions, respectively. –ΔΔCt method was utilized for data analysis that was expressed as mean ± standard deviation (SD).

Li S., Liu Z., Fang X.D., Wang X.Y, & Fei B.Y. (2019). MicroRNA (miR)-597-5p Inhibits Colon Cancer Cell Migration and Invasion by Targeting FOS-Like Antigen 2 (FOSL2). Frontiers in Oncology, 9, 495.

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RNA Extraction and qRT-PCR Analysis

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Dissected brain tissue was homogenized by sonication for 10sec in lysis buffer from Qiagen (Chatsworth, CA) RNeasy Mini kit. Tubes were spun briefly to pellet the mixture. The lysate was transferred to new 1.5-ml tubes. Total RNA was extracted by using the Qiagen RNeasy Mini kit following the manufacturer’s instructions including a DNase treatment. Quality and concentration of RNA was obtained using a Nanodrop and only samples with corresponding 260/280 and 260/230 ratios greater than 1.7 were further processed. Approximately, 200ng of isolated RNA was then reversed transcribed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Cheshire, UK). The target cDNA was amplified by polymerase chain reaction (PCR). All qPCR assays were carried out in triplicate using the Brilliant III Ultra-Fast QPCR Master Mix (Agilent Technologies; Santa Clara, CA, USA) and using TaqMan Gene Expression Probes (ThermoFisher Scientific). The assay identification of each gene was IL-1β (Rn99999009_m1) and GAPDH (Rn01775763_g1). The cDNA quantities were normalized by using the housekeeping gene GAPDH as a reference and the 2^−ΔΔct method was used to analyze relative gene expression as previously described (Porterfield et al., 2011 (link)).

Barnard D.F., Gabella K.M., Kulp A.C., Parker A.D., Dugan P.B, & Johnson J.D. (2019). Sex Differences in the Regulation of Brain IL-1β in Response to Chronic Stress. Psychoneuroendocrinology, 103, 203-211.

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Transcriptional Profiling of Cellular Pathways

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100 ng of total RNA was reverse-transcribed and amplified with SIAH1 (Hs02339360_m1)–, SMURF1 (Hs00410929_m1)–, SMURF2 (Hs00224203_m1)–, IL-6 (Hs00985639_m1)–, IL-8 (Hs00174103_m1)–, IFIT1 (Hs01911452_s1)–, IFIT2 (Hs00533665_m1)–, or beta-actin (Hs99999903_m1)–specific Taqman gene expression probes (Thermo Fisher) and Taqman RNA-to-CT 1-Step reagents (Applied Biosystems) on a StepOne Plus real-time PCR instrument (Applied Biosystems) based on the manufacturer’s recommendations. Relative transcript expression of the genes of interest was calculated using the ΔΔCt method (Schmittgen and Livak, 2008 (link)) with beta-actin serving as the endogenous control transcript.

Murphy Schafer A.R., Smith J.L., Pryke K.M., DeFilippis V.R, & Hirsch A.J. (2020). The E3 Ubiquitin Ligase SIAH1 Targets MyD88 for Proteasomal Degradation During Dengue Virus Infection. Frontiers in Microbiology, 11, 24.

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Quantification of Sirt Transcripts in Rat Hippocampus

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Total hippocampal RNA (1 μg) was extracted (Qiagen, United States), reverse transcribed (Applied Biosystems, United States), and relative mRNA levels were detected by quantitative PCR (StepOnePlus, Thermo Fisher, United States). Predesigned rat Sirt1, Sirt6, Sirt7, and β-actin TaqMan Gene Expression probes (Assay IDs: Rn01428096_m1, Rn01408249_m1, Rn01471420_m1, and Rn00667869_m1, respectively) were used with TaqMan Gene Expression Master Mix (Thermo Fisher, United States). Analyses were performed using the standard curve method with Sirt transcripts normalized to β-actin as the endogenous control.

Elamin M., Ruskin D.N., Masino S.A, & Sacchetti P. (2018). Ketogenic Diet Modulates NAD+-Dependent Enzymes and Reduces DNA Damage in Hippocampus. Frontiers in Cellular Neuroscience, 12, 263.

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RNA Isolation and qRT-PCR Analysis

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RNA was isolated using the ReliaPrep RNA Miniprep System (Promega, Wisconsin, USA) according to the manufacturer’s instructions. cDNA was synthesised from isolated RNA using Tetro cDNA Synthesis Kit (BioLine Meridian Bioscience, Ohio, USA) in accordance with the manufacturer’s instructions. qRT-PCR was performed using TaqMan gene expression probes (ThermoFisher) All target genes were normalised to 18S as a housekeeping gene.

Jamieson S., Mawdesley A., Deehan D., Kirby J., Holland J, & Tyson-Capper A. (2021). Inflammatory responses to metal oxide ceramic nanopowders. Scientific Reports, 11, 10531.

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Quantitative RNA Expression Analysis

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Frozen TRIzol samples were thawed, and RNA was extracted with chloroform, precipitated using isopropanol and 75% ethanol, and reconstituted in DNase/RNase-free water as previously described (49 (link)). cDNA was synthesized with random hexamers using an Omniscript RT kit (Qiagen). cDNA was quantified using TaqMan gene expression probes (ThermoFisher Scientific) and data collected using an Applied Biosystems 7500 real-time PCR instrument. The ΔΔC_T method was used to quantify relative numerical units (RNU), normalized to endogenous 18S RNA, relative to saline.

Piergallini T.J., Scordo J.M., Pino P.A., Schlesinger L.S., Torrelles J.B, & Turner J. (2021). Acute Inflammation Confers Enhanced Protection against Mycobacterium tuberculosis Infection in Mice. Microbiology Spectrum, 9(1), 10.1128/spectrum.00016-21.

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Quantification of IDO and TDO Gene Expression

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RNA was isolated using the Qiagen RNAeasy Plus RNA isolation kit (Qiagen, Venlo, the Netherlands) according to manufacturer’s instructions. cDNA was synthesized with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) using random hexamers. qRT-PCR for IDO was performed in 2 duplicated serial dilutions, on an ABI 7000 thermal cycler with SYBR Green PCR Mastermix (Eurogentec, Cologne, Germany). Primer sequences: IDO: fwd: 5′-GCTTTGCTCTACCACATCCAC-3′, rev: 5′-CAGGCGCTGTAACCTGTGT-3′, GAPDH: fwd: 5′-GCCTTCCGTGTTCCTACCC-3′. rev: 5′-CAGTGGGCCCTCAGATGC-3′. qRT-PCR for TDO was performed using TaqMan Gene Expression probes and TaqMan Fast Universal PCR Master Mix (ThermoFisher Scientific, Waltham, MA, USA), TDO2: Mm00451269_m1, GAPDH: Mm99999915_g1, detector: FAM. Data were evaluated with AB 7000 System SDS software. All results were normalized to GAPDH.

Lanz T.V., Williams S.K., Stojic A., Iwantscheff S., Sonner J.K., Grabitz C., Becker S., Böhler L.I., Mohapatra S.R., Sahm F., Küblbeck G., Nakamura T., Funakoshi H., Opitz C.A., Wick W., Diem R, & Platten M. (2017). Tryptophan-2,3-Dioxygenase (TDO) deficiency is associated with subclinical neuroprotection in a mouse model of multiple sclerosis. Scientific Reports, 7, 41271.

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Quantification of Aire Expression in Aged Murine B Cells

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Splenic B cells from young (5 weeks) or aged (12 months) Adig mice were magnetically enriched using a mouse B cell Isolation Kit with a QuadroMACS separator and LS columns according to the manufacturer’s instructions. Enriched B cells were suspended in DAPI in FACS buffer and sorted on Aire-GFP⁻ viable singlets. Subsequently, 2 × 10⁵ sorted B cells were cultured for 3 days with or without agonistic anti-CD40 monoclonal antibody (FGK45; Bio X Cell) (10 μg/mL) and recombinant mouse IL-4 (PeproTech) (5 ng/mL) in flat-bottom 96-well plates. For qRT-PCR, RNA extraction, cDNA synthesis, and qPCR were performed on 2 × 10⁵ cells using Taqman Gene Expression Probes (Thermo Fisher Scientific) for Aire and Hprt and gene expression values normalized as previously described.
For flow cytometry analysis, 6 × 10⁵ to 1 × 10⁶ stimulated B cells were stained with CD19-A594 (6D5; BioLegend) and suspended in DAPI in FACS buffer. Aire-GFP expression was analyzed on CD19⁺ viable singlets using a BD LSRII flow cytometer with BD FACSDiva and FlowJo^® flow cytometry analysis software.

Cepeda S., Cantu C., Orozco S., Xiao Y., Brown Z., Semwal M.K., Venables T., Anderson M.S, & Griffith A.V. (2018). Age-Associated Decline in Thymic B Cell Expression of Aire and Aire-Dependent Self-Antigens. Cell reports, 22(5), 1276-1287.

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Gene Expression Validation by qPCR

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The remaining cDNA generated from the sorted cells was used for transcript-level validation. qPCR was performed using the TaqMan Universal Master Mix II, with UNG (4440038) and TaqMan gene expression probes (4331182) on a Quantstudio 3 Real-Time PCR system (A28137; all products from Thermo Fisher Scientific). Samples were run in technical duplicate, using 1 ng as the input amount, and analysed using the equation: 2^{Cq(mean (control))−Cq(sample)}.
The probes used were: Bst2 (mm1609165_g1), C5ar1 (mm00500292_s1), Cd44 (mm01277161_m1), Fas (mm01204974_m1), Lair1 (mm00618113_m1), Mertk (mm00434920_m1), Milr1 (mm01242703_m1), P2ry12 (mm01950543_s1), Siglech (mm00618627_m1), Slamf1 (mm00443317_m1).

Bell O.H., Copland D.A., Ward A., Nicholson L.B., Lange C.A., Chu C.J, & Dick A.D. (2020). Single Eye mRNA-Seq Reveals Normalisation of the Retinal Microglial Transcriptome Following Acute Inflammation. Frontiers in Immunology, 10, 3033.

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Quantitative PCR for Gene Expression

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Snap-frozen skin tissue or purified cells were homogenized in Trizol and RNA was extracted using a Direct-zol RNA Miniprep kit (Zymo Research) following manufacturer’s instructions. mRNA was reverse-transcribed into cDNA using a Superscript III kit (Invitrogen) followed by RNase digestion. Quantitative PCR (qPCR) was performed using an EagleTaq mastermix (Roche) and Taqman gene expression probes (Thermo Fisher) corresponding to each gene of interest on a StepOnePlus RT-PCR system (Applied Biosystems). Gene expression was normalized to Actb using the ΔCt method. The same samples were measured for multiple mRNAs. RT-PCR of Mrgpra2 and Gapdh was done using primers listed in the Key Reagent Table.

Dong X., Limjunyawong N., Sypek E.I., Wang G., Ortines R.V., Youn C., Alphonse M.P., Dikeman D., Wang Y., Lay M., Kothari R., Vasavda C., Pundir P., Goff L., Miller L.S., Lu W., Garza L.A., Kim B.S., Archer N.K, & Dong X. (2022). Keratinocyte-derived defensins activate neutrophil-specific receptors Mrgpra2a/b to prevent skin dysbiosis and bacterial infection. Immunity.

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