Pre designed taqman assays

Manufactured by Thermo Fisher Scientific

Sourced in United States

Pre-designed Taqman assays are a set of optimized real-time PCR assays for the detection and quantification of specific gene targets. These assays are pre-formulated and pre-validated, providing a reliable and consistent solution for gene expression analysis.

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TaqMan pre-designed gene-expression assays TaqMan assays

5 protocols using pre designed taqman assays

Colon RNA Isolation and RT-qPCR

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For RNA isolation, 0.5 cm long colon pieces were mechanically disrupted in a homogenization solution containing 1-Thioglycerol (Promega, Madison, WI, USA) in gentleMACS tissue disrupter tubes (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer’s instructions. RNA was isolated using the Maxwell RSC simplyRNA Tissue Kit (Promega) according to the manufacturer’s instructions. RNA concentration was estimated by measuring absorbance at 260 and 280 nm. Complementary DNA (cDNA) was synthesized using the high-capacity cDNA reverse transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s instructions. RT-PCR was performed using FAST qPCR Master Mix and predesigned Taqman assays (Thermo Fisher Scientific) on a QuantStudio 6 system using QuantStudio software (Thermo Fisher Scientific). Mouse Actb was used as endogenous control, measurements were performed in triplicate, and relative expression levels were calculated according to the ΔΔCT method.

Hering L., Katkeviciute E., Schwarzfischer M., Niechcial A., Riggs J.B., Wawrzyniak M., Atrott K., van de Sande M., Lang S., Becher B., Rogler G., Scharl M, & Spalinger M.R. (2021). Macrophages Compensate for Loss of Protein Tyrosine Phosphatase N2 in Dendritic Cells to Protect from Elevated Colitis. International Journal of Molecular Sciences, 22(13), 6820.

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Digital PCR Analysis of POLR1D and ERBB2

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SCNAs of POLR1D and ERBB2 were analyzed using digital PCR (dPCR) and performed on the QuantStudio 3D platform (Life Technologies, Carlsbad, CA, USA). Pre-designed TaqMan assays specific for the detection of the copy number of POLR1D (Hs02926936_cn), ERBB2 (Hs00450668_cn), and a reference assay (TERT; 4403315) were purchased from Thermo Fisher. For dPCR, a total amount of 3–5 ng plasma DNA was used as input and samples were run in duplicate using the QuantStudio™ 3D Digital PCR 20 K Chip Kit v2 and a QuantStudio 3D instrument (Life Technologies, Carlsbad, CA, USA). Raw data were analyzed using the relative quantification module of the QuantStudio 3D Analysis Suite Software, including a Poisson correction. The confidence level was set to 95%, and the desired precision value was 10%.

Zhou Q., Perakis S.O., Ulz P., Mohan S., Riedl J.M., Talakic E., Lax S., Tötsch M., Hoefler G., Bauernhofer T., Pichler M., Gerger A., Geigl J.B., Heitzer E, & Speicher M.R. (2020). Cell-free DNA analysis reveals POLR1D-mediated resistance to bevacizumab in colorectal cancer. Genome Medicine, 12, 20.

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Quantitative RNA Expression Analysis

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The total RNA from biopsies that passed QC was transcribed to complementary DNA (cDNA) using a High-Capacity cDNA Archive RT kit (Applied Biosystems, Foster City, CA, USA) and was then used to perform real-time quantitative polymerase chain reaction (qPCR) in triplicate wells with a TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA) containing the probe of interest and β-actin (TaqMan primers and probes; Applied Biosystems). Predesigned TaqMan Assays (Applied Biosystems, Foster City, CA, USA) for 19 different genes that are primarily expressed by individual cell types in the intestine (Supplementary Table S2) were used. The PCRs were performed using an Applied Biosystems 7500 Fast Real-Time PCR detection system. Relative quantification (x) was calculated using the formula

x = 2^{- Δ C t} \times 1000

, where

Δ C t = C t_{t a r g e t g e n e} - C t_{β a c t i n}

Aguilar D., Revilla L., Garrido-Trigo A., Panés J., Lozano J.J., Planell N., Esteller M., Lacerda A.P., Guay H., Butler J., Davis J.W, & Salas A. (2021). Randomized Controlled Trial Substudy of Cell-specific Mechanisms of Janus Kinase 1 Inhibition With Upadacitinib in the Crohn’s Disease Intestinal Mucosa: Analysis From the CELEST Study. Inflammatory Bowel Diseases, 27(12), 1999-2009.

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Quantitative RNA Expression Analysis

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Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Equal amounts of RNA template were reverse transcribed using the Verso cDNA synthesis kit (Thermo Scientific). Differential mRNA expression of type 1 collagen (Col1a1), lysyl oxidase (Lox) and 18s was measured using TaqMan Mastermix and pre-designed Taqman assays (Applied Biosystems). Four μl of cDNA from tumor samples and independent passages of each cell line were run in triplicate on a Bio-Rad CFX96. Q-Gene software (28 (link)) was used to determine relative normalized expression to 18s. Data analysis was based on the Ct method.

Jolly L.A., Novitskiy S., Owens P., Massoll N., Cheng N., Fang W., Moses H.L, & Franco A.T. (2016). Fibroblast-mediated collagen remodeling within the tumor microenvironment facilitates progression of thyroid cancers driven by BrafV600E and Pten loss. Cancer research, 76(7), 1804-1813.

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Quantification of Mcsf and 18s mRNA

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Total RNA was extracted using the RNeasy Plus Mini Kit (Qiagen). Equal amounts of RNA template were reverse transcribed using the Verso cDNA synthesis kit (Thermo Scientific). Mcsf and 18s mRNA expression was measured using TaqMan Mastermix and pre-designed Taqman assays (Applied Biosystems). Four μl of cDNA from tumor samples and independent passages of each cell line were run in triplicate on a Bio-Rad CFX96. Q-Gene software [26 (link)] was used to determine relative normalized expression to 18s. Data analysis was based on the Ct method.

Jolly L.A., Massoll N, & Franco A.T. (2016). Immune Suppression Mediated by Myeloid and Lymphoid Derived Immune Cells in the Tumor Microenvironment Facilitates Progression of Thyroid Cancers Driven by HrasG12V and Pten Loss. Journal of clinical & cellular immunology, 7(5), 451.

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