Single tube taqman gene expression assays

Manufactured by Thermo Fisher Scientific

Sourced in United States

Single Tube TaqMan Gene Expression Assays are a type of real-time PCR assay designed to quantify gene expression levels. The assays are pre-designed and pre-optimized, containing all necessary components for gene expression analysis in a single tube format.

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Single Tube Custom TaqMan Gene Expression Assays Single-tube TaqMan assays TaqMan Gene Expression Assays TaqMan expression assays Taqman gene assays Gene Expression Assays TaqMan assays

13 protocols using single tube taqman gene expression assays

Cutaneous Gene Expression Analysis

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Total RNA was extracted from skin samples using the TRIzol Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer’s instructions. The quantity of RNA was verified on a NanoDrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). The quality of RNA was analyzed by performing agarose gel electrophoresis. The cDNA was synthesized from 500 ng of total RNA using a High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Thermo Fisher Scientific, Waltham, MA, USA). To measure the mRNA expression of Hprt1; mCherry; eGFP, Pparγ; Fapb4; Mest; Zfp 423; Igf2; Bmp2; Srebp1c; Fasn; Glut1; Glut4 and Atgl, Single Tube TaqMan^® Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) were used (Table S2). Hprt1 was chosen as the most stable housekeeping gene during cutaneous wound healing, after an analysis described previously [28 (link)]. Amplification was performed using a 7900HT Fast Real-Time PCR System (Applied Biosystems by Thermo Fisher Scientific, Waltham, MA, USA) under the following conditions: initial denaturation for 10 min at 95 °C, followed by 40 cycles of 15 sec at 95 °C and 1 min at 60 °C. Each run included a standard curve based on aliquots of pooled skin sample RNA. All samples were analyzed in duplicate. The mRNA expression levels were normalized to the reference gene Hprt1 and multiplied by 10.

Gawronska-Kozak B., Walendzik K., Machcinska S., Padzik A., Kopcewicz M, & Wiśniewska J. (2021). Dermal White Adipose Tissue (dWAT) Is Regulated by Foxn1 and Hif-1α during the Early Phase of Skin Wound Healing. International Journal of Molecular Sciences, 23(1), 257.

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Quantitative Real-Time PCR for Gene Expression Analysis

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Quantitative real-time PCR was performed according to the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA, USA) according to the Applied Biosystems StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). included The following primers were used: The housekeeping genes GAPDH (Mm99999915_g1) and GUSB (Mm00446953_m1) as well as TGFβ (Mm01178820_m1), HGF (Hs04329698_m1), CCL17 (Mm01244826_g1), IFNγ (Hs00989291_m1) and TNFα (HS01113624_g1) (Single Tube TaqMan Gene Expression Assays, Thermo Fisher Scientific, Waltham, MA, USA). The analysis was performed using StepOnePlus™ Software v2.3.
The mean cycle threshold (CT) value was calculated for the housekeeping genes. Relative expression values for the analyzed genes were then calculated as the difference between the mean cycle threshold (CT) of the housekeeping genes and the analyzed gene (delta CT) and depicted as the logarithmic value.

Jodeleit H., Palamides P., Beigel F., Mueller T., Wolf E., Siebeck M, & Gropp R. (2017). Design and validation of a disease network of inflammatory processes in the NSG-UC mouse model. Journal of Translational Medicine, 15, 265.

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Quantitative RT-PCR Analysis of Gene Expression

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According to the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA) quantitative RT-PCR was performed using the Applied Biosystems StepOnePlus RT-PCR system (Thermo Fisher Scientific, Waltham, MA). Single-tube TaqMan gene expression assays (Thermo Fisher Scientific, Waltham, MA) included the housekeeping genes GAPDH (Mm99999915_g1) and GUSB (Mm00446953_m1), as well as TGFβ (Mm 01178820_m1), HGF (Hs04329698_m1), CCL17 (Mm01244826_g1), IFNγ (HS00989291_m1) and TNFα (HS01113624_g1) (Thermo Fisher Scientific assay IDs are given in brackets). Analysis was performed using StepOnePlus™ Software v2.3. For expression analysis, a mean value of cycle threshold values was calculated for two housekeeping genes. Relative expression values for the respective analyzed genes were calculated as the difference between the mean cycle threshold (CT) value of the housekeeping genes and the respective analyzed gene (delta CT). Relative expression is depicted as the logarithmic value of the delta CT.

Palamides P., Jodeleit H., Föhlinger M., Beigel F., Herbach N., Mueller T., Wolf E., Siebeck M, & Gropp R. (2016). A mouse model for ulcerative colitis based on NOD-scid IL2R γnull mice reconstituted with peripheral blood mononuclear cells from affected individuals. Disease Models & Mechanisms, 9(9), 985-997.

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Quantitative RT-PCR Analysis of Gene Expression

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Quantitative real-time polymerase chain reaction (RT-PCR) was performed according to the TaqMan Fast Advanced Master Mix protocol (Thermo Fisher Scientific, Waltham, MA, USA) using the Applied Biosystems StepOnePlus real-time PCR system (Thermo Fisher Scientific, Waltham, MA, USA). Single Tube TaqMan Gene Expression Assays (Thermo Fisher Scientific, Waltham, MA, USA) included primers for the housekeeping genes GAPDH (Mm99999915_g1), GUSB (Mm00446953_m1), and TGFβ (Mm01178820_m1), HGF (Hs04329698_m1), CCL17 (Mm01244826_g1), and IFNγ (Hs00989291_m1). Analysis was performed using StepOnePlus Software v2.3.
The mean cycle threshold (CT) value was calculated for the housekeeping genes. Relative expression values for the analyzed genes were calculated as the difference between the mean cycle threshold (CT) of the housekeeping genes and the analyzed gene (delta CT) and depicted as the logarithmic value.

Al-amodi O., Jodeleit H., Beigel F., Wolf E., Siebeck M, & Gropp R. (2018). CD1a-Expressing Monocytes as Mediators of Inflammation in Ulcerative Colitis. Inflammatory Bowel Diseases, 24(6), 1225-1236.

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Quantifying Immune Signaling Genes

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To measure the levels of ISGs (MX1, OAS1, IRF7, ISG15, IFIT1), MTs (MT2A), HMOX1 and HIV RNA with HPRT1 as an internal control, THP-1 cells and primary cells were harvested for RNA extraction and making of cDNA using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). Real-time PCR was performed using the following primers and probes:
Single Tube TaqMan Gene Expression Assays (Thermo Fisher Scientific): human HPRT1 (Hs01003267_m1), MX1 (Hs00895608_m1), IRF7 (Hs01014809_g1), OAS1 (Hs00973635_m1). Relative mRNA expression was calculated by normalizing each gene to HPRT1 mRNA expression.

Tomer S., Mu W., Suryawanshi G., Ng H., Wang L., Wennerberg W., Rezek V., Martin H., Chen I., Kitchen S, & Zhen A. (2022). Cannabidiol modulates expression of type I IFN response genes and HIV infection in macrophages. Frontiers in Immunology, 13, 926696.

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Quantitative RT-PCR of Gastric Tissue

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The gastric corpus was removed and freeze clamped in liquid nitrogen followed by storage at −80°C. Prior to use, samples were weighed and crushed via mortar and pestle. Total RNA was extracted from the samples by homogenizing in QIAzol Lysis Reagent (Qiagen, Hilden, Germany). Total RNA was purified using the RNeasy Lipid Tissue Mini Kit (Qiagen) with DNase treatment followed by spectrophotometry with Nanodrop 1000 (Wilmington, DE). 1μg of RNA was quantitatively converted into cDNA (High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor, Applied Biosystems, Foster City, CA) using a Veriti 96-well thermal cycler (Applied Biosystems). cDNA was prepared for qPCR using TaqMan Universal Master Mix II and single-tube Taqman Gene Expression Assays (ThermoFisher, Leesport, PA) and cycled in a QuantStudio 12k flex thermal cycler (Applied Biosystems) according to the manufacturers instructions.
Samples were prepared in triplicate. Any sample that did not superimpose upon the triplicate was excluded from analysis. Data were normalized to α-actin (Actb) and expressed as fold-change. Data were analyzed using QuantStudio 12k Flex software (Applied Biosystems) and GraphPad Prism 6 (La Jolla, CA).

McMenamin C.A., Clyburn C, & Browning K.N. (2018). High fat diet during the perinatal period induces loss of myenteric nitrergic neurons and increases enteric glial density, prior to the development of obesity. Neuroscience, 393, 369-380.

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Quantifying Skin Gene Expression

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Total RNA was extracted from skin samples using TRI Reagent (Sigma-Aldrich by Merck) according to the manufacturer’s instructions. Quantity and quality of RNA was verified on NanoDrop 1000 (Thermo Fisher Scientific) and through analysis of agarose gel after electrophoresis. cDNA was synthesized from 500 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems by Thermo Fisher Scientific). To measure the levels of Collagen 1, Collagen 3, Hprt-1, Mmp-9, Mmp-13, Tgfβ-1, Tgfβ-3 and Timp-1, mRNA expression, Single Tube TaqMan® Gene Expression Assays (Life Technologies Thermo Fisher Scientific) were used. Amplification was performed using 7900HT Fast Real-Time PCR System under conditions: initial denaturation for 10 min at 95°C, followed by 40 cycles of 15 sec at 95°C and 1 min at 60°C. Each run included standard curve based on aliquots of pooled skin RNA. All samples were analyzed in duplicates. Hprt-1 was chosen as the most stable housekeeping gene during cutaneous wound healing after analysis described previously [67 (link)]. mRNA expression levels were normalized to the reference gene Hprt-1 and multiplied by 10.

Kopcewicz M., Walendzik K., Bukowska J., Kur-Piotrowska A., Machcinska S., Gimble J.M, & Gawronska-Kozak B. (2020). Cutaneous wound healing in aged, high fat diet-induced obese female or male C57BL/6 mice. Aging (Albany NY), 12(8), 7066-7111.

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Real-time RT-qPCR for Adipogenic Gene Expression

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Real-time RT-qPCR was performed as described previously [11 (link)]. Total RNA was isolated using the TRIzol Reagent (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA). RNA (500 ng) was reverse transcribed with the High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems, Thermo Fisher Scientific) according to the manufacturer’s instructions. The mRNA levels of adipogenic-related genes were measured with Single Tube TaqMan Gene Expression Assays (Life Technologies, Thermo Fisher Scientific) (Supplementary Table S8). The levels of gene expression were quantified relative to the level of Hprt1.

Walendzik K., Bukowska J., Kopcewicz M., Machcinska S., Gimble J.M, & Gawronska-Kozak B. (2020). Age, Diet and Epidermal Signaling Modulate Dermal Fibroblasts’ Adipogenic Potential. International Journal of Molecular Sciences, 21(23), 8955.

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Quantitative RT-PCR Analysis of Endocytic Genes in Cancer

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Quantitative RT-PCR was performed on patient samples from the Irish PCRC cohort. RNA was extracted from non-malignant (n = 11), indolent (n = 5) and aggressive cancer tissues (n = 6). Reverse transcription of 500 ng of total RNA was performed using a high-capacity cDNA Reverse Transcription Kit (Life Technologies Pty. Ltd., Cat# 4368814). Real-time PCR was performed using 2 μL cDNA for each of the following TaqMan^® gene expression assays (Single Tube TaqMan^® Gene Expression Assays Life Technologies); APPL1 (Hs00179382_m1), RAB5A (Hs00991290_m1), EEA1 (Hs00929215_m1), PDCD6IP (Hs00994346_m1), NOX4 (Hs00418356_m1), SORT1 (Hs00361760_m1) and TaqMan^® Gene Expression Master Mix in a total volume of 10 μL. Three biological replicates were performed consisting of three technical replicates. Expression of each gene was normalized to an average of ALAS1 (Hs00963534_m1) and HPRT1 (Hs02800695_m1) reference genes and relative quantities calculated by ΔCt.

Johnson I.R., Parkinson-Lawrence E.J., Keegan H., Spillane C.D., Barry-O'Crowley J., Watson W.R., Selemidis S., Butler L.M., O'Leary J.J, & Brooks D.A. (2015). Endosomal gene expression: a new indicator for prostate cancer patient prognosis?. Oncotarget, 6(35), 37919-37929.

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Real-time RT-qPCR for Gene Expression

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Real-time RT-qPCR was performed as previously described⁹. Briefly, total RNA was extracted from cell culture using TRI Reagent (Sigma-Aldrich). cDNA was synthesized from 500 ng of total RNA using High-Capacity cDNA Reverse Transcription Kit with RNase Inhibitor (Applied Biosystems). mRNA levels of Foxn1, Mmp-9, hypoxanthine phosphoribosyltransferase 1 (Hprt1), and TATA-binding protein (Tbp) were measured with Single-Tube TaqMan® Gene Expression Assays (Life Technologies). The levels of gene expression were quantified relative to the level of Hprt1 or Tbp using the standard curve method.

Kur-Piotrowska A., Bukowska J., Kopcewicz M.M., Dietrich M., Nynca J., Slowinska M, & Gawronska-Kozak B. (2018). Foxn1 expression in keratinocytes is stimulated by hypoxia: further evidence of its role in skin wound healing. Scientific Reports, 8, 5425.

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