Taqman gene expression assay kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Japan, Canada

The TaqMan Gene Expression Assay kits are a set of reagents designed for real-time quantitative PCR (RT-qPCR) analysis of gene expression. The kits include pre-designed and validated gene-specific primers and TaqMan probes to enable accurate and sensitive detection and quantification of target gene transcripts.

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TaqMan assays (1810 citations) Gene Expression Assays (111 citations) TaqMan expression assays (54 citations) TaqMan Gene Expression Assays kit (20 citations) TaqMan kits (18 citations)

191 protocols using taqman gene expression assay kit

Quantitative Analysis of Brain Injury Biomarkers in Macaques

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2x Fast Taqman Universal PCR Master Mix, No AmpErase UNG and Taqman Gene Expression Assay kits (Invitrogen) were used for semi-quantitative analysis of cytokines, chemokines, proteases, and traumatic brain injury markers (MMP-9, GFAP, LIF, IL-1β, MCP-1, IP-10, IFNγ. IL-6, IL-8) within whole brain tissues, following manufacturer’s instructions. Probes were labeled at the 5’ end with 6-FAM and quenched with BHQ1, with a modified 3’ end to prevent probe extension by Taq polymerase. Thermocycling (QuantStudio 6 Real-Time PCR Flex System; Applied Biosystems) parameters comprised the following: hold step, 50°C for 120 seconds; initial denaturing, 95°C for 120 seconds; and cycling PCR amplification, 95°C for 1 second and 60°C for 20 seconds (40 cycles). All kits were targeted to cynomolgus macaque-specific, highly-conserved amplicons that spanned one or more exons to guarantee desired mRNA transcript specificity. Endogenous controls comprised Taqman Gene Expression Assay kits (Invitrogen) for GAPDH and β-actin, with corresponding brain tissue from a mock-infected macaque serving as the reference tissue. Inter-run calibration to normalize inter-run/inter-plate variability was carried out according to best practices [31 (link),32 (link)].

Ma H., Albe J.R., Gilliland T., McMillen C.M., Gardner C.L., Boyles D.A., Cottle E.L., Dunn M.D., Lundy J.D., Salama N., O’Malley K.J., Pandrea I., Teichert T., Barrick S., Klimstra W.B., Hartman A.L, & Reed D.S. (2022). Long-term persistence of viral RNA and inflammation in the CNS of macaques exposed to aerosolized Venezuelan equine encephalitis virus. PLoS Pathogens, 18(6), e1009946.

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Quantitative RT-PCR Analysis of BMP-2 Expression

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Quantitative RT-PCR for BMP-2 was performed with total RNA extracted from periosteum-derived osteoblastic cells at indicated times. First-strand cDNA was generated using random hexamer primers provided in the first-strand cDNA synthesis kit (Applied Biosystems Inc., Waltham, MA, USA). Primers and probes [glyceraldehyde 3-phosphate dehydrogenase (GAPDH) Cat. #Hs02758991-g1; BMP-2 Cat. #Hs00154192-m1] were obtained commercially (TaqMan® Gene Expression Assay Kit, Applied Biosystems Inc., USA) and amplified using the same kit and following the manufacturer's instructions (TaqMan® Gene Expression Assay Kit, Gene Expression Master Mix, Applied Biosystems Inc.). Amplification conditions were as follow: 50 °C, 2 min; 95 °C, 10 min; followed by 40 cycles of 94 °C, 15 s and 60 °C, 1 min in 96-well plates using the ViiA™ 7 Real-Time PCR System (Applied Biosystems Inc.). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. All experiments were performed in triplicate. Quantitative RT-PCR for BMP-2 was examined at 2 weeks of culture.

Chung J.E., Park J.H., Yun J.W., Kang Y.H., Park B.W., Hwang S.C., Cho Y.C., Sung I.Y., Woo D.K, & Byun J.H. (2016). Cultured Human Periosteum-Derived Cells Can Differentiate into Osteoblasts in a Perioxisome Proliferator-Activated Receptor Gamma-Mediated Fashion via Bone Morphogenetic Protein signaling. International Journal of Medical Sciences, 13(11), 806-818.

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cDNA Quantification via Taqman Assay

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cDNA was synthesized with a kit (Qiagen, Germantown MD). Gene expression was quantified with the Taqman Gene Expression Assay kits (Thermo Fisher, Walkersville MD).

Claudio E., Wang H., Kamenyeva O., Tang W., Ha H.L, & Siebenlist U. (2019). IL-25 orchestrates activation of Thelper cells via conventional dendritic cells in tissue to exacerbate chronic HDM-induced asthma pathology. Journal of immunology (Baltimore, Md. : 1950), 203(8), 2319-2327.

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Quantitative Analysis of Lung Epithelial Gene Expression

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Total RNA was extracted from YAC-treated HPAEpiCs and A549 cells (passage 3) cultured under submerged and ALI conditions using an RNeasy Mini Kit (Qiagen, Hilden, Germany). The cDNA was synthesized using SuperScript IV VILO Mastermix (Thermo Fisher Scientific). Quantitative real-time PCR was performed using a 7900HT Fast Real-Time PCR system (Thermo Fisher Scientific) with the following TaqMan gene expression assay kits (Thermo Fisher Scientific): Hs00387048_m1 (AQP5), Hs00831305_s1 (SFTPA1), Hs00167036_m1 (SFTPB), Hs00161628_m1 (SFTPC), and Hs01108490_m1 (SFTPD). The following thermal cycling profile was applied to all samples: 2 min at 50 °C, 20 s at 95 °C followed by 40 cycles of 1 s at 95 °C and 20 s at 60 °C. Relative expression levels were calculated by the ΔΔCt method using 18 S rRNA (4319413E, Thermo Fisher Scientific) as an internal control. When cycle threshold (Ct) values were undetermined, calculations were performed with a Ct value of 40. If the Ct values were undetermined for an entire set of samples, expression of the gene was considered to be undetectable under those conditions. HPAEpiCs (lot #27000) cultured under submerged conditions (passage 1) were used as a calibrator (relative expression = 1).

Tanabe I., Ishimori K, & Ishikawa S. (2024). Development of an in vitro human alveolar epithelial air-liquid interface model using a small molecule inhibitor cocktail. BMC Molecular and Cell Biology, 25, 9.

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Real-time PCR Analysis of IFT88, COX-2

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Real time PCR was performed using TaqMan gene expression assay kits (Thermo Fisher) and the Quant Studio 7 flex real time PCR system (Thermo Fisher). Gene expression was analyzed by quantitative real‐time PCR using primers and probes (Life Technologies) for analysis of intraflagellar transport 88, IFT88 (Mm00493675_m1); cyclooxygenase‐2, COX‐2 (Mm00478374_m1) and GAPDH (4 351 309). GAPDH was used as a housekeeping gene endogenous control and relative fold change in gene expression was calculated using the 2^−ΔΔCT method.

Verbruggen S.W., Nolan J., Duffy M.P., Pearce O.M., Jacobs C.R, & Knight M.M. (2023). A Novel Primary Cilium‐Mediated Mechanism Through which Osteocytes Regulate Metastatic Behavior of Both Breast and Prostate Cancer Cells. Advanced Science, 11(2), 2305842.

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Quantification of Gene Expression in Induced Motor Neurons

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Total RNA was isolated from DIV15 iMNs using the Qiagen RNAeasy Mini KIT (74104). Using the High Capacity cDNA reverse transcription kit (Applied Biosystems, 436814), RNA was reverse transcribed. Quantitative real-time PCR was performed in the Bio Rad CFX384 Real Time System with C1000 Touch Thermal Cycler using the TaqMan Fast Advanced Master Mix (ThermoFisher, 4444556) and TaqMan Gene Expression Assay KITS (FAM, Hs01028461_s1, Hs04194669_sH, Hs00259032_s1, Hs01115690_m1, Hs00388055_m1, Hs00956610_mH, Hs02786624_g1). For relative expression, ΔΔCq method was employed, using GAPDH as an internal control. For each differentiation, quantitative RT-PCR was measured in triplicate and the mean for each differentiation used for analysis. A two-way ANOVA followed by Sidak’s multiple comparison test was performed to determine significance.

Baron D.M., Fenton A.R., Saez-Atienzar S., Giampetruzzi A., Sreeram A., S.h.a.n.k.a.r.a.c.h.a.r.y.a., Keagle P.J., Doocy V.R., Smith N.J., Danielson E.W., Andresano M., McCormack M.C., Garcia J., Bercier V., Van Den Bosch L., Brent J.R., Fallini C., Traynor B.J., Holzbaur E.L, & Landers J.E. (2022). ALS-associated KIF5A mutations abolish autoinhibition resulting in a toxic gain of function. Cell reports, 39(1), 110598.

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Measuring Gene Expression Using Real-Time PCR

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Applied Biosystems QuantStudio 7 Flex Real-Time PCR was used to measure gene expression using Taqman Gene Expression Assay Kits (Thermo Fisher). Seven different housekeeping genes were tested using Normfinder and the three most stable genes between the three cell lines were used to normalise gene expression across samples as previously described [18 (link)]. Relative fold change in gene expression was calculated using the 2^−ΔΔCT method and represented relative to a control sample set to 1 for each experiment. Data presented is from three independent experiments displayed as mean +/− SEM.

Mahdi A.F., Malacrida B., Nolan J., McCumiskey M.E., Merrigan A.B., Lal A., Tormey S., Lowery A.J., McGourty K, & Kiely P.A. (2020). Expression of Annexin A2 Promotes Cancer Progression in Estrogen Receptor Negative Breast Cancers. Cells, 9(7), 1582.

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Quantifying IFN Response in RVFV-Infected Livers

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cDNA was made from RNA isolated from RVFV-infected or uninfected liver homogenate of Lrp1^f/f and Lrp1^f/fAlb-Cre tissue at 3 dpi as previously described (40 (link)). Using TaqMan Multiplex Master Mix (Applied Biosystems), TaqMan Gene Expression Assay kits were used for IFNα and IFNβ (IFNα: Thermo Fisher Scientific, 4331182 and IFNβ: Thermo Fisher Scientific, 4331182), and data were normalized to GAPDH (Thermo Fisher Scientific, 4448489) and from uninfected liver tissue of both genotypes. A QuantStudio 6 (Thermo Fisher Scientific) was used for amplification using the following parameters: 20-s hold step at 95°C, followed by a 1-s 95°C amplification step, and a 20-s 60° hold step repeated 40×.

Schwarz M.M., Ganaie S.S., Feng A., Brown G., Yangdon T., White J.M., Hoehl R.M., McMillen C.M., Rush R.E., Connors K.A., Cui X., Leung D.W., Egawa T., Amarasinghe G.K, & Hartman A.L. (2023). Lrp1 is essential for lethal Rift Valley fever hepatic disease in mice. Science Advances, 9(28), eadh2264.

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Quantitative Gene Expression Analysis of Gingival Biopsies

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Total RNA from CHX and NT gingival biopsies of the three enrolled patients were extracted using TRIzol reagent (Cat# 15596026, Thermo Fisher Scientific, Carlsbad, CA, USA) following the manufacturer’s instructions, and was reverse transcribed using High Capacity RNA to cDNA Kit (Cat# 4387406, Thermo Fisher Scientific, Carlsbad, CA, USA). cDNAs were then used for amplification of BAX, Col1a1, αSMA, RAC1, SERPINE1 and TIMP1, using the appropriate TaqMan gene expression assay kits (Assay IDs: Hs00180269_m1 (BAX); Hs00164004_m1 (Col1a1); Hs00559403 (αSMA); HS00167155-M1 (SERPINE1); HS01902432_S1 (RAC1); HS01092512_ G1 (TIMP1); Thermo Fisher Scientific, Carlsbad, CA, USA). A total of 2 µl/well of template was added to the sample wells along with TaqMan Universal PCR master mix (Cat# 4305719, Thermo Fisher Scientific, Carlsbad, CA, USA) at a concentration of 1 × and water to a volume of 25 µL/well. Assays were conducted in triplicate on an ABI 7500 Real Time instrument (Thermo Fisher Scientific, Carlsbad, CA, USA) using the following conditions: 50 °C for 2 min, 95 °C for 10 min, and then 95 °C for 15 s, and 60 °C for 1 min, repeated 40 times. Relative quantification was performed using GAPDH mRNA as an endogenous control.

Pilloni A., Ceccarelli S., Bosco D., Gerini G., Marchese C., Marini L, & Rojas M.A. (2021). Effect of Chlorhexidine Digluconate in Early Wound Healing of Human Gingival Tissues. A Histological, Immunohistochemical and Biomolecular Analysis. Antibiotics, 10(10), 1192.

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Quantitative Gene Expression Assay

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TaqMan Gene Expression Assay kits (ThermoFisher) were used to target genes of interest (GFAP Rh00909240_m1; MMP9 Mf02856273_g1; LIF Mf04365516_m1). Probes were labeled at the 5’ end with the reporter molecule 6-carbocyfluorescein (6-FAM) and quenched internally at a modified “T” residue with BHQ1 (Black Hole Quencher), with a modified 3’ end to prevent probe extension by Taq polymerase. Thermocycling parameters comprised the following: hold step, 50°C for 120 seconds; initial denaturing, 95°C for 120 seconds; and cycling PCR amplification, 95°C for 1 second and 60°C for 20 seconds (40 cycles). Endogenous controls comprised TaqMan Gene Expression Assay kits for GAPDH, with thalamus tissue from a mock-infected macaque serving as the reference tissue; endogenous controls for genes of circadian regulation employed 18S rRNA as a time-invariant housekeeping gene. Inter-run calibration to normalize inter-run/inter-plate variability was carried out according to best practices [43 (link), 44 (link)].

Albe J.R., Ma H., Gilliland T.H., McMillen C.M., Gardner C.L., Boyles D.A., Cottle E.L., Dunn M.D., Lundy J.D., O’Malley K.J., Salama N., Walters A.W., Pandrea I., Teichert T., Klimstra W.B., Reed D.S, & Hartman A.L. (2021). Physiological and immunological changes in the brain associated with lethal eastern equine encephalitis virus in macaques. PLoS Pathogens, 17(2), e1009308.

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