Taqman fast cells to ct kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The TaqMan Fast Cells-to-CT kit is a rapid and efficient tool for direct reverse transcription and real-time PCR analysis of gene expression from cellular samples. The kit streamlines the process of RNA extraction, cDNA synthesis, and quantitative PCR, providing a simple and reliable workflow for gene expression studies.

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8 protocols using taqman fast cells to ct kit

Measuring eNOS Gene Expression

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Complimentary DNA (cDNA) was prepared from cell lysates using the TaqMan Fast Cells-to-Ct kit (Product #: 4399003; Life Technologies, Carlsbad, CA) according to manufacturer instructions. cDNA samples were subsequently used in two-step RT-PCR experiments using TaqMan gene expression assays (Applied Biosystems; Bedord, MA, USA) for eNOS (assay ID: Hs01574659_m1) and GAPDH (assay ID: Hs99999905_m1) on 96-well TaqMan Plates in a QuantStudio 3 RT-PCR System (Applied Biosystems). All samples were run in duplicate for genes of interest and endogenous control (GAPDH) and analyzed using the comparative threshold (ΔΔCt) method.

Hammond S.T., Baumfalk D.R., Parr S.K., Butenas A.L., Scheuermann B.C., Turpin V.R., Behnke B.J., Hashmi M.H, & Ade C.J. (2023). Impaired microvascular reactivity in patients treated with 5-fluorouracil chemotherapy regimens: Potential role of endothelial dysfunction. International Journal of Cardiology. Heart & Vasculature, 49, 101300.

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Analyzing Gene Expression Changes

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The TaqMan Fast Cells-to-C T Kit (Life Technologies) was used to extract total RNA and reverse transcribe RNA to cDNA according to the manufacturer's specifications. Four microliters of cDNA from the RT reaction was used to set up TaqMan qRT-PCR on a QuantStudio 7 Flex Real-Time PCR System (Life Technologies). The TaqMan Gene Expression Assays used for this study include BCL2 (Hs00608023_m1), BAX (Hs00180269_m1), MCL1 (Hs01050896_m1), BTK (Hs00975865_m1), MAP3K7 (Hs00177373_m1), IRAK4 (Hs00211610_m1), GAPDH (Hs02758991_g1), and ACTB (Hs01060665_g1).

Kuo H.P., Ezell S.A., Schweighofer K.J., Cheung L.W.K., Hsieh S., Apatira M., Sirisawad M., Eckert K., Hsu S.J., Chen C.T., Beaupre D.M., Versele M, & Chang B.Y. (2017). Combination of Ibrutinib and ABT-199 in Diffuse Large B-Cell Lymphoma and Follicular Lymphoma. Molecular cancer therapeutics, 16(7).

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Quantitative Analysis of Hedgehog Pathway

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Cells were grown to confluence and plated on 24-well plates (50,000 cells/well, 500 μL). After 24 h, growth media were removed and replaced with corresponding low FBS media (500 μL per well). Cells were treated with DMSO, OHCs, OHCs plus compound, or compound alone, depending on the assay. Cells were incubated (37 °C, 5 % CO₂) for the indicated time period, and total RNA was extracted using Ambion by Life Technologies TaqMan Fast Cells-to-CT kit following the manufacturer’s instructions. Synthesis of cDNA was performed using a Bio-Rad MyCycler. Quantitative PCR was performed on an Applied Biosystems (ABI) 7500 Fast system using the following TaqMan Gene Expression Primer/Probe solutions (ABI): mouse ActB (Mm00607939s1), mouse Gli1 (Mm00494645m1), mouse Gli2 (Mm01293117_m1), mouse Ptch1 (Mm00436026_m1), and mouse Hhip (Mm00469580_m1). Relative gene-expression levels were computed by the ΔΔCt method. Values represent mRNA expression relative to OHCs (C3H10T1/2, set to 100%) or DMSO (set to 1.0). Data were analyzed using GraphPad Prism 5, and all values are the mean ± SEM for at least two separate experiments performed in triplicate.

Dash R.C., Wen J., Zaino A.M., Morel S.R., Chau L.Q., Wechsler-Reya R.J, & Hadden M.K. (2021). Structure-based virtual screening identifies an 8-hydroxyquinoline as a small molecule GLI1 inhibitor. Molecular Therapy Oncolytics, 20, 265-276.

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Quantitative Analysis of SULF-2 and Cyclin D1 in RCC

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RNA isolation from clinical RCC samples was performed using ISOGEN (Nippon Gene, Tokyo, Japan) in accordance with the manufacturer's instructions. cDNA was constructed using the SuperScript III First‐strand Synthesis System for RT‐PCR (Invitrogen, Carlsbad, CA, USA). RNA isolation and cDNA construction from human RCC cell lines were performed using the TaqMan Fast Cells‐to‐CT Kit (Ambion/Life Technologies, Carlsbad, CA, USA). Each cell line was plated at a density of 8000 cells/well on a 96‐well culture plate. TaqMan PCR reagents for SULF‐2 (Hs01016476_m1) and cyclin D1 (Hs00765553_m1) were purchased from Applied Biosystems (Foster city, CA, USA). Quantitative real‐time PCR was carried out using the TaqMan Master Mix Reagent Kit protocol with a StepOne real‐time PCR system (Applied Biosystems). The data were standardized against β‐actin gene expression using Pre‐Developed TaqMan Assay reagents (Applied Biosystems). The expression level of SULF‐2 mRNA was determined by the ΔΔC_t method based on the normal tissue from one patient as a control. All experiments were conducted at least three times.

Kumagai S., Ishibashi K., Kataoka M., Oguro T., Kiko Y., Yanagida T., Aikawa K, & Kojima Y. (2016). Impact of Sulfatase‐2 on cancer progression and prognosis in patients with renal cell carcinoma. Cancer Science, 107(11), 1632-1641.

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Quantitative Real-Time PCR Protocol for Gene Expression Analysis

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Total RNA was extracted and reverse transcribed using the Taqman Fast Cells-to-CT Kit (Thermo Fisher, 4399003). Real-time quantitative RT-PCR was performed using the Taqman PreAmp Master Mix (Thermo Fisher, 4391128), Taqman Fast Universal PCR Master Mix (Thermo Fisher, 4366072), and a QuantStudio 3 Real-Time PCR System (Thermo Fisher). In some experiments, total RNA was extracted using the RNeasy Micro Kit (Qiagen, 74004) and reverse transcribed using the EvoScript Reverse Transcriptase (Roche, 07912315001). Real-time quantitative RT-PCR was performed using the TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara, RR820A) and a Takara Thermal Cycler Dice Realtime System (Takara). Relative expression was calculated for each gene by the comparative CT method and with Actb for normalization. Probes were shown in Supplementary Fig. 2.

Matsumura T., Totani H., Gunji Y., Fukuda M., Yokomori R., Deng J., Rethnam M., Yang C., Tan T.K., Karasawa T., Kario K., Takahashi M., Osato M., Sanda T, & Suda T. (2022). A Myb enhancer-guided analysis of basophil and mast cell differentiation. Nature Communications, 13, 7064.

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Quantification of PDGFD and EGFR Expression

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Cells were seeded in 6-well plates (1×10⁶/well). Total RNA was extracted using the TaqMan^® Fast Cells-to-CT™ kit (Thermo Fisher Scientific, Inc.) and then reverse transcribed into cDNA according to the manufacturer's instructions. quantitative PCR (qPCR) was performed on a QuantStudio 7 Flex Real-Time PCR System (Thermo Fisher Scientific, Inc.). Amplification was performed in a three-step cycle procedure, 95°C (denaturation) 10 sec, 60°C (annealing) 30 sec, and 72°C (extension) 30 sec, for 40 cycles. The primers were: PDGFD, forward 5′-GAACAGCTACCCCAGGAACC-3′, reverse 5′-CTTGTGTCCACACCATCGTC-3′; EGFR, forward 5′-CCCTCCTGAGCTCTCTGAGT-3′, reverse 5′-GTTTCCCCCTCTGGAGATGC-3′; β-actin, forward 5′-TTGTTACAGGAAGTCCCTTGCC-3′; reverse 5′-ATGCTATCACCTCCCCTGTGTG-3′. mRNA levels were quantified using the 2^−ΔΔCq method (40 (link)) and normalized to the internal reference gene β-actin. All experiments were repeated three times.

Jin J., Wang L., Tao Z., Zhang J., Lv F., Cao J, & Hu X. (2020). PDGFD induces ibrutinib resistance of diffuse large B-cell lymphoma through activation of EGFR. Molecular Medicine Reports, 21(5), 2209-2219.

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Quantification of Immune Markers in Mouse Brain

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Mice were euthanized under isoflurane anesthesia and perfused with chilled PBS. Brains were removed and immersed in a solution containing RNAlater (Thermo Fisher Scientific, Tokyo, Japan) and incubated overnight at 4 °C. Immersed brains were homogenized using Micro Smash (Tomy Seiko, Tokyo, Japan). Total RNA was extracted from tissue homogenates using the RNeasy mini kit (Qiagen, Germantown, MD, USA), and cDNA was synthesized from 1 µg of RNA using the TaqMan Fast Cells-to-CT kit (Thermo Fisher Scientific). The concentration of RNA in each sample was determined using NanoDrop (Thermo Fisher Scientific). The TaqMan real-time polymerase chain reaction assays using probes and primers for Tlr2, Tnf and 18S rRNA (Applied Biosystems, Waltham, CA, USA) were performed using the QuantStudio 7 Flex system (Applied Biosystems). For an endogenous reference gene, 18S rRNA was used. Fold changes were determined using the ΔΔCT method.

Koyanagi Y., Kassai M, & Yoneyama H. (2024). The Impact of Intestinal Microbiota and Toll-like Receptor 2 Signaling on α-Synuclein Pathology in Nontransgenic Mice Injected with α-Synuclein Preformed Fibrils. Microorganisms, 12(1), 106.

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Evaluating Hedgehog Pathway Inhibition in BCC Cells

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Protocols for general cell culture, qPCR, and Hh inhibition in ASZ cells have been previously described [17 , 20 ]. All PSZ analogues were tested for Hh inhibitory activity by measuring their inhibition of endogenous Gli1 mRNA expression in a murine BCC cell line (Ptch^+/−), ASZ. Cells at 80% confluence were plated in 154CF growth media (2% FBS, ThermoFisher) in a 96-well plate (10,000 cells/well). Cells were incubated for 24 hours (37°C, 5% CO₂) before media without fetal bovine serum (FBS) was added. Cells were again incubated for 24 hours (37°C, 5% CO₂) before being treated with DMSO (vehicle control), PSZ, or PSZ analoguea at varying concentrations (10 – 0.001 μM). Compounds were incubated with the cells for 48 hours (37°C, 5% CO₂). Cells were visually examined to ensure there was no non-specific cytotoxicity present for the concentrations evaluated. Cells were lysed, mRNA was extracted, and Act B (internal control) and Glil expression levels were measured by qRT-PCR using the TaqMan Fast Cells-to-C_T kit (ThermoFisher). Compound concentrations that significantly decreased actin B levels were excluded form our qRT-PCR calculations. Data was analyzed using GraphPad Prism 5 and IC₅₀ values computed as mean ± SEM from at least three separate experiments performed in triplicate.

Teske K.A., Dash R.C., Morel S.R., Chou L.Q., Wechsler-Reya R.J, & Hadden M.K. (2018). Development of Posaconazole-based Analogues as Hedgehog Signaling Pathway Inhibitors. European journal of medicinal chemistry, 163, 320-332.

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