Taqman gene expression cells to ct kit

Manufactured by Thermo Fisher Scientific

Sourced in United States, Australia

The TaqMan Gene Expression Cells-to-CT Kit is a lab equipment product designed for the direct quantification of gene expression from cultured cells. It enables the extraction, reverse transcription, and real-time PCR amplification of RNA in a single tube, streamlining the workflow for gene expression analysis.

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Cell-to-CT kit (23 citations) TaqMan kits (18 citations) Taqman® Gene Expression Cells-to-CTTM Kit (4 citations)

116 protocols using taqman gene expression cells to ct kit

Quantitative RT-PCR Analysis of Ovarian Cancer Cells

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Primary ovarian cancer cells were plated at 30000 cells/well in 96-well plates and cultured for 24 h. RNA was extracted and reverse-transcribed with the TaqMan^® Gene expression Cells-to-CT kit (Applied Biosystems, Waltham, MA USA), according to manufacturer’s instructions. For spheroid cultures, RNA was isolated using the mirVana miRNA Isolation Kit (AM1560, Thermofisher, Waltham, MA USA), as per the manufacturer’s instructions. RNA from spheroid culture was quantified using NanoDrop (Thermofisher), and 20 ng were added in complementary DNA cDNA synthesis reactions using TaqMan^® Gene expression Cells-to-CT kit. qRT-PCR reactions were performed on triplicate cDNA samples using TaqMan^® primer sets in Table S3 using Quantstudio 12 K flex real time PCR machine (Applied Biosystems) as described [58 (link)]. CT values were normalised to the house keeping gene β-actin and calibrator using the 2^−∆∆CT method.

Lokman N.A., Price Z.K., Hawkins E.K., Macpherson A.M., Oehler M.K, & Ricciardelli C. (2019). 4-Methylumbelliferone Inhibits Cancer Stem Cell Activation and Overcomes Chemoresistance in Ovarian Cancer. Cancers, 11(8), 1187.

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Evaluating XIST and X-Linked Gene Expression

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XIST expression (probe XIST ID: Hs01079824_m1; Ambion, Austin, TX, USA) was assessed in the hESCs and HFFs, the hBES-1, hPES-1 and hPES-2 cells at P20, P40 and P60, the hPES-2′ undifferentiated cells at P50, P60 and P70, as well as in EBs of hPES-1 (P60), hPES-2 (P60) and hPES-2′ (P70) cells. X-linked gene expression was assessed in the hBES-1, hPES-1 and hPES-2 cells at P20, P40 and P60, and in the hPES-2′ undifferentiated cells at P50, P60 and P70. Real-time PCR was carried out using TaqMan gene expression Cells-to CT kits (Cat. no. 4399002; Ambion) as described in the TaqMan gene expression Cells-to CT kit protocol (http://www.lifetechnologies.com/order/catalog/product/4399002?ICID=search-product). The target X-linked genes were alpha thalassemia/mental retardation, X-Linked (ATRX; assay ID: Hs00230877_m1) and cysteine-rich hydrophobic domain 1 (CHIC1; assay ID: Hs01371424_m1) (both from Ambion). Relative XIST gene and other X-linked gene expression levels were calculated using the 2^−∆∆CT method following normalization to GAPDH (assay ID: Hs02758991_m1; Ambion) expression levels.

QI Q., DING C., HONG P., YANG G., XIE Y., WANG J., HUANG S., HE K, & ZHOU C. (2014). X chromosome inactivation in human parthenogenetic embryonic stem cells following prolonged passaging. International Journal of Molecular Medicine, 35(3), 569-578.

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Compound Dose-Response Assay for Gene Expression

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Eight thousand cells of indicated genotypes were seeded in each well of 96-well plate and allowed to settle overnight. On the second day, 11 pt serial dilution (1:4 fold dilution across) of indicated compound with a top dosage of 10 μM final was added to the culturing medium. Four hours post compound addition, culturing medium was decanted and washed once with PBS. PBS was then decanted completely from the plate and Lysis buffer (plus DNase I) from TaqMan Gene Expression Cells-to-CT Kit (Thermo Fisher, cat # AM1729) was added according to the manual. After 5 min incubation at RT on the shaker, stop solution was added to each well and incubated for 2 min. Reverse transcription was set up immediately using the Cells-to-CT Kit and cDNAs were used for quantitative real-time PCR analysis using Viia7 (Thermo Fisher). Each reaction is multiplexed with an FAM-labelled probe targeting specific target gene splicing isoforms and a VIC-labelled probe targeted 18S rRNA as loading control. Therefore, the FAM Ct value in each well was first normalized to the VIC Ct value in the same well before further normalization to the FAM/VIC ratio of DMSO-treated control samples to calculate fold change over DMSO. Graphs were generated using Graphpad Prism 6, n=2. Taqman gene expression probes used in these assays are listed in Supplementary Table 1.

Teng T., Tsai J.H., Puyang X., Seiler M., Peng S., Prajapati S., Aird D., Buonamici S., Caleb B., Chan B., Corson L., Feala J., Fekkes P., Gerard B., Karr C., Korpal M., Liu X., T. Lowe J., Mizui Y., Palacino J., Park E., Smith P.G., Subramanian V., Wu Z.J., Zou J., Yu L., Chicas A., Warmuth M., Larsen N, & Zhu P. (2017). Splicing modulators act at the branch point adenosine binding pocket defined by the PHF5A–SF3b complex. Nature Communications, 8, 15522.

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Intestinal Gene Expression Profiling

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Total RNA was extracted from small intestines tissue samples using the RNeasy plus mini kit (QIAGEN) following manufactures instructions. An on-column DNase treatment was included. cDNA was generated from each RNA sample using a Taqman Gene Expression Cells-to-CT kit (Thermo Fisher Scientific). Gene expression assay for Tnf (Mm00443260_g1), Ccl2 (Mm00441242_m1), Birc3 (Mm01168413_m1), Nfkbia (Mm00477800_g1), Cxcl1 (Mm04207460_m1) and GAPDH (Mm99999915_g1) were from Thermo Fisher Scientific and for Icam1 (Mm.PT.58.43714327) was from IDT. All mRNA expression levels were normalized to GAPDH gene expression.

Witt A., Goncharov T., Lee Y.M., Kist M., Dohse M., Eastham J., Dugger D., Newton K., Webster J.D, & Vucic D. (2023). XIAP deletion sensitizes mice to TNF-induced and RIP1-mediated death. Cell Death & Disease, 14(4), 262.

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Transcriptomic Profiling of FACS-Sorted Islet Cells

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FACS-sorted islet cells were washed, and lysed in a minimum of 5 μL Cells-to-CT buffer containing 1% DNAse I (Taqman Gene Expression Cells-to-CT Kit, Thermo Fisher Scientific), or a volume adjusted for a final cell concentration of ~ 1000 cells/μL for 5 min at RT, and stopped by addition of 10% Stop Solution, according to manufacturer instructions. Transcriptome analysis was performed via Nanostring nCounter XT (Nanostring Technologies, Seattle, USA) with the XT_PGX_MmV1_Immunology Code Set. Due to low cell input from 3-month old islets, pre-amplification of RNA was performed via Nanostring Low RNA Input Kit following manufacturer instructions. Briefly, RNA was diluted 1:2 in water, and 4 μL sample was reverse transcribed, followed by 8 rounds of amplification with M Immunology v1 Primers. All other samples were assessed without pre-amplification. Cell lysate or pre-amplified cDNA (5 μL) was added to hybridization mixtures for 18 h at 65 °C with a 70 °C-heated lid. Gene expression was measured with a Nanostring SPRINT Profiler, and data were analyzed via Nsolver 4.0 software.

Denroche H.C., Miard S., Sallé-Lefort S., Picard F, & Verchere C.B. (2021). T cells accumulate in non-diabetic islets during ageing. Immunity & Ageing : I & A, 18, 8.

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Quantifying Gene Expression in Cell Lines and Tissues

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For Caco-2 cells and mouse ileal mucosal scrapings, total RNA was purified using RNeasy Mini Kit following the manufacturer’s instructions (Qiagen, Valencia, CA) and cDNA synthesized using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). For enteroids, total RNA isolation and cDNA synthesis were performed using TaqMan™ Gene Expression Cells-to-CT™ Kit (Thermo Fisher, Grand Island, NY) following the instruction. qPCR was performed using a TaqMan Gene Expression Master Mix and TaqMan probes (Thermo Fisher) according to the manufacturer’s protocol. Mouse β-actin and human GAPDH probes were used as the internal controls for mouse tissues and Caco-2 cells, respectively. Expression levels were assessed by evaluating threshold cycle (Ct) values. The relative amount of mRNA expression was calculated by the comparative ΔΔCt method. Each group included 3 wells in the qPCR assay.

Li J., Li X., Song J., Yan B., Rock S., Jia J., Liu J., Wang C., Weiss T., Weiss H.L., Gao T., Alam A, & Evers B.M. (2020). Absence of neurotensin attenuates intestinal dysbiosis and inflammation by maintaining Mmp7/α-defensin axis in diet-induced obese mice. FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 34(6), 8596-8610.

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Optimization of Fetal Bovine Serum Protocols

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Fetal bovine serum (FBS), TaqMan Gene Expression Cells-to-CT Kit (product no. AM1728), electrophoresis markers, Lipofectamine 3000 (product no. L3000001) and GRP78 siRNA were purchased from ThermoFisher Scientific (Waltham, MA, USA). Phosphate-buffered saline (PBS) was purchased from BioWest (Riverside, MO, USA). Medium M199 (product no. M2154), antibiotic–antimycotic solution, Tris, trypsin, FSH (product no. F4021), LH (product no. L5259), IGF1 (product no. I3769), human vaspin (product no. SRP4915), KT5720 (product no. K3761), Laemmli buffer (product no. 38733), Na-deoxycholate, Nonidet NP-40, sodium dodecyl sulfate (SDS), protease inhibitors (ethylene diamine tetraacetic acid-free), dithiothreitol (DTT), Tween 20, bromophenol blue, and 1 bromo-3-chloro-propane were obtained from Sigma-Aldrich (St. Louis, MO, USA). Pig vaspin was not available at the beginning of experiments; therefore, human vaspin was used (there is 90% homology of vaspin nucleotides between these species; sequences were analyzed against the NCBI, USA database using the BLAST program). PD98059 (product no. 1213) was obtained from Tocris (Bristol, GB). Bradford protein assay kit, 4–20% gels (product no. 456-1093) and membranes (product no. 1704156) were obtained from Bio-Rad (Hercules, CA, USA).

Kurowska P., Mlyczyńska E., Dawid M., Dupont J, & Rak A. (2020). Role of vaspin in porcine ovary: effect on signaling pathways and steroid synthesis via GRP78 receptor and protein kinase A. Biology of Reproduction, 102(6), 1290-1305.

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RNA Extraction and qRT-PCR Analysis of Cancer Cells

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RNA was extracted from cancer cells and fibroblasts using the RNeasy Kit from Qiagen. Total RNA and first-strand cDNA synthesis was performed using TaqMan Gene Expression Cells-To-Ct Kit (ThermoFisher) as previously described (Dragoi et al., 2014 (link); Abshire et al., 2016 (link)). mRNA levels were determined by quantitative real-time PCR using the universal ProbeLibrary (Roche, Life Science) and LightCycler 480 Probes Master (Roche, Life Science) or PowerUp SYBR Green Master Mix (Thermo Fisher). For the LightCycler 480 Probes Master the thermal cycling was carried out using a LightCycler 96 instrument (Roche Diagnostics) under the following conditions: 95°C for 5 min and 40 cycles at 95°C for 10 s and 60°C for 25 s. For the PowerUp SYBR Green (for ITGA5 and ITGB1) the thermal cycling was carried out under these conditions: preamplification: 95°C for 10 min; amplification: 40 cycles at 95°C for 10 s, 60°C for 10 s and 72°C at 10 s; melting: 95°C for 10 s, 65°C for 60 s and 97°C for 1 s. Relative quantification was performed using 2^−ΔΔCT method. Gene expression was normalized to GAPDH as reference gene. The fold increase is represented as relative values to the Control condition. A complete list of primers and probes used in the study are presented in Supplementary Table S10.

Dhungel N., Youngblood R., Chu M., Carroll J, & Dragoi A.M. (2023). Assessing the epithelial-to-mesenchymal plasticity in a small cell lung carcinoma (SCLC) and lung fibroblasts co-culture model. Frontiers in Molecular Biosciences, 10, 1096326.

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SLC7A11 mRNA Expression Quantification

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SFN (20 µM) 96 h-treated and untreated control cells (1×10⁴ cells/sample) were seeded into 96-well plates. To investigate the changes in mRNA expression levels of SLC7A11, RT-qPCR was performed using the TaqMan^® Gene Expression Cells-to-CT™ kit (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The following temperature conditions were used: Incubation at at 37°C for 60 min then at 95°C for 5 min. RT-qPCR was performed using an Applied Biosystems 7500 Fast Real-Time PCR System (Thermo Fisher Scientific, Inc.). The target mRNA was amplified using a TaqMan gene expression master mix (Thermo Fisher Scientific, Inc.) and the following TaqMan gene expression assay (probe and primer): SLC7A11, Hs00921938_m1 and GAPDH, Hs02758991_g1 (both from Thermo Fisher Scientific, Inc.).
The RT-qPCR thermocycling conditions were as follows: Initial denaturation at 50°C for 2 min, 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 1 min. The expression levels of target mRNA were calculated using the 2^−∆∆Cq method and normalized to those of GAPDH (30 (link)).

Iida Y., Okamoto-Katsuyama M., Maruoka S., Mizumura K., Shimizu T., Shikano S., Hikichi M., Takahashi M., Tsuya K., Okamoto S., Inoue T., Nakanishi Y., Takahashi N., Masuda S., Hashimoto S, & Gon Y. (2020). Effective ferroptotic small-cell lung cancer cell death from SLC7A11 inhibition by sulforaphane. Oncology Letters, 21(1), 71.

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Comprehensive Molecular Profiling Protocol

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RNA extraction, reverse transcription, and qRT-PCR were performed using the TaqMan^® Gene Expression Cells-to-CT Kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's guidelines. Relative fold transcript change was determined using the ΔΔCq method. The TaqMan^® probe assays used for specific transcripts are BRAF (Hs00269944_m1), KRAS (Hs00364284_g1), PAX8 (Hs01015257_g1), NKX2-1 (Hs00968940_m1), and ACTB (Hs01060665_g1).

Danysh B.P., Rieger E.Y., Sinha D.K., Evers C.V., Cote G.J., Cabanillas M.E, & Hofmann M.C. (2016). Long-term vemurafenib treatment drives inhibitor resistance through a spontaneous KRAS G12D mutation in a BRAF V600E papillary thyroid carcinoma model. Oncotarget, 7(21), 30907-30923.

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