Cells to ct kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Cells-to-CT kit is a laboratory product developed by Thermo Fisher Scientific. It is designed to facilitate the direct lysis of cells and the subsequent detection of gene expression levels using real-time PCR technology. The kit provides a streamlined workflow for RNA extraction and cDNA synthesis from small cell samples.

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Cell-to-CT kit (23 citations) Power SYBRTM Green Cells-to-CTTM Kit (4 citations) Taqman® Gene Expression Cells-to-CTTM Kit (4 citations)

109 protocols using cells to ct kit

RORC Expression in PBMCs and PHA Blasts

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Freshly thawed PBMCs were dispended into a U-bottomed 96-well plate at a density of 2×10⁵cells/well and were stimulated for 24 hours with Dynabeads Human T-Activator CD3/CD28 (Gibco, Cat: 111.61D, 8×10⁴ beads/well). Cells were lysed and reverse-transcribed with the Cells-to-Ct kit (Thermo Fisher Scientific). Two FAM-MGB TaqMan probes for RORC (Hs01076122_m1 and Hs00172860_m1) were used to assess the expression of RORC. The VIC-TAMRA probe for GUSB (Applied Biosystems) was used as an endogenous control. Real-time qPCR was performed as described above.
PHA blasts were dispended into a U-bottomed 96-well plate at a density of 5×10⁴cells/well without further stimulation. Cells were lysed and reverse-transcribed with the Cells-to-Ct kit (Thermo Fisher Scientific). A FAM-MGB TaqMan probe for RORC (Hs00172860_m1) was used to assess the expression of RORC. The VIC-TAMRA probe for GUSB (Applied Biosystems) was used as an endogenous control. Real-time qPCR was performed as described above.

Ogishi M., Yang R., Aytekin C., Langlais D., Bourgey M., Khan T., Ali F.A., Rahman M., Delmonte O.M., Chrabieh M., Zhang P., Gruber C., Pelham S.J., Spaan A.N., Rosain J., Lei W.T., Drutman S., Hellmann M.D., Callahan M.K., Adamow M., Wong P., Wolchok J.D., Rao G., Ma C.S., Nakajima Y., Yaguchi T., Chamoto K., Williams S.C., Emile J.F., Rozenberg F., Glickman M.S., Rapaport F., Kerner G., Allington G., Tezcan I., Cagdas D., Hosnut F.O., Dogu F., Ikinciogullari A., Rao V.K., Kainulainen L., Béziat V., Bustamante J., Vilarinho S., Lifton R.P., Boisson B., Abel L., Bogunovic D., Marr N., Notarangelo L.D., Tangye S.G., Honjo T., Gros P., Boisson-Dupuis S, & Casanova J.L. (2021). Inherited PD-1 deficiency underlies tuberculosis and autoimmunity in a child. Nature medicine, 27(9), 1646-1654.

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Measuring Metabolic Syndrome Markers

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Total RNA was isolated from samples using the RNeasy kit (Qiagen, Valencia, CA, USA) or the Cells to Ct kit (Invitrogen, Carlsbad, CA, USA). RNA was reverse transcribed using the Thermoscript RT-PCR kit (Invitrogen, Carlsbad, CA, USA) or the Cells to Ct kit, and semi-quantitative PCR was performed with the primers in Table 1; primers selected based on established involvement in metabolic syndrome and diabetes [21 (link)]. All reactions were performed in triplicate, and cycle threshold values were averaged over triplicates. Relative mRNA expression was determined using the 2^−ΔΔCT method, with GAPDH as the endogenous control gene. In addition, IL-6, TNF-α, and VEGF cytokine protein release under all tested conditions was measured by ELISA, as specified by manufacturer’s instruction (R&D Systems, Minneapolis, MN, USA)

Chokpaisarn J., Urao N., Voravuthikunchai S.P, & Koh T.J. (2017). Quercus infectoria Inhibits Set7/NF-κB Inflammatory Pathway in Macrophages Exposed to a Diabetic Environment. Cytokine, 94, 29-36.

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Androgen Receptor Signaling Assay

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Example 2

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LNCaP (ATCC) or LNCaP-EnzR (MR49F is received from Dr. Martin Gleave, University of British Columbia) cells are plated in 96 well plates at 15,000-20,000 cells/well in RPMI+1% csFBS without phenol red. Cells are treated 2 days after plating and harvested 18 hours after treatment (for TMPRSS2) or 24 hours after treatment (for PSA and FKBP5). RNA is isolated (cells to ct kit, Life Technologies), cDNA synthesized (cells to ct kit), and expression of TMPRSS2, PSA or FKBP5 and expression of GAPDH are measured using realtime PCR primers and probes (TaqMan probes, Life Technologies) by realtime PCR (ABI 7900, Life Technologies). Relative expression is calculated using ddct method.

US11034669B2. Pyrrole and pyrazole compounds and methods of use thereof (2021-06-15). Nuvation Bio Inc. [US]. Inventors: Sarvajit Chakravarty [US], Son Minh Pham [US], Jiyun Chen [US], Jayakanth Kankanala [US], Jeremy D. Pettigrew [CA], Anjan Kumar Nayak [IN], Anup Barde [IN], Brahmam Pujala [IN].

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Quantification of siRNA-Ago2 Binding

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siRNAs were transfected into Hepa 1-6 cells and primary hepatocytes plated at 3,500 and 16,000 cells per well, respectively, using 0.9 μL RNAiMax (Lipofectamine; Invitrogen). Cells were incubated for 18–24 h prior to isolating RNA, using the Cells to Ct kit (Ambion) according to the product protocol. TaqMan Gene Expression Assays (Applied Biosystems) were performed as described within the product protocol using the following primer probes: Mm00838341_m1 (Ago2), Mm00431814_m1 (Apoa4), Mm01545154_g1 (ApoB), Mm00432327_m1 (Serpina6), Mm00447374_m1 (SSB), and 4352339E (Gapdh). All reactions were performed in duplicate, and data were analyzed using the ddCt method, with GAPDH serving as the internal control.⁴⁷ (link) The relative amount of siRNA bound to Ago2 was quantitated using stem-loop RT-PCR of anti-mouse Ago2 immunoprecipitate, as described previously.38 (link), 39 (link)

Mihaila R., Ruhela D., Galinski B., Card A., Cancilla M., Shadel T., Kang J., Tep S., Wei J., Haas R.M., Caldwell J., Flanagan W.M., Kuklin N., Cherkaev E, & Ason B. (2019). Modeling the Kinetics of Lipid-Nanoparticle- Mediated Delivery of Multiple siRNAs to Evaluate the Effect on Competition for Ago2. Molecular Therapy. Nucleic Acids, 16, 367-377.

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In vivo siRNA Delivery and Analysis

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All in vivo work was performed according to an approved animal protocol, as set by the Institutional American Association for the Accreditation of Laboratory Animal Care. C57BL/6 male mice (Charles River) ∼8 weeks of age were administered siRNAs by intravenous (i.v.) injection. Animals were euthanized by CO₂ inhalation. Immediately after euthanasia, sections from the right medial lobe of each liver were excised, placed in RNALater (for TaqMan Gene Expression Analysis), and stored at 4°C or flash frozen (for siRNA quantification) and stored at −80°C until further use. Quantification of the siRNA concentration in the liver was determined as described previously.38 (link), 39 (link) For TaqMan Gene Expression Analysis, RNA was isolated using QIAGEN’s RNeasy96 Universal Tissue Kit, according to the supplied product protocol. An on-column DNase I treatment was performed, and samples were washed three times prior to elution with 100 μL of RNase-free water. Reverse transcription was performed with the Cells to Ct kit (Ambion) in a 20 μL volume with 350 ng RNA in 1× reverse transcriptase and buffer incubated at 37°C for 1 h. TaqMan Gene Expression Assays (Applied Biosystems) were performed as described within the product protocol, using the primer-probes listed above.

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Single-cell qPCR Analysis Workflow

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Real-time PCR was performed using the StepOnePlus real-time PCR system and Fast SYBR Green Master Mix (Applied Biosystems). Expression of the target genes was normalized to those of the rabbit TATA-box binding protein (Tbp) and Gapdh genes. For single-cell qPCR, cells were dissociated using Accutase. Single cells were captured on the C1 Array IFC (10–17 μm) and subjected to reverse transcription and specific target amplification using components from the Cells-to-Ct kit (Ambion) and C1 Single-Cell Auto Prep Modules kit (Fluidigm). These pre-amplified products were subsequently analyzed with Universal PCR TaqMan Master Mix (Applied Biosystems) and coupled with a DNA Binding Dye Sample Loading Reagent (Fluidigm) and Evagreen (Biotium 31000) in 96.96 Dynamic Arrays on a BioMark system.

Osteil P., Moulin A., Santamaria C., Joly T., Jouneau L., Aubry M., Tapponnier Y., Archilla C., Schmaltz-Panneau B., Lecardonnel J., Barasc H., Mouney-Bonnet N., Genthon C., Roulet A., Donnadieu C., Acloque H., Gocza E., Duranthon V., Afanassieff M, & Savatier P. (2016). A Panel of Embryonic Stem Cell Lines Reveals the Variety and Dynamic of Pluripotent States in Rabbits. Stem Cell Reports, 7(3), 383-398.

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Quantitative PCR for mRNA Expression

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To detect mRNA expression, mRNA of three independent samples was extracted using the Cells-to-Ct kit (Ambion, Austin, TX, USA). Quantitative PCR was subsequently performed using the Universal Probe Library system (Roche, Branchburg, NJ, USA) with the following primer/probe combinations: CENPF-UPL74–forward: 5′-gagtcctccaaaccaacagc-3′, CENPF-UPL74–reverse: 5′-tccgctgagcaactttgac-3′, FOXM1-UPL11–forward: 5′-actttaagcacattgccaagc-3′, FOXM1-UPL11–reverse: 5′-cgtgcagggaaaggttgt-3′, Pin1-UPL1–forward: 5′-gaagatcacccggaccaag-3′, Pin1-UPL1–reverse: 5′-aagtcctcctctcccgactt-3′, and Tubulin-UPL58–forward: 5′-cttcgtctccgccatcag-3′ and Tubulin-UPL58–reverse: 5′-ttgccaatctggacacca-3′.

Kruiswijk F., Hasenfuss S.C., Sivapatham R., Baar M.P., Putavet D., Naipal K.A., van den Broek N.J., Kruit W., van der Spek P.J., van Gent D.C., Brenkman A.B., Campisi J., Burgering B.M., Hoeijmakers J.H, & de Keizer P.L. (2015). Targeted inhibition of metastatic melanoma through interference with Pin1-FOXM1 signaling. Oncogene, 35(17), 2166-2177.

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Transcriptional Regulation on Hydrogels

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U373-MG cells were grown on dual gradient patterned hydrogels overnight, after which total mRNA was isolated from the cells with an Ambion Cells-to-Ct kit, as per the manufacturer's instructions. Reverse transcription was performed with primers for miR18a (Applied Biosystems) or universal amplification (iScript; BioRad), with three thermal stages on a BioRad C1000 Thermal Cycler 1(6°C for 30 min, 42°C for 30 min, 85°C for 5 min). Quantitative PCR was performed on either the target or control sample with a BioRad iQ5 quantitative PCR machine with primers for either miR18a (Applied Biosystems) or for 18S ribosomal RNA (control; Applied Biosystems). The Ct of each sample was computed with BioRad software, after which differences between soft and stiff samples were calculated using the ΔΔCt method.

Rape A.D., Zibinsky M., Murthy N, & Kumar S. (2015). A synthetic hydrogel for the high-throughput study of cell–ECM interactions. Nature Communications, 6, 8129.

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Real-Time PCR Analysis of Gene Expression

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RNA was purified using the Cells-to-CT Kit (Ambion, ThermoFisher, 4402953). Complementary DNA was obtained by reverse transcription with the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, 4368814). cDNAs were analyzed by real-time PCR using the Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659). Amplification, detection and data analysis were carried out with an ABI PRISM 7900HT Sequence Detection System and normalized to GusB and Gapdh expression. Changes in mRNA expression are noted as ×-fold change relative to the control. qPCR primers are listed in Table S2.

Herrero D., Cañón S., Albericio G., Carmona R.M., Aguilar S., Mañes S, & Bernad A. (2019). Age-related oxidative stress confines damage-responsive Bmi1+ cells to perivascular regions in the murine adult heart. Redox Biology, 22, 101156.

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Anti-viral Gene Expression Assay

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Cells were plated at 1 × 10⁴ per well in 96 well plates. After 24 hr, viruses were mixed 1:1 with antibody and incubated for 30 mins at room temperature prior to infection. AdV was used at 1.25 × 10⁹ pts per well mixed with 10 mg/ml human serum IgG (Sanquin). HRV was used at 2.5 × 10² TCID₅₀ units per well mixed with 5 mg/ml human serum IgG (Sanquin). Cells were incubated at 37° C for 3.5 hr, before washing with PBS and preparation of cDNA using Cells to CT Kit (Ambion). Gene expression was monitored by TaqMan Gene Expression Assays (Applied Biosystems) on a StepOnePlus Real Time PCR System (Life Technologies). Gene expression assays were: mouse β-actin (4352933E), CxCL10 (Mm00445235_m1), TNF (Mm00443260_g1), IL-6 (Mm00446190_m1), IFIT1 (Mm00515153_m1). Relative expression was quantified using the 2^−ΔΔCt method.

Dickson C., Fletcher A.J., Vaysburd M., Yang J.C., Mallery D.L., Zeng J., Johnson C.M., McLaughlin S.H., Skehel M., Maslen S., Cruickshank J., Huguenin-Dezot N., Chin J.W., Neuhaus D, & James L.C. (2018). Intracellular antibody signalling is regulated by phosphorylation of the Fc receptor TRIM21. eLife, 7, e32660.

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