This test was performed using TaqMan® Fast Advanced Cells-to-CT Kit (Thermo Fisher Scientific) and TaqMan® Gene Expression Assay (Thermo Fisher Scientific). The predesigned human-specific primers with TaqMan probes that were used in this study were ACTB TaqMan® Gene Expression Assay (FAM) (assay ID: Hs99999903_m1, Thermo Fisher Scientific); NFE2L2 TaqMan® Gene Expression Assay (FAM) (assay ID: Hs00975961_g1, Thermo Fisher Scientific); AHR TaqMan® Gene Expression Assay (FAM) (assay ID: Hs00169233_m1, Thermo Fisher Scientific); and PPARA TaqMan® Gene Expression Assay (FAM) (assay ID: Hs00947536_m1, Thermo Fisher Scientific) (Table I ). The ∆∆Cq method was used to calculate the fold gene expression of NFE2L2 or AhR. ACTB was used as a housekeeping gene to normalize the Ct values (17 (link)). The formulas used in this study were as follows: ∆Cq=Cq (gene of interest)-Cq (housekeeping gene); ∆∆Cq=∆Cq (Sample)-∆Cq (Control average); Fold gene expression=2−(∆∆Cq).
Taqman fast advanced cells to ct kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The TaqMan Fast Advanced Cells-to-Ct Kit is a quick and convenient solution for gene expression analysis directly from cultured cells. The kit provides a simple workflow for isolating RNA and performing real-time RT-PCR, enabling efficient and reliable quantification of target gene expression.
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Lab products found in correlation
Taqman gene expression assay, by Thermo Fisher Scientific (2 mentions)
Powerup sybr green master mix, by Thermo Fisher Scientific (2 mentions)
Revertra ace qpcr rt master mix, by Toyobo (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Thunderbird probe qpcr mix, by Toyobo (2 mentions)
Taqman ids, by Thermo Fisher Scientific (2 mentions)
Endo porter, by Gene Tools (1 mentions)
Real time pcr system, by Thermo Fisher Scientific (1 mentions)
Quantstudio 1, by Thermo Fisher Scientific (1 mentions)
Aurora a, by Thermo Fisher Scientific (1 mentions)
Cyclin b1, by Thermo Fisher Scientific (1 mentions)
Ccnb1, by Thermo Fisher Scientific (1 mentions)
Hs99999903 m1, by Thermo Fisher Scientific (1 mentions)
Simpliamp thermal cycler, by Thermo Fisher Scientific (1 mentions)
Specific pcr primers, by Thermo Fisher Scientific (1 mentions)
8 protocols using taqman fast advanced cells to ct kit
1
Quantifying Gene Expression via qRT-PCR
2
Antisense Oligomer Transdifferentiation Assay
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Four days after onset of transdifferentation of P1 cells, antisense oligomer (specifically, PMO) treatments were carried out in 96-well plates for an additional 2 days. PMOs were rationally designed to target the nucleotide of interest, but avoid both the native splice acceptor and branch site. PMOs were purchased from GeneTools, LLC including a standard control and were reconstituted as 1-mM stock solutions in water. The oligomer solutions were added to each well at a final concentration of 10 μM and the plate was gently swirled after each oligomer addition. Endo-Porter (8 μM, Gene Tools, SKU:OT-EP-PEG1) was added directly to each well, and the plate was vigorously swirled after each addition. Cells were lysed at the end of the treatment period using Taqman Fast Advanced Cells-to-Ct Kit (Thermo Fisher Scientific, Cat #A35378). PMO sequences were: PMO 1 [5′-GCCTAGCGAGAAGGACATGGTCACC-3′], PMO 2 [5′-CTAGCGAGAAGGACATGGTCACCGC-3′], PMO3[5′-AGGCCTAGCGAGAAGGACATGGTCA-3′], PMO 4 [5′-AGCGAGAAGGACATGGTCACCGCCA-3′], PMO 5 [5′-CGAGAAGGACATGGTCACCGCCACC-3′].
3
RT-qPCR Analysis of Lipid Metabolism Genes
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RNA was extracted and converted to cDNA in one-step using a TaqMan™ Fast Advanced Cells-to-CT™ Kit (Thermo). Relative expression levels of mRNAs encoding the selected genes were measured according to the manufacturer’s protocol by RT-qPCR using specific TaqMan gene expression assays (Thermo, Australia), the following genes were analyzed on a ViiATM 7 Real-Time PCR System using MicroAmp Endura Plates with optical adhesive covers: Lrp8 (Cat. #: 4331182; Assay ID: Mm00474030_m1), Gpx4 (Cat. #: 4351372; Assay ID: Mm04411498_m1), Notch1 (Cat. #: 4331182; Assay ID: Mm00627185_m1) and β-actin (Cat. #: 4448489; Assay ID: Mm01205647_g1). The expression levels of Lrp8 were normalized to β-actin and calculated as fold-change vs WT by the 2–∆∆Ct method.
4
Quantitative Analysis of Cell Cycle Regulators
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HeLa S3 cells were transfected with siCtrl, siIGF1R #1, or #2, and 40 h later, cells were treated with 4 mM thymidine (T1895-10G, Merck) for 24 h. Subsequently, cells were washed and then cultured with prewarmed fresh medium for 8 h. Cells were lysed, treated with DNaseI, and reverse-transcribed into cDNA using the TaqMan Fast Advanced Cells-to-Ct kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions. Quantitative PCR was performed using TaqMan probe-based gene expression analysis and QuantStudio 1 (Thermo Fisher Scientific). The primers and probes purchased from Thermo Fisher Scientific were as follows: cyclin B1 (CCNB1, Hs01030099_m1), Aurora A (AURKA, Hs01582072_m1), Aurora B (AURKB, Hs00945858_g1), CDC25B (CDC25B, Hs01582335_m1), GAPDH (GAPDH, Hs02786624_g1), CENPB (CENPB, Hs00374196_s1), CENPF (CENPF, Hs01118845_m1), CDK1 (CDK1, Hs00938777_m1), PLK1 (PLK1, Hs00983227_m1), and β-actin (ACTB, Hs01060665_g1). Expression of the housekeeping gene ACTN or GAPDH was used as internal control. Relative expression was analyzed by the ∆∆Ct method using Ct values.
5
Quantification of ER Stress Markers in HT-29 Cells
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Total mRNA from HT-29 cells was extracted and reverse transcribed using TaqMan Fast Advanced Cells-to-Ct Kit (Thermo Fisher, A35374) according to the manufacturer’s instructions. Briefly, cells were washed in PBS and lysed for five minutes at room temperature with DNase treatment. Cell lysates (45% v/v for RT reaction) were used for reverse transcription to generate cDNA. 1-2 μL of cDNA was used for Taqman Gene Expression Assays. Expression of mRNA was calculated after normalization to ACTB mRNA. Probes against XBP1s (Hs03929085_g1), XBP1u (Hs02856596_m1), IRE1α(Hs00176385_m1), PERK (Hs00984005_m1), HSPA5 (Hs00607129_gH), DDIT3 (Hs00358796_g1), and ACTB (Hs99999903_m1) were purchased from Thermo Fisher Scientific.
6
RNA Extraction and RT-qPCR Analysis
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Total RNA extraction, reverse transcription, and real-time quantitative PCR (RT-qPCR) amplification was performed using the TaqMan Fast Advanced Cells-to-Ct Kit following the manufacturer's instructions (Thermo Fisher). Briefly, cells were washed in PBS and lysed in solution containing DNase for 5 minutes at room temperature. Lysis was terminated at room temperature by a 2-minute incubation with Stop Solution. Lysates were reverse transcribed in a SimpliAmp Thermal cycler (Life Technologies, Carlsbad, California, USA). Quantification of selected genes was done using specific primers for Ifit2, Ifit3, Irf7, Spp1, Axl, Trem2 and Gapdh (Life Technologies) in a 7500 Fast Real-Time PCR system (Applied Biosystem, Life Technologies). Gapdh was used as housekeeping gene.
7
Total RNA Extraction and qPCR Analysis
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Total RNA was extracted from cells using TRIzol® Reagent (Life Technologies, Cat. No. 15596018), then cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (TOYOBO CO., LTD, Cat. No. FSQ-201). Quantitative real-time PCR was performed using Power Up™ SYBR™ Green Master Mix (Applied Biosystems, Cat. No. A25742) or THUNDERBIRD® Probe qPCR Mix (TOYOBO CO., LTD, Cat. No. QPS-101). As another procedure, using TaqMan™ Fast Advanced Cells-to-CT™ Kit (Invitrogen) in accordance with the manufacturer’s protocol, after cells were lysed cDNA was synthesized and then quantitative real-time PCR was performed. The TaqMan™ IDs (Applied Biosystems) of the gene expression assay were as follows: Gapdh (Mm99999915_g1), Alp (Mm00475834_m1), and Osteocalcin (Mm03413826_mH). Following are the sequences of the specific PCR primers (Life Technologies): Gapdh: 5ʹ-AAATGGTGAAGGTCGGTGG-3ʹ and 5ʹ-TGAAGGGGTCGTTGATGG-3ʹ, Npnt: 5ʹ-CACGAGTAATTACGGTTGACAACAG-3ʹ and 5ʹ-CTGCCGTGGAATGAACACAT-3ʹ.
8
Comprehensive RNA Extraction and qPCR Analysis
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Total RNA was extracted from cells using TRIzol® Reagent (Life Technologies, Cat. No. 15596018), then cDNA was synthesized using ReverTra Ace® qPCR RT Master Mix (TOYOBO CO., LTD, Cat. No. FSQ-201).
Quantitative real-time PCR was performed using Power Up™ SYBR™ Green Master Mix (Applied Biosystems, Cat. No. A25742) or THUNDERBIRD® Probe qPCR Mix (TOYOBO CO., LTD, Cat. No. QPS-101). As another procedure, using TaqMan™ Fast Advanced Cells-to-CT™ Kit (Invitrogen) in accordance with the manufacturer's protocol, after cells were lysed cDNA was synthesized and then quantitative realtime PCR was performed. The TaqMan™ IDs (Applied Biosystems) of the gene expression assay were as follows: Gapdh (Mm99999915_g1), Alp (Mm00475834_m1), and Osteocalcin (Mm03413826_mH). Following are the sequences of the speci c PCR primers (Life Technologies): Gapdh: 5'-AAATGGTGAAGGTCGGTGG-3' and 5'-TGAAGGGGTCGTTGATGG-3', Npnt: 5'-CACGAGTAATTACGGTTGACAACAG - 3' and 5'-CTGCCGTGGAATGAACACAT-3'.
Quantitative real-time PCR was performed using Power Up™ SYBR™ Green Master Mix (Applied Biosystems, Cat. No. A25742) or THUNDERBIRD® Probe qPCR Mix (TOYOBO CO., LTD, Cat. No. QPS-101). As another procedure, using TaqMan™ Fast Advanced Cells-to-CT™ Kit (Invitrogen) in accordance with the manufacturer's protocol, after cells were lysed cDNA was synthesized and then quantitative realtime PCR was performed. The TaqMan™ IDs (Applied Biosystems) of the gene expression assay were as follows: Gapdh (Mm99999915_g1), Alp (Mm00475834_m1), and Osteocalcin (Mm03413826_mH). Following are the sequences of the speci c PCR primers (Life Technologies): Gapdh: 5'-AAATGGTGAAGGTCGGTGG-3' and 5'-TGAAGGGGTCGTTGATGG-3', Npnt: 5'-CACGAGTAATTACGGTTGACAACAG - 3' and 5'-CTGCCGTGGAATGAACACAT-3'.
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