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Steponeplus 96 well real time pcr

Manufactured by Thermo Fisher Scientific
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The StepOnePlus 96-well Real-Time PCR System is a qPCR instrument designed for precise, sensitive, and reliable gene expression analysis. It features a 96-well thermal block, a high-performance optical system, and advanced software for data analysis.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (4 mentions) 18s ribosomal rna, by Thermo Fisher Scientific (3 mentions) Eagletaq universal master mix, by Roche (3 mentions) Protoscript 2 rt kit, by New England Biolabs (2 mentions) Slc2a1, by Thermo Fisher Scientific (2 mentions) Hif 1α, by Thermo Fisher Scientific (2 mentions) Protoscript 2 reverse transcriptase, by New England Biolabs (2 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Protoscript 2 rt, by New England Biolabs (1 mentions) Trizol, by Thermo Fisher Scientific (1 mentions) Cytofix cytoperm fixation and permeabilization solution, by BD (1 mentions) Facscelesta, by BD (1 mentions)

5 protocols using steponeplus 96 well real time pcr

1

Quantitative RT-PCR Analysis of Hypoxia Markers

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Total RNA was extracted from cells at indicated time points using TRIzol reagent (Life Technologies). cDNA was generated with ProtoScript II RT Kit (New England BioLabs) and real-time PCR was performed using Eagle Taq Universal Master Mix (Roche) and the Applied Biosystems StepOnePlus™ 96-well Real-Time PCR. Predesigned TaqMan® Assays were purchased from Applied Biosystems: Hif1α (Mm00468869_m1), Slc2a1 (Mm00441473_m1), Hk2 (Mm00443394_m1), Sell (Mm00441291_m1). 18s ribosomal RNA (Life Technologies) was used as an endogenous control.
Xu W., Patel C.H., Zhao L., Sun I.H., Oh M.H., Sun I.M., Helms R.S., Wen J, & Powell J.D. (2023). GOT1 regulates CD8+ effector and memory T cell generation. Cell reports, 42(1), 111987.
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2

Quantitative RNA Expression Analysis

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Total RNA from cells was extracted with TRIzol Reagent (Life Technologies, 15596018). RNA was converted to cDNA using the ProtoScript II Reverse Transcriptase (New England BioLabs, M0368). qRT-PCR was performed with PowerSYBR® Green PCR Master Mix (ThermoFisher). 5’ ETS pre-rRNA, Ifng, and Actb primers were ordered from IDT:
5’ ETS: F 5’-TTTTGGGGAGGTGGAGAGTC-3’ | R 5’-AGAGAACTCCGGAGCACCAC-3’. Ifng: F 5’-CGGCACAGTCATTGAAAGCCTA-3’ | R 5’-GTTGCTGATGGCCTGATTGTC-3’. Actb: F 5’-CATTGCTGACAGGATGCAGAAGG-3’ | R 5’- TGCTGGAAGGTGGACAGTGAGG-3’. Reactions were run using an Applied Biosystems StepOnePlus 96-well Real-Time PCR. ΔΔCt values were normalized to β-actin and a control group of interest.
Claiborne M.D., Sengupta S., Zhao L., Arwood M.L., Sun I.M., Wen J., Thompson E.A., Mitchell-Flack M., Laiho M, & Powell J.D. (2022). Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge. Science immunology, 7(71), eabh4271.
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3

Gata6 expression in IL-4-stimulated peritoneal cells

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Attached peritoneal exudate cells (0.5 × 106) were incubated with/without recombinant IL-4 (10ng/mL) or with/without 1μM FOXO inhibitor AS184285 (Calbiochem) and collected at indicated time points. Total RNA was extracted with Trizol (Life Technologies) and was converted to cDNA using the ProtoScript II RT (New England BioLabs). Predesigned TaqMan® Assays were purchased from Applied Biosystems: Gata6 (Mm00802636_m1). qPCR was performed using Eagle Taq Universal Master Mix (Roche) and Applied Biosystems StepOnePlus 96-well Real-Time PCR. Ct values were normalized to 18S ribosomal RNA (Life Technologies) and relative quantification of gene expression ratio was shown in comparison to control.
Oh M.H., Collins S.L., Sun I.H., Tam A.J., Patel C.H., Arwood M.L., Chan-Li Y., Powell J.D, & Horton M.R. (2017). mTORC2 signaling selectively regulates generation and function of tissue-resident peritoneal macrophages. Cell reports, 20(10), 2439-2454.
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4

Quantitative RT-PCR Analysis of Hypoxia Markers

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Total RNA was extracted from cells at indicated time points using TRIzol reagent (Life Technologies). cDNA was generated with ProtoScript II RT Kit (New England BioLabs) and real-time PCR was performed using Eagle Taq Universal Master Mix (Roche) and the Applied Biosystems StepOnePlus™ 96-well Real-Time PCR. Predesigned TaqMan® Assays were purchased from Applied Biosystems: Hif1α (Mm00468869_m1), Slc2a1 (Mm00441473_m1), Hk2 (Mm00443394_m1), Sell (Mm00441291_m1). 18s ribosomal RNA (Life Technologies) was used as an endogenous control.
Xu W., Patel C.H., Zhao L., Sun I.H., Oh M.H., Sun I.M., Helms R.S., Wen J, & Powell J.D. (2023). GOT1 regulates CD8+ effector and memory T cell generation. Cell reports, 42(1), 111987.
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5

Comprehensive Flow Cytometry and qPCR Analysis

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Flow cytometry experiments were performed using a FACS Celesta (BD Biosciences) and analyzed using FlowJo software (v.10.3). Intracellular staining for cytokines, pCAD, and pS6 was performed with BD Cytofix/Cytoperm Fixation and Permeabilization solution (BD Biosciences). For PRIME Flow® studies, samples were prepared in accordance with the provided protocol (ThermoFisher). qPCR Total RNA from cells was extracted with TRIzol Reagent (Life Technologies 15596018) RNA was converted to cDNA using the ProtoScript II Reverse Transcriptase (New England BioLabs M0368). qRT-PCR was performed with PowerTrack SYBRGreen Mix (ThermoFisher). 5' ETS pre-rRNA, Ifng, and β-actin probes were ordered from IDT: 5' ETS: F 5'-TTTTGGGGAGGTGGAGAGTC-3' | R 5'-AGAGAACTCCGGAGCACCAC-3' Ifng: F 5'-CGGCACAGTCATTGAAAGCCTA-3' | R 5'-GTTGCTGATGGCCTGATTGTC-3' β-actin: F 5'-CATTGCTGACAGGATGCAGAAGG-3' | R 5'-TGCTGGAAGGTGGACAGTGAGG-3' Reactions were run using an Applied Biosystems StepOnePlus 96-well Real-Time PCR. ΔΔCt values were normalized to β-actin and a control group of interest.
Claiborne M.D., Sengupta S., Zhao L., Arwood M.L., Sun I., Wen J., Thompson E.A., Mitchell-Flack M., Laiho M., & Powell J.D. (2021). Persistent CAD activity in memory CD8+ T cells supports rRNA synthesis and ribosomal biogenesis required at rechallenge.
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