Quantitative RT-PCR Analysis of Cytokine and Chemokine Expression

We performed the experiment protocol described below, referring to a previous report.¹⁴ (link) Total RNA from cultured cells was extracted using an RNA Blood Mini Kit (Qiagen, Hilden, Germany) and was reverse-transcribed into first-strand cDNA (cDNA) using a QuantiTect Reverse Transcription Kit (Qiagen). Quantitative PCR was performed using a Step One Plus (Applied Biosystems, Carlsbad, CA, USA) according to the manufacturer's protocol. Primers and probes were selected from the ABI TaqMan Gene Expression Assay catalog (for human targets: CXCL9, Hs00171065_m1; CXCL10, Hs01124251_g1; CXCL11, Hs04187682_g1; IFNγ, Hs00989291_m1; TNFα, Hs01113624_g1; CCL3, Hs00234142_m1; CCL4, Hs00237011_m1; CCL5, Hs00174575_m1; TRAIL, Hs00921974_m1; and IL-13, Hs00174379_m1; for mouse targets: CXCL9, Mm00434946_m1; CXCL10, Mm00445235_m1; CXCL11, and Mm00444662_m1). Expression of each gene was normalized to that of GAPDH. Fold change was determined by the ΔΔCt method. Each experiment was performed in triplicate.

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Fujii K., Miyahara Y., Harada N., Muraoka D., Komura M., Yamaguchi R., Yagita H., Nakamura J., Sugino S., Okumura S., Imoto S., Miyano S, & Shiku H. (2017). Identification of an immunogenic neo-epitope encoded by mouse sarcoma using CXCR3 ligand mRNAs as sensors. Oncoimmunology, 6(5), e1306617.

Publication 2017

RNA Blood Mini Kit QuantiTect Reverse Transcription Kit Step One Plus

Assay Blood Ccl3 Ccl4 Ccl5 Cdna Cultured cells Cxcl11 Cxcl9 Expression gene Gapdh Human Ifnγ Il 13 Mouse Primers Reverse transcription Step one Tnfα Trail

Corresponding Organization : Mie University

Other organizations : University of Shizuoka, Juntendo University

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Variable analysis

independent variables

Cell culture conditions

dependent variables

Expression of CXCL9, CXCL10, CXCL11, IFNγ, TNFα, CCL3, CCL4, CCL5, TRAIL, and IL-13 genes

control variables

GAPDH gene expression (used for normalization)

controls

No controls explicitly mentioned

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