Quantitative Analysis of Inflammatory Gene Expression

Cells were washed with prechilled PBS and homogenized in TRIzol reagent (Invitrogen, Carlsbad, CA). mRNA was extracted according to manufacturer's protocol followed by cDNA synthesis using MultiScribe Reverse Transcriptase (Invitrogen) and qPCR using StepOne Real-Time PCR Systems (Applied Biosystems, Foster City, CA). Predeveloped probes for Tnf (Mm00443258_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), Ccl2 (Mm00441242_m1), and Hprt1 (Mm01545399_m1) (all from Applied Biosystems) were used for mRNA analyses. Threshold cycle values for each sample were used to calculate the number of cell equivalents using the standard curve method.^{11 (link)} The data were normalized to the housekeeping Hprt1 mRNA levels and expressed as percentage change of the control group.

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Agalave N.M., Rudjito R., Farinotti A.B., Khoonsari P.E., Sandor K., Nomura Y., Szabo-Pardi T.A., Urbina C.M., Palada V., Price T.J., Erlandsson Harris H., Burton M.D., Kultima K, & Svensson C.I. (2020). Sex-dependent role of microglia in disulfide high mobility group box 1 protein-mediated mechanical hypersensitivity. Pain, 162(2), 446-458.

Publication 2020

TRIzol reagent MultiScribe Reverse Transcriptase StepOne Real-Time PCR Systems Mm00443258_m1 (Mm00434228_m1 (Mm00446190_m1), (Mm00441242_m1), Mm01545399_m1)

Ccl2 Cdna Cells Il1b Mrna Reverse transcriptase Synthesis Trizol

Corresponding Organization :

Other organizations : Karolinska Institutet, The University of Texas at Dallas, Uppsala University

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

MRNA levels of Tnf, Il1b, Il6, Ccl2

control variables

Cells were washed with prechilled PBS
Hprt1 mRNA levels were used for normalization

controls

Positive control: None mentioned
Negative control: None mentioned

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