Quantitative Transcriptome Analysis of Immune Cells in Myocarditis

Mouse hearts were perfused and digested with collagenase II (10,000 U/mL, Worthington, CLS-2) to release immune cells using a gentleMACs Dissociator. Immune cells were isolated using MACs magnetic microbeads for CD11b or CD45 (Miltenyi Biotec, Auburn, CA). Trizol Reagent and the PureLink Micro-to-Midi system (Invitrogen, Carlsbad, CA) were used for purification of RNA from total hearts or immune cells. Human RNA was isolated from heart biopsies of formalin-fixed, paraffin-embedded (FFPE) blocks from patients with histology-verified myocarditis. Human RNA was processed using a PureLink FFPE RNA Isolation Kit (Invitrogen, Carlsbad, CA). Quantitative RT-PCR (qRT-PCR) of gene expression was measured using Assay-on-Demand probe sets (Applied Biosystems, Foster City, CA) or RT²qPCR Primer Assay (Qiagen, Venlo, Netherlands) and reactions analyzed using the ABI 7000 Taqman system, as previously [13 (link), 21 (link)]. Hypoxanthine phosphoribosyltransferase (Hprt) was used for normalization.

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Fairweather D., Coronado M.J., Garton A.E., Dziedzic J.L., Bucek A., Cooper LT J.r., Brandt J.E., Alikhan F.S., Wang H., Endres C.J., Choi J., Pomper M.G, & Guilarte T.R. (2014). Sex differences in translocator protein 18 kDa (TSPO) in the heart: implications for imaging myocardial inflammation. Journal of cardiovascular translational research, 7(2), 192-202.

Publication 2014

collagenase II Trizol Reagent PureLink Micro-to-Midi system PureLink FFPE RNA Isolation Kit RT2qPCR Primer Assay

Assay Biopsies Cd11b Cells Collagenase Embedded paraffin Formalin Gene expression Heart blocks Hearts Human Hypoxanthine phosphoribosyltransferase Isolation Microbeads Mouse Myocarditis Paraffin Patients Primer Rt pcr Trizol

Corresponding Organization : Columbia University

Other organizations : Mayo Clinic

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Variable analysis

independent variables

Collagenase II concentration (10,000 U/mL)

dependent variables

Gene expression measured by quantitative RT-PCR (qRT-PCR)

control variables

Hypoxanthine phosphoribosyltransferase (Hprt) used for normalization

controls

Positive control: Not specified
Negative control: Not specified

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