Cardiac Tissue RNA Isolation and Analysis

Total RNA from cardiac human tissue was homogenized in RNA-Bee (Amsbio, Abbingdon, UK) and the Phenol/Chloroform (Roth, Karlsruhe, Germany) extraction protocol was used for the RNA isolation. The Caliper LabChip bioanalyzer (Agilent Technologies, Ratingen Germany) was used to analyze the purity of the isolated RNA. Quantitative real-time PCR were performed using the Brilliant SYBR Green qPCR master mix (Applied Biosystems, Foster City, CA, USA). The relative amount of target mRNA was determined using the comparative threshold (Ct) method as previously described [34 (link)]. The mRNA contents of target genes were normalized to the expression of hypoxanthine phosphoribosyl transferase (HPRT).

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Barcena M.L., Pozdniakova S., Haritonow N., Breiter P., Kühl A.A., Milting H., Baczko I., Ladilov Y, & Regitz-Zagrosek V. (2020). Dilated cardiomyopathy impairs mitochondrial biogenesis and promotes inflammation in an age- and sex-dependent manner. Aging (Albany NY), 12(23), 24117-24133.

Publication 2020

RNA-Bee Phenol/Chloroform Caliper LabChip bioanalyzer Brilliant SYBR Green qPCR master mix

Brilliant green Cardiac Chloroform Genes Human Hypoxanthine phosphoribosyl transferase Mrna Phenol Quantitative real time pcr Tissue

Corresponding Organization : German Centre for Cardiovascular Research

Other organizations : Charité - Universitätsmedizin Berlin, Barcelona Institute for Global Health, Humboldt-Universität zu Berlin, Freie Universität Berlin, Ruhr University Bochum, Heart and Diabetes Center North Rhine-Westphalia, University of Szeged, University Hospital of Zurich, University of Zurich

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Variable analysis

independent variables

RNA isolation protocol (Phenol/Chloroform extraction)

dependent variables

Purity of isolated RNA (measured by Caliper LabChip bioanalyzer)
Relative amount of target mRNA (measured by qPCR)

control variables

Tissue type (cardiac human tissue)
RNA homogenization method (RNA-Bee)
QPCR master mix (Brilliant SYBR Green qPCR master mix)
Normalization of target mRNA to HPRT expression

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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