Anne-Marie Koop

4 181 Organs and weights After catheterization, blood and organs were taken out. The heart was prepared and divided into the right atrium, left atrium, left ventricle (LV) plus septum, and RV and individually weighted. The liver lobe was weighted as well before and after overnight at 65 °C. The liver wet/dry weight ratio was determined. The heart was snap-frozen and stored at -80 °C for further analyses. One third of the RV and LV including septum, lung and liver were fixated in formalin and paraffin sections were made. Real-time polymerase chain reaction (qPCR) RNA was extracted from nitrogen snap-frozen RV’s using TRIzol reagent (Invitrogen Corporation, Carlsbad, CA, USA). RNA concentration and purity was assessed using Nanodrop spectrophotometer (Nanodrop 1000, Thermo Scientific, Breda, The Netherlands), where after cCNA was derived. Gene expression was measured with Absolute qPCR SYBR Green ROX mix (abgene, Epsom, UK) in the present of 7.5ng cDNA and 200 nM forward and reverse primers. Real time quantitative reverse transcription (qRT-PCR) was performed by the Biorad CFX384 (Bio-Rad, Veenendaal, the Netherlands) according standard protocol. Measured mRNA expression was corrected for expression of housekeeping gene GAPDH.