15283-B-Blokker

123 PM tissue biopsies obtained at MIA: an RNA-quality analysis 7 Tissue sampling During theMIA tissue sampleswere obtainedwith CT guided biopsies, using a 12-gauge needle. Immediately after each biopsy the sampled tissue was snap frozen in a 50 ml tube filled with pre-cooled isopentane on dry ice. 190 The samples were then placed in a pre-cooled aluminum vial, stored temporarily in a -80°C freezer and finally transferred into liquid nitrogen storage. Immediately after MIA the body was returned to the mortuary with an ambient temperature of 4°C. The following day tissue samples of approximately 0.5 cm 3 were collected from the same organs in the same subject during CA. Due to logistics at CA, snap freezing immediately after harvesting was not possible in all cases. In two cases the tissue samples were temporarily stored at 4°C before snap freezing. All collected samples were eventually stored in liquid nitrogen until RNA could be isolated. Fresh frozen tissues were either derived from surgically resected tissues or from biopsies (heart). These tissue samples were stored in liquid nitrogen in the Erasmus MC Tissue Bank after being snap frozen using pre-cooled isopentane and liquid nitrogen. Two extra heart samples (1 fromMIA, 1 fresh frozen from the Erasmus MC Tissue Bank) were collected for training purposes. To prevent selection bias, these extra samples were both included in the analyses. RNA extraction, RIN measurement and frozen H&E sections Depending on the size of the sample, 10 to 20 10 μm thick frozen sections were cut on a cryostat microtome (Microm HM560, Adamas, The Netherlands). The sections were transferred to 700 μL Qiazol (Qiagen, Hilden, Germany) and RNA was extracted from these sections using the miRNeasy kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA was eluted in 40 μL RNase-free water and 1 μL of RNase inhibitor (20 U/μL; AB, California, USA) was added to avoid RNA degradation during further handling. TheRNA integrity number (RINvalue) 191 of the extractedRNAand theRNA concentration (ng/μl) was assessed by on-chip-electrophoresis, using the BioAnalyzer (Agilent RNA 6000 Nano kit and BioAnalyzer 2100 Expert, Agilent Technologies, USA). 5μmsectionswerecut andstainedwithHaematoxylinandEosin (H&E) formorphological investigation of the samples used for RNA isolation. The morphologic tissue quality assessment comprised of 1) the representativeness of the sample (yes or no); 2) the presence of necrosis (yes or no); 3) scoring the degree of autolysis: no (morphology unaffected), moderate (mild loss of staining of nuclei; some detachment of endothelium in vessels), or severe (severe/complete loss of staining of nuclei; complete detachment of endothelium in vessels).