Franny Jongbloed

71 3 PROTECTION OF FASTING ON IRINOTECAN TOXICITY EDTA. Samples were directly centrifuged (3,500 rpm; 10 min) after which the serum was transferred to a separate tube. Serum aspartate transaminase (AST), alanine transaminase (ALT), lactate dehydrogenase (LDH), urea, and creatinine levels were analyzed at the Central Clinical Chemical Laboratory of the Erasmus University Medical Center. Fifty µL of blood was used to measure the number of leukocytes with a Z-series Coulter Counter (Beckman Coulter, Woerden, The Netherlands). Tissue sampling Livers and tumors were collected and weighed. The median liver lobe was isolated for array analysis and directly stored in RNAlater® Solution (Life Technologies Europe BV, Bleijswijk, the Netherlands) and stored at 4ºC until further analysis. Parts of viable tumor border were identified and also directly stored in RNAlater® until further analysis. RNA isolation Tumor and liver samples were kept at 4ºC in the RNAlater® Solution (Thermo Fisher Scientific™, Breda, the Netherlands) until further analyses. RNA isolation took place between 24 hours and 96 hours after sample collection. Total RNA was extracted via the QIAzol lysis Reagent and miRNAeasy Mini Kits (QIAGEN, Hilden, Germany), according to Qiagen protocol. Concentrated buffers RPE and RWT (QIAGEN) for washing of membrane-bound RNA and purification were added mechanically by using the QIAcube (QIAGEN, Hilden, Germany) via the miRNeasy program. Subsequently, isolated RNA was stored at -80°C. RNA concentrations were measured using the Nanodrop (Thermo Fisher Scientific™, Breda, the Netherlands) and RNA quality was assessed using the 2100 Bio-Analyzer (Agilent Technologies, Amstelveen, the Netherlands), according to manufacturer’s instructions. The RNA quality was quantitatively expressed as the RNA Integrity Number (RIN, range 0-10). Out of the six tumor samples and six liver samples per group, the four samples with the highest RIN were used for microarray analyses. RIN-values of the tumor samples ranged between 7.8 and 10, the RIN-values of the liver samples ranged between 7.6 and 8.6. Array analysis Microarray hybridization was done at the Microarray Department of the University of Amsterdam (the Netherlands) to Affymetrix HT MG-430 PM Array Plates, according to the Affymetrix protocols. For each group, four biological replicates were used. The output of the hybridization contained raw mean expression data put into CEL files. Subsequent quality control and normalization were done using the pipeline at the www.arrayanalysis. org website (Maastricht University, the Netherlands) 32 . Normalization was performed via the Robust Multichip Average (RMA) algorithm, and the output of the normalization