Bioanalyzer assays

Total RNA was isolated from sections of snap-frozen E478 xenograft tissue, human tumor tissues and from 80% confluent SKRC7 and SKRC7-VHL^HA cells using TRIzol reagent (Life Technologies, ThermoFisher Scientific, Waltham, MA, USA) according to the manufacturers’ instructions. RNA quality was estimated based on relative levels of 28S, 18S and 5S rRNA bands on agarose gel and with Bioanalyzer assays (Agilent Technologies, Amstelveen, The Netherlands). RNA was reverse transcribed to cDNA using Superscript II reverse transcriptase (Invitrogen, ThermoFisher Scientific, Waltham, MA, USA) and random hexamer primers (Promega, Madison, WI, USA) according to standard protocols. Next, cDNA was purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel, Düren, Germany). For quality control, cDNA was subjected to PCR for reference gene hydroxymethylbilane Synthase (HBMS) with forward primer HMBSFw (5′-CTGGTAACGGCAATGCGGCT-3′) and reverse primer HMBSRv (5′-TTCTTCTCCAGGGCATGTTC-3′) using AmpliTaq Gold 360 master mix (Applied Biosystems, ThermoFisher Scientific, Waltham, MA USA).

For the analysis of brain and PBMC mRNA, mice were killed by decapitation while trunk blood was collected in EDTA tubes. Brains were subsequently dissected, deprived of cerebellum and pons-medulla, and stored at −80 °C. For correlation analysis, blood (200 μl) was instead collected via submandibular puncture. PBMCs were extracted from whole blood using RBC lysis buffer according to the manufacturer's protocol and RNA was subsequently isolated using the Total RNA purification Plus Kit (Norgen Biotek, Thorold, Ontario, Canada). Brain total RNA was isolated using standard Trizol (Invitrogen, Carlsbad, CA, USA) protocol and subsequently purified with DNAse treatment. RNA quantity was determined by absorbance at 260 nm using a NanoDrop UV-VIS spectrophotometer and the quality of RNA was controlled in random samples by running bioanalyzer assays (Agilent Technologies, Palo Alto, CA, USA).