Both positive and negative control samples were also measured simultaneously in each assay for quality control.
In addition, a 477-bp amplicon for the IGF2 region was included in the assay for quality assessment of bisulfite-treated DNA.
Prior to microarray hybridizations, amplified products were quantified using a spectrophotometer (Nanodrop, Wilmington, Delaware, USA) and subjected to BioAnalyzer to assay for quality (Agilent Technologies, Santa Clara, California, USA).
Total RNA samples were assayed for quality using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA) and for yield using Ribogreen (Ambion, Austin, TX) metrics prior to amplification.
Extracted total RNAs were further assayed for quality by Agilent 2100 bioanalyzer.
A total of 68 samples from a single blood pool was assayed for quality control purposes.
Isolated total RNA samples were then assayed for quality (Agilent Bioanalyzer) and yield (Ribogreen) metrics prior to amplification.
RNA was assayed for quality and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON) and RNA 6000 Nano LabChip kit (Caliper Technologies, Hopkinton, MA).
Resultant RNA was quantified via spectrophotometry and assayed for quality on Agilent (Santa Clara, CA, USA) and Bio-Rad (Hercules, CA, USA) bioanalyzers at the Rocky Mountain Regional Center for Excellence RMRCEE) Genomics Core at CSU.
For the ICGC cohort, tumour cells were enriched by macro-dissection, then RNA was extracted from tumors using Qiagen Allprep® (Qiagen, Valencia, CA) in accordance with the manufacturer's instructions, assayed for quality on an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA), and hybridized to Illumina Human HT-12 V4 microarrays.
Total RNA from a pooled sample was prepared using TRIZOL Reagent Invitrogen Life Technologiess, Carlsbad, CA) following the manufacturer's instructions and was assayed for quality and quantified using an Agilent 2100 Bioanalyzer (Agilent Technologies, Mississauga, ON) and RNA 6000 Nano LabChip kit (Caliper Technologies, Hopkinton, MA).
