After surgical excision, tissue samples for nucleic acids extraction were immediately sliced into aliquots of about 100 mg, snap-frozen in liquid nitrogen and stored at -80°C until use. For RNA extraction, each liver tissue aliquot (100 mg) was kept on ice, immediately homogenized in 2 ml of TriReagent® (Sigma-Aldrich, St. Louis, MO, USA) and the homogenate stored at -80°C until use. RNA was extracted by guanidinium thiocyanate-phenol-chloroform-based method using TRIReagent® (Sigma-Aldrich, St. Louis, MO, USA)
Label
Cy3
Label protocol
10 ug of total RNA were utilized to synthesize double-stranded cDNA by Superscript® Double-Stranded cDNA Synthesis Kit (Invitrogen, Carlsbad, CA, USA); 1ug of cDNA was labeled by One-Color DNA Labeling Kit (NimbleGen-Roche, Madison, WI, USA) and 4 ug of Cy3-labeled cDNA were hybridized on the array.
Hybridization protocol
Samples were hybridized on the Human Gene Expression 12x135K Arrays (Nimblegen-Roche, Madison, WI, USA) following manufacturer instructions
Scan protocol
Microarrays were scanned with Axon GenePix 4400A microarray scanner setting laser power to 100%. PMT gain was set in order to obtain about 1e-5 normalized counts at 65.000 intensity level for both channels.
Description
Expression analysis of non neoplastic liver tissue
Data processing
Feature data were extracted, summarized using RMA algorithm and quantile normalized using NimbleScan 2.5 software.
