Patients had been fasting over night. Blood was collected in CPT-tubes (Becton Dickinson, USA, #362782). Samples were centrifuged at room temperature for 20 minutes at 1800 g. The cell layer was collected, mixed with PBS (Invitrogen, #14190-094) and centrifuged at room temperature for 15 minutes at 300 g. The supernatant was removed and the pellet was mixed with RLT buffer (Qiagen, RNeasy Mini Kit, #74106, , Valencia, CA, USA) and β-mercaptoethanol (Sigma M-3148) and stored at -70°C until RNA extraction.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Mini Kit (Qiagen).
Label
biotin
Label protocol
Total RNA were amplified and biotin labeled using the Whole Transcript (WT) Sense Target Labeling Protocol
Hybridization protocol
From non-degraded high-quality total RNA we prepared and hybridized biotinylated complementary RNA to Gene 1.1 ST Arrays, and then washed, stained and scanned slides using standardized protocols (Affymetrix Inc., Santa Clara, CA).
Scan protocol
We used standardized Affymetrix protocols (Affymetrix Inc., Santa Clara, CA)
Data processing
Subsequent data analyses were performed using the Affymetrix Expression Console version 1.1. Method = med-polish (rma-bg,quant-norm, sketch=-1, bioc=true, lowprecision=false, usepm=true, target=0, doavg=false)) To allow comparisons of transcript levels between samples, all samples were subjected to an all-probe set scaling to target signal 100.
