Twenty ml of peripheral blood from patients and normal volunteers was collected in EDTA tubes and centrifuged at 150 x g for 10 minutes and platelet rich plasma (PRP) was carefully aspirated and re centrifuged at 150 x g for 5 minutes to remove remaining red and white cells. PRP was centrifuged again at 1500 x g for 10 min to pellet the platelets. The cell pellet was lysed twice with erythrocyte lysis buffer to remove traces of contaminating red blood cells. The pellet was than washed with phosphate buffered saline and checked for purity in a Cell-Dyn coulter counter (Abbott Diagnostics, Abbott Park, IL).
Extracted molecule
total RNA
Extraction protocol
Total Platelet RNA was extracted using RNAqueous micro RNA isolation kit (Ambion, Austin, TX) following the manufacturer’s directions. Platelets were lysed in lysis buffer containing guanidinium thiocyanate and the cell lysate was mixed with ethanol and applied to a silica based filter that selectively binds RNA. Genomic DNA was removed by DNase treatment. The concentration of the isolated RNA was determined using the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Quality and integrity of the total RNA isolated was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA).Total Platelet RNA was extracted using RNAqueous micro RNA isolation kit (Ambion, Austin, TX) following the manufacturer’s directions. Platelets were lysed in lysis buffer containing guanidinium thiocyanate and the cell lysate was mixed with ethanol and applied to a silica based filter that selectively binds RNA. Genomic DNA was removed by DNase treatment. The concentration of the isolated RNA was determined using the Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). Quality and integrity of the total RNA isolated was assessed on the Agilent 2100 bioanalyzer (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
T7 based RNA amplification was carried out on 10 ng of the isolated platelet total RNA (corresponding to ~ 4-5 ml of collected blood) using Riboamp OA two-round amplification kit as suggested by the manufacturer (Arcturus, Mountain View, CA). Briefly, total RNA was incubated with oligo dT/T7 primers and reverse transcribed into double stranded cDNA. In vitro transcription of the purified cDNA was performed using T7 RNA polymerase at 42ºC for 6 hours. The amplified RNA was purified and subjected to a second round of amplification and biotin labeling using Affymetrix’s IVT labeling kit following the manufacturer’s directions. The yield and integrity of the biotin labeled cRNA were determined using the Nanodrop ND-1000 spectrophotometer and the Agilent 2100 bioanalyzer.
Hybridization protocol
Twenty μg of biotin-labeled RNA was fragmented to ~200 bp size by incubating in fragmentation buffer containing 200 mM Tris-acetate pH 8.2, 500 mM potassium acetate and 500 mM magnesium acetate for 35 minutes at 94ºC prior to hybridization. Fragmented RNA was assessed for relative length on Agilent 2100 bioanalyzer and hybridized to Affymetrix HG-U133 plus 2.0 chips for 16 hours, washed, stained on an Affymetrix fluidics station.
Scan protocol
Hybridized chips were scanned using Affymetrix genechip scanner 3000
Description
two-round amplification
Data processing
Affymetrix GCOS version 1.4 was used to calculate the signal intensity and the percent present calls on the hybridized Affymetrix chip. To select genes differentially expressed between patients and normals, the signal intensity values obtained for probe sets in the microarrays were transformed using an adaptive variance-stabilizing, quantile-normalizing transformation (Munson, P.J., GeneLogic Workshop of Low Level Analysis of Affymetrix GeneChip Data, 2001, software available at http://abs.cit.nih.gov/geneexpression.html.) The transform, termed S10 is scaled to match the logarithm transform, base 10.
