The clinical protocol called for participants to be treated for up to 48 weeks with peginterferon alpha-2a (Pegasys™, Roche Pharmaceuticals Nutley, NJ) in a dose of 180 µg weekly by self-administered subcutaneous injection and ribavirin (Copegus™, Roche) orally in a dose of 1000 or 1200 mg daily based on body weight of less than 75 kg or equal to or greater than 75 kg.
Extracted molecule
total RNA
Extraction protocol
Samples were shipped overnight by express courier at 4 degrees C to a central repository where RNA was isolated on arrival The PBMC were lysed in 1 ml of TRI reagent (Molecular Research Center Inc: Cincinnati, OH). The PBMC lysate was mixed with 1-Bromo-3-chloropropane (BCP)-phase separation agent for 1 minute, and incubated at room temperature for 15 minutes. After centrifugation for 15 minutes at 12,000 rpm and 4 degrees C, RNA was precipitated from the supernatant overnight at -20 degrees C with an equal volume of isopropanol and 1/10 volume of 7.5 M-ammonium acetate. The precipitate was washed twice with 75% ethanol, and then with 95% ethanol. RNA was briefly air-dried and then further purified using RNeasy columns (Qiagen: Valencia, CA).
Label
biotin
Label protocol
Preparation of cDNA, cRNA, and labeling were carried out according to the protocols recommended by Affymetrix in the GeneChip® Expression Analysis Technical Manual (Affymetrix, Santa Clara, CA)
Hybridization protocol
Following fragmentation, 15 microg of cRNA were mixed with spike controls and 2/3 was hybridized for 17 hr at 45C on GeneChip Human Genome 133A Array. GeneChips were washed and stained in the Affymetrix Fluidics Station per manufacturer's instructions.
Scan protocol
The microarrays were scanned using a dedicated Model 3000 scanner controlled by GCOS software.
Description
Gene Expression for human subject. Human PBMC collected from whole blood. PBMC were collected in sodium heparin-CPT tubes at day 0 (before treatment) and days 1, 2, 7, 14 and 28 after initiation of treatment. Whole blood was diluted with an equal volume (8 ml) of phosphate buffered saline (PBS), carefully layered over a 10 ml Ficoll-Hypaque gradient (Amersham/Pharmacia, Piscataway, NJ) and centrifuged at 800 rpm for 20 minutes at room temperature. The buffy coat layer was transferred to a 15ml RNAse-free tube, diluted with PBS, and centrifuged at 100 x g for 15 minutes at room temperature. The supernatants were discarded and the PBMC were retained.
Data processing
GCOS was used for data processing. The average intensity on each array was normalized by global scaling to a target intensity of 1000. Data were extracted using the Affymetrix Microarray Suite 5 (MAS5) algorithm
