To examine gene expression in purified cellular fractions, normal human blood was collected from 2 donors as per institutional policy. The granulocyte (CD15+), monocyte (CD14+), T cell (CD3+), B cell (Non-PC gated, CD19+), and PC (CD27++CD38++) fractions from peripheral blood were separated. White blood cells were washed with FACS buffer (PBS + 0.5%BSA + 2mM EDTA (Gibco)) and incubated with 20% heat-inactivated FBS for 10-15 minutes on ice. The following mAbs were added directly to the cells: CD15 (HI98); CD14 (M5E2), CD3 (UCHT1), CD27 (M-T271), CD38 (HB7), and DAPI (Molecular probes). Cells were sorted on a Becton Dickinson FACS Aria II flow cytometer. All sorted fractions were collected in FACS buffer, centrifuged, and the resulting cell pellet was suspended in RNA lysis buffer (Ambion).
Extracted molecule
total RNA
Extraction protocol
Zymo RNA MicroPrep kit extraction of total RNA was performed according to the manufacturer's instructions.
Label
biotin
Label protocol
Biotinylated cRNA were prepared according to the standard Ambion MessageAmp II-Biotin Enhanced Single Round aRNA Amplification protocol from 75 ng total RNA.
Hybridization protocol
Following fragmentation, 10 ug of cRNA were hybridized for 16 hr at 45C on HGU133 Plus 2.0 Array. GeneChips were washed and stained in an Affymetrix Fluidics Station 450.
Scan protocol
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000 7G.
