Morning leukopheresis, 100ml, followed by standard Ficoll protocol, buffy coat separation and isolation of CD8+ T cells using negative selection kit from StemCell (StemSep Human CD8+ T Cell Enrichment Cocktail).
Extracted molecule
total RNA
Extraction protocol
Qiagen RNeasy kit
Label
biotin
Label protocol
5-6 µg of total RNA was reverse-transcribed using SuperScript II reverse transcriptase (Invitrogen) and second strand synthesis was performed using SuperScript Double-Stranded cDNA Synthesis Kit (cat. # 11917-010, Invitrogen) per manufacturer’s instructions. cDNA was purified using cDNA cleanup columns of the GeneChip Sample Cleanup Module (cat. # 900371, Affymetrix). In vitro transcription of biotin-labeled cRNA was performed with the BioArray HighYield RNA Transcript Labeling kit (cat. # 900182, Enzo Life Sciences) following manufacturer’s instructions. cRNA was purified using IVT cleanup columns of the GeneChip Sample Cleanup Module (cat. # 900371, Affymetrix)
Hybridization protocol
20 µg of biotin-labeled cRNA was fragmented following GeneChip Sample Cleanup Module protocol (cat. # 900371, Affymetrix). Fragmented cRNA was hybridized to Hu133A microarrays (Affymetrix) for 16hr in GeneChip Hybridization Oven 640 (Affymetrix) in hybridization cocktail. Washing, staining and scanning were performed on-site using Fluidics Station 400 (Affymetrix) for washes.
Scan protocol
GeneArray Scanner (Agilent) was used for scans. Initial image processing was performed using GCOS ver. 1.2 (Affymetrix) with the following settings Alpha1: 0.05, Alpha2: 0.065, Tau: 0.015, Gamma1H: 0.0045, Gamma1L: 0.0045, Gamma2H: 0.006, Gamma2L: 0.006, Target: 500, NF: 1. Scaling factors ranged from 1.2 to 4.8
