Peripheral blood mononuclear cells (PBMC) were collected using standard density centrifugation. After 24h plastic adherence, cells were counted and adjusted to 1-2 x 106 cells/ml in complete medium (RPMI + 10% FCS + 1% Penicillin/Streptomycin). Cell activation was per-formed with CD3/CD28-nanomatrix (TransAct, Miltenyi Biotec, Bergisch-Gladbach, GER) according to manufacturer’s instructions in presence of interleukin 2 (IL-2).
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the Qiagen (Qiagen, Venlo, NED) RNeasy Plus mini kit following the manufacturer's recommendations. The protocol includes gDNA elimination. RNA was quantified using a NanoDrop (ND-1000, Thermo Fisher) spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeled cRNA was prepared from 100 ng RNA using the Quick Amp Labeling kit (Agilent) according to the manufacturer's instructions, using T7 priming and MMLV-RT to generate cDNA. cRNA is then prepared using T7 RNA Polymerase according to the manufacturer's instructions, and labeled with Cy3. RNA was purified by RNAeasy column purification (Qiagen). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol
Samples were hybridized using Agilent 10x blocking agent and 25x fragmentation buffer, and 2xGEx hybridization buffer according to the manufacturer's instructions for 17 hours at 65C.
Scan protocol
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505C) using one color scan setting Agilent HD GX 1Color (Scan Area 61x21.6 mm, Scan resolution 10um, Dye channel is set to Green).
Description
gene expression
Data processing
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE2_107_Sep09 and Grid: 026652_D_F_20100430) to obtain background subtracted and spatially detrended Processed Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
