CD4 T cells sorted from total PBMC of healthy subject
Treatment protocol
PBMC were thawed in the presence of 10 mg/ml DNase I (Roche Diagnostics GmbH, Mannheim, Germany), then resuspended in Hanks’ medium without CaCl2 (supplemented with 1% fetal calf serum and 1mM EDTA) and stained with FITC-labeled CD4, APC-labeled CD20 and PE-labeled CD11c monoclonal antibodies (all purchased from BD-Pharmingen, Erembodegem, Belgium). The CD4 and CD20 positive cell populations were sorted out by flow cytometry using a FacsVantageTM cell sorter (BD-Pharmingen). Typically, 25 to 35% of the total PBMC population was positive for the CD4 staining versus 5 to 15% for the CD20 marker.
Growth protocol
None
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from both cell subsets using the Nucleospin® RNA II extraction kit (Macherey-Nagel GmbH & Co, Düren, Germany), including DNase treatment of the samples. RNA quality was assessed by capillary electrophoresis using RNA 6000 Pico LabChips on an Agilent 2100 Bioanalyzer (Agilent Technologies Inc, Palo Alto, CA, USA).
Label
SA-PE
Description
Labeling of RNA (cRNA synthesis) was performed according to a standard Affymetrix® procedure (One-Cycle Target Labeling kit, Affymetrix UK Ltd, High Wycombe, United Kingdom); briefly total RNA was first reverse transcribed into single-stranded cDNA using a T7-Oligo(dT) Promoter Primer and Superscript II reverse transcriptase. Next, Rnase H was added together with E. Coli DNA polymerase I and E. Coli DNA ligase, followed by a short incubation with T4 DNA polymerase in order to achieve synthesis of the second-strand cDNA. The double-stranded cDNA was purified and served as a template for the overnight in vitro transcription reaction, carried out in the presence of T7 RNA polymerase and a biotinylated nucleotide analog/ribonucleotide mix. At the end of this procedure, the biotinylated complementary RNA (cRNA) was cleaned, and fragmented by a 35 minute incubation at 95°c. Because a minimal amount of 1 ug total RNA is required for the synthesis of cRNA, not all the CD20 cell samples could be processed, accounting for the lower number of CD20 cell hybridizations.
Data processing
Data were retrieved on GCOS software for the initial normalization and analysis steps. The number of positive genes was between 40 and 51% on each slide. After scaling on all probe set (to a value of 100), the amplification scale was reported between 1.5 and 2.5 for the CD4 slides, and between 5 and 10 for the CD20 slides. The signals given by the poly-A RNA controls, hybridization controls and housekeeping/control genes were indicative of the good quality of the amplification and hybridization procedures.
