disease state: Healthy Control cell type: CD4+ T cells gender: male age: 63 Stage: 0 (Healthy Control) time-point post activation: 4 h
Treatment protocol
CD4+ T cells were isolated from peripheral blood samples. The isolated cells were incubated in 25 mm flasks overnight (RPMI 1640 medium with 10 % v/v heat inactivated fetal bovine serum and 1 % v/v penicillin-streptomycin (100 U/ml)) . At the following day, the cells were seeded in a 96 well format (350,000 cells/well) and were in vitro activated by αCD2/αCD3/αCD28 beads. Cellular samples were collected from different wells at 0, 2, 4, 8, 12 and 24 h after activation for subsequent RNA extraction.
Extracted molecule
total RNA
Extraction protocol
RNA was prepared using the miRNeasy Micro Kit (Qiagen, Hilden, Germany) following the manufacturer's recommendations.
Label
Cy3
Label protocol
Cyanine-3 (Cy3) labeling of cRNA was performed using 25 ng total RNA and following the instructions from Low Input Quick Amp Labeling Kit (One-Color) (Agilent Technologies, Santa Clara, CA, USA). The cRNA samples were purified by RNeasy Mini Kit (Qiagen, Hilden, Germany) and concentrations of cRNAS were determined using a NanoDrop™ 2000c Spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Hybridization protocol
labeled samples were hybridized to Human SurePrint G3 Gene Expression Microarrays (V3, G4851C; Agilent Technologies, Santa Clara, CA, USA) following the inctructions from Gene Expression Hybridization Kit (Agilent Technologies)
Scan protocol
Array slides were scanned with a resolution of 3 μm.
Data processing
Raw data was extracted using Feature Extraction software (Agilent Technologies, Santa Clara, CA, USA). Raw expression values were processed using the limma R-package. A background correction (method = normexp, offset = 16) was conducted
