cell type: T cells gender: female subject status: heathly control
Treatment protocol
Freshly drawn peripheral blood from healthy controls and vitiligo patinets. Leukocyte-enriched supernatants were separated using a Ficoll-Hypaque density gradient solution (specific gravity 1.077; Pharmacia Biotech, Uppsala, Sweden). The mononuclear cells were then aspirated from the interface, after centrifugation at 250 × g for 25 min. Finally, T cells were obtained using anti-human CD3-coated magnetic beads by the IMag Cell Separation System (BD Bioscience, Franklin Lakes, NJ, USA).
Extracted molecule
total RNA
Extraction protocol
Total RNA extraction was performed using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The extracted RNA was quantified by a ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) at OD260 nm and then qualitied by a Bioanalyzer 2100 (Agilent Technology, USA) using an RNA 6000 labchip kit (Agilent Technologies, Santa Clara, CA, USA).
Label
Cy3
Label protocol
0.2 μg of total RNA was amplified by a Low Input Quick-Amp Labeling kit (Agilent Technologies, USA) and labeled with Cy3 (CyDye, Agilent Technologies, USA) during the in vitro transcription process.
Hybridization protocol
0.6 μg of Cy3-labled cRNA was fragmented to an average size of about 50-100 nucleotides by incubation with fragmentation buffer at 60°C for 30 minutes.
Scan protocol
After washing and drying by nitrogen gun blowing, microarrays are scanned with an Agilent microarray scanner (Agilent Technologies, USA) at 535 nm for Cy3.
Description
T cells isolation from heathly controls PBMC
Data processing
Scanned images are analyzed by Feature extraction10.7.3.1 software (Agilent Technologies, USA), an image analysis and normalization software is used to quantify signal and background intensity for each feature. Raw signal data was normalized by quantile normalization for differential expressed genes discovering.
