Tonsils are cut in small pieces with cleaned surgical scissors on an agar plate in a small volume of culture media. They are then minced through a cell strainer sitting on an agar plate, using the handle-end of a 5 mL syringe. • The resulting suspension on the floor of the agar plate is pipetted (via sterile transfer pipette) through another cell strainer. The cell suspension is layered onto room-temperature FicollPaque PLUS (‘Ficoll’): typically, 3 mL cell suspension is layered onto 9-10 mL Ficoll in a 15 mL (red-top) Falcon tube. • These tubes are centrifuged (2000 rpm / 20°C / 20 minutes, without braking on deceleration). The lymphocyte layer (often somewhat adherent to the Falcon tube wall, and generally less well-defined than with density gradient separation of blood) is removed by pipetting, and lymphocytes are washed twice in cold culture media. • After resuspension (e.g. in 10 mL WM), cells are passed through a cell strainer before counting. Cells are then stained for surface markers and FACS sorted for different T cell population.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using miRVANA mRNA isolation kit (Ambion) according to the manufacturer's protocol
Label
Cy3
Label protocol
not provided
Hybridization protocol
not provided
Scan protocol
In association with the Peter Wills Bioinformatics Centre (PWBC) at the Garvan Institute, NSW, the data was scanned on the Ramaciotti Centre's Affymetrix scanner
Description
microRNA expression in Naïve T cells The provided raw data has been summarized at the miRNA level. Probe-level raw data are not available.
Data processing
Following scanning of the arrays, the raw images are processed by “Feature Extractor” which produces raw (‘TXT’), and summarised (‘Gene View’) data for each array, as well a QC report. On this array, there are 13,737 probes, representing 821 miR’s. Of these, there are 723 human miR’s, with the remaining 98 miR’s being derived from other species, or controls. The summarised data was imported into GeneSpring. Since this processed data contains negative values, it was ensured that all measurements are above 1.0, and then further normalise the data, by normalising each miR to the median of the measurements for that miR. That is, expression level of each miR into was converted in to an expression ratio.
