Leukocyte concentrates obtained from healthy donors (DRK, Berlin, Germany)
Treatment protocol
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Hypaque density gradient centrifugation from leukocyte concentrates obtained from healthy donors (DRK, Berlin, Germany). NK cell subsets were separated by MACS to enrich CD56+ cells (Miltenyi Biotech) and CD3- CD56bright CD62L+, CD3- CD56dim CD62L+ and CD3- CD56dim CD62L- NK cells were sorted using a FACSAria (BD Biosciences).
Growth protocol
Peripheral blood mononuclear cells (PBMC) were isolated by Ficoll Hypaque density gradient centrifugation from leukocyte concentrates obtained from healthy donors (DRK, Berlin, Germany). NK cell subsets were separated by MACS to enrich CD56+ cells (Miltenyi Biotech) and CD3- CD56bright CD62L+, CD3- CD56dim CD62L+ and CD3- CD56dim CD62L- NK cells were sorted using a FACSAria (BD Biosciences).
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using the RNeasy Mini Kit (Quiagene) according to the munufacterers instruction. The amount, purity, and integrity of RNA was assessed for each sample using an Agilent 2100 Bioanalyzer with the RNA 6000 Nano LabChip and a NanoDrop ND-1000 spectrophotometer. Contaminating genomic DNA was removed by an on-column DNA digestion step (Qiagen).
Label
biotin
Label protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacterers instruction. Briefly, after total RNA extraction for three samples per group, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to the 9 GeneChip arrays. The HG-U133_Plus_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Hybridization protocol
Labeling and hybridization of total RNA was performed using the GeneChip 3’ IVT Express Kit from Affymetrix according to the manufacterers instruction. Briefly, after total RNA extraction for three samples per group, 100 ng of total RNA from each cell sample was reverse transcribed, cDNA was extracted, biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized to the 9 GeneChip arrays. The HG-U133_Plus_2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 °C for 16 h in a hybridization oven 640 (Affymetrix), washed and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400.
Scan protocol
Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Description
Gene expression profiles of FACSAria sorted (a) CD3- CD56bright CD62L+, (b) CD3- CD56dim CD62L+ and (c) CD3- CD56dim CD62L- NK cells from human peripheral blood of three donors were compared using Affymetrix GeneChip HG-U133_Plus_2 arrays. After total RNA extraction, reverse transcription, cDNA extraction, the biotinylated cRNA was transcribed, fragmented, and 15 µg cRNA hybridized in triplicates for each of the three groups to the GeneChip arrays. Group1: CD62L+ CD56bright CD3-,. Group2: CD62L+ CD56dim CD3-,. Group3: CD62L- CD56dim CD3-. CD56dim CD62L+ NK cells have an intermediate gene expression profile between CD56bright and CD56dim CD62L- NK cells.
Data processing
Data were analyzed according to High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen) with the Bioretis database (http://www.bioretis-analysis.de/) using the default filter parameters for decreased and increased gene lists (description of database c.f. open access paper Menssen et al., 2009; PMID: 19265543). Chip data included in the Bioretis database were analyzed using the GeneChip Operating Software (GCOS, Affymetrix), version 1.4. Microarrays were globally normalized and scaled to a trimmed mean expression value of 150. Quality checks were performed according to the manufacturer's recommendations. All three chips of one group were compared to any of the three chips of a second group (9 comparisons,between two groups) and all chips were additionally compared within each group,(6 comparisons within each group). The following parameters of absolute and comparative analysis were included in the Bioretis database: expression heights (Signals) and mean, median and standard deviation of Signals of both groups, call for presence of transcripts (Absent, A; Marginal, M; Present, P), p value for presence or absence of transcripts, log2 value of fold change (Signal Log Ratio, SLR) and the calculated fold change as mean values, call for the significance of differentially expression (Change Call: Increase, I; Marginal I, MI; Decrease, D; Marginal D, MD; No Change, NC), and the p value for that call. Additionally – not with GCOS calculated – t tests of log Signals and SLRs were included in the database. For each present transcript the significance of differential expression between the groups of arrays was either calculated using (a) strict Bonferroni corrected Welch t tests between 9 SLR values of group 1 vs group 2 and 6 plus 6 SLR values within both groups (the latter 12 giving a mean SLR value of zero; p-value had to be <= 5.279E-08) or (b) more than 50% of non parametrically calculated Change calls (Mann-Whitney U test, GCOS) i.e. 5 or more of all 9 Change calls have to be in the same direction. Significantly differentially expressed genes were filtered using the both default parameter sets (for up- and downregulated genes separately) of filter criteria; these are a combination of four different queries. Filter criteria were developed with various data sets of GeneChips and validated with the Affymetrix Latin Square dataset as shown in Menssen et al., 2009; PubMedID: 19265543.
