Naive CD4+CD62L+ Th lymphocytes from T cell receptor transgenic mice (DO11.10) were cultured in complete RPMI1640 (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, 0.1 mg/ml streptomycin, and 10 µM -mercaptoethanol. Naive CD4+CD62L+ lymphocytes from 6-8 week old DO11.10 mice were isolated as described. Irradiated (30 Gy) BALB/c splenocytes were used as antigen-presenting cells (APC) at a ratio of 5:1. The cognate peptide ova323-339 (R. Volkmer-Engert, Humboldt University of Berlin, Berlin, Germany) was added at 0.5ßµM. For Th1 differentiation, cells were stimulated in the presence of 5 ng/ml recombinant murine IL-12 (R&D Systems) and 5 µg/ml anti-IL-4 antibody (11B11). Dead cells were removed by Ficoll-Histopaque separation. Every 6 days viable Th1 cells were harvested and restimulated under the original conditions, except that 10 ng/ml murine IL-2 (R&D Systems) was added.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted using Trizol reagent (Invitrogen). RNA concentration, purity and integrity were assessed with Agilent 2100 Bioanalyzer, and the RNA 6000 Nano LabChip. Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 oC for 16 h in a rotisserie motor, washed, and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400. Arrays were scanned on an Affymetrix GeneChip Scanner 3000 (MG430Av2 arrays).
Label
biotin
Label protocol
blocking: according to manufactures recommendations - probes were subsequently hybridised with the GeneChip 430A version 2 for 16 hrs at 45 oC- after washing the hybridisation signals were visualised by staining with streptavidin-phycoerythrin and amplification with an anti-streptavidin antibody
Hybridization protocol
Total RNA was extracted using Trizol reagent (Invitrogen). RNA concentration, purity and integrity were assessed with Agilent 2100 Bioanalyzer, and the RNA 6000 Nano LabChip. Ten micrograms of total RNA was reverse-transcribed, followed by cDNA extraction using a PhaseLock gel (Eppendorf), and precipitation with ethanol and ammonium acetate. Biotinylated cRNA was in vitro transcribed using the MEGAscript high yield transcription kit (Ambion) according to the manufacturer's recommendations. Biotinylated cRNA was fragmented, and the hybridization cocktail was prepared according to Affymetrix protocols (15 µg fragmented biotin-labeled cRNA spiked with Eukaryotic Hybridization control). The Murine Genome 430A version 2 GeneChip arrays were loaded with the hybridization cocktail, hybridized at 45 oC for 16 h in a rotisserie motor, washed, and stained with streptavidin-phycoerythrin using the Affymetrix GeneChip Fluidics Workstation 400. Arrays were scanned on an Affymetrix GeneChip Scanner 3000 (MG430Av2 arrays).
Scan protocol
Arrays were scanned on an Affymetrix GeneChip Scanner 3000
Description
no additional information
Data processing
High Performance Chip Data Analysis (HPCDA, unpublished - Joachim R. Gruen); Data were analyzed using the Microarray Suite 5.0 software (Affymetrix). Microarrays were globally normalized and scaled to a trimmed mean expression value of 200. Quality checks were performed according to the manufacturer's recommendations. All arrays were compared to each other, and a relational database was generated using Microsoft Access software, including the following parameters: expression heights, call for presence of transcripts, p value for presence or absence of transcripts, log2 value of fold change and 95% confidence intervals, call for the significance of differentially expression, and the p value for that call. For each transcript the significance of differential expression between the groups of arrays was calculated using strict Bonferroni corrected Welch t-tests. Significantly differentially expressed genes were filtered according to the following criteria: mean fold change >= 2; difference of means >= 200; p-value <= 0,05; and immunoglobulin genes were excluded.
