ratio experiment by combining dye-reversal ratio profiles
CD4+ alpha beta T cells were positively sorted from LatY136F or from wild-type spleen using a MACS and anti-CD4 beads. Gamma delta T cells were sorted from the spleen of Lat3YF and of Eb-/- mice using MACS and anti-CD5 beads. In vitro differentiated Th1 and Th2 cells were generated by culturing naive wild-type CD4+ T cells for 4 days in complete RPMI in the presence of anti-CD3 (1 µg/ml) plus anti-CD28 (1 µg/ml) with the addition of IL-12 (10ng/ml) and of anti-IL-4 (5µg/ml) for Th1-polarizing conditions, and of IL-4 (20ng/ml) plus anti-IFN-gamma (10µg/ml) for Th2-polarizing conditions. RNA was prepared using the Trizol method followed by DNAse digestion. Microarray experiments were carried out as two-color ratio hybridizations. RNA labeling was performed with a Fluorescent Linear Amplification Kit (Agilent Technologies). In brief, cDNA was reverse transcribed from 4 µg total RNA with an oligo-dT-T7 promoter primer and MMLV-RT. Second strand synthesis was carried out with random hexamers. Fluorescent antisense cRNA was synthesized with either cyanine 3-CTP (Cy3-CTP) or cyanine 5-CTP (Cy5-CTP) and T7 polymerase. The fluorescent-labeled antisense cRNA was precipitated over night with LiCl, ethanol washed and resuspended in water. The purified products were quantified at A552nm for Cy3-CTP and A650nm for Cy5-CTP and labeling efficiency was verified with a Nanodrop photometer (Kisker, Steinfurt, Germany). Before hybridization, 1.25 µg labeled cRNA of each product were fragmented and mixed with control targets and hybridization buffer according to the supplier's protocol (Agilent Technologies). Hybridizations were done over night for approximately 17 h at 60°C. The slides were washed according to the manufacturer's manual and scanning of microarrays was performed with 5 µm resolution using a DNA microarray laser scanner (Agilent Technologies). In order to compensate dye specific effects, and to ensure statistically relevant data, a color swap dye reversal was performed. Features were extracted with an image analysis tool Version A 6.1.1 (Agilent Technologies) using default settings. Data analysis was carried out on the Rosetta Inpharmatics platform Resolver Built 4.0. Expression patterns were identified by stringent data analysis using anti-correlation of the dye reversal ratio profiles and a 2fold expression cut-off of the ratio experiments. By combining the first and the second criteria of analysis we filtered out data points with low P-value (P-value < 0.01), making the analysis robust and reproducible. Additionally, by using this strategy we did the data selection independent of error models implemented in the Rosetta Resolver system. Keywords: other
