Animals were sacrificed and organs were rapidly frozen in liquid nitrogen, and subsequently stored at <ETH>80oC. Tissues were mechanically homogenized with a polytron and total cellular RNA was extracted using a commercially available reagent (TMTRIzol; Invitrogen). The poly-(A)+ fraction of the RNA was isolated by chromatography with oligo d(T) cellulose beads. For cDNA labeling, either 25 mg of total RNA or 2.5 mg of poly-(A) RNA was converted into cDNA using reverse transcriptase (RT; Super Script II, Invitrogen) and alternatively Cy5 or Cy3-labeled dCTP (1mM, Perkin Elmer-Cetus/NEN, Boston) in a reaction mixture containing 1.5 mL oligo (dT) (100pmol/ mL), 3 mL dNTP-dCTP (6.67mM each), 1 mL dCTP (2mM), 4 mL DTT (100mM), 8 mL 5 X RT Buffer (Invitrogen, California). The reactions were carried out at 42¼C for 3 hrs, after which the RNA was degraded by the addition of 0.5 mL RNase A (1 mg/mL) and 1.5 mL RNaseH 5 units/mL). Labeled cDNA was separated from unincorporated nucleotides (Qiagen column) and further concentrated by evaporation under vacuum. The arrays were pre-hybridized for 1-2hrs with DIGEasy hybridization buffer (Roche) containing 10ug/ml denatured salmon sperm DNA, and 10ug/ml yeast tRNA. The Cy5 and Cy3 labelled cDNAs were combined and hybridized in the same medium and incubated with the arrays for 16-18hrs at 37Co. Finally, the arrays were washed 3 x 10 mins in 0.1 x SSC (20 X SSC is 3M sodium chloride, 0.3 M sodium citrate, pH 7.0), 0.1%SDS at 50Co, and 4 x 3min in 0.1 x SSC at room temperature, and dried by centrifugation. Fluorescent array images were collected for both Cy3 and Cy5 with a ScanArray fluorescent scanner and image intensity data were extracted and analyzed with QuantArray 3.0 analysis software. Quantification data was imported in GeneSpring (Silicon Genetics). The import format was modified to maintain information relevant to user-contributed flagging as well as QC parameter furnished by the array manufacturer. Dye swap and Intensity-based normalization (Lowess) were performed using the default settings. Keywords = spleen Keywords = malaria
