ABSTRACT: The origin of biological samples (In vitro infection of HCT-8 cells with Cryptosporidium parvum); Monolayers of the HCT-8 cells (ATCC CCL-244, American Type Culture Collection) were cultured in RPMI 1640 medium containing 10% fetal bovine serum and additional nutritional supplements. Cells in log phase were plated at 2Ã106 cells per 150 mm tissue culture dish and infected with sterilised Cryptosporidium parvum oocysts (Iowa strain). Purified oocysts were suspended in phosphate buffered saline, sterilised in 33% bleach for 7 min on ice, washed in Hank's buffered saline solution, and added to HCT-8 cultures at a ratio of one oocyst per cell. Following a 2 h excystation period at 37 °C, cells were washed with warm Hank's buffered saline solution, and cells were incubated in fresh supplemented media. Mock-infected cultures were treated identically, with the exception that oocysts were not added to the cultures. Three independent mock- and C. parvum-infected cultures were prepared for analysis. The origin of mRNA samples; At 6, 12, 24, 48, and 72h post-inoculation, cell culture media were removed and cultures were immediately lysed by the addition of 3.5 ml of Trizol® reagent (GIBCO BRL Life Technologies) directly to the culture plate. Total RNA was prepared as described by the manufacturer, and poly(A) mRNA was isolated using oligo-dT cellulose columns (Amersham Pharmacia Biotech). The qualities of total RNA and poly(A) mRNA preparations were assessed by Northern blot. Protocols for conducting microarray hybridization; Poly(A) mRNA was converted intotarget suitable for hybridisation to Affymetrix microarray chips according to protocols provided by Affymetrix Inc. (Santa Clara, CA). Briefly, 2.0 g of poly(A) mRNA was reverse-transcribed to prepare double-stranded cDNA using T7-(dT)24 primer (GENSET Corp., La Jolla, CA) and Superscript II reverse transcriptase (GIBCO). Approximately 1 g of cDNA was used for in vitro transcription in the presence of biotinylated UTP and CTP using the Enzo BioArray High Yield RNA Transcript Labelling Kit (Enzo Diagnostics, Inc). This labelled cRNA was fragmented in 40 mM Trisâacetate (pH 8.1), 100 mM potassium acetate, and 30 mM magnesium acetate at 94 °C for 35 min. The integrity of cRNA and the efficiency of fragmentation were monitored by Northern blot analysis (not shown). Fragmented biotinylated cRNA targets were hybridised to HG-U95Av2 chips (Affymetrix) that contain probe sets for 12,600 human gene/transcripts. Fifteen micrograms of fragmented cRNA and appropriate controls were hybridised to the chips at 45 °C for 16 h with constant rotation at 60 rev./min. The chips were subsequently washed and stained with streptavidinâphycoerythrin conjugate using the GeneChip Fluidics station protocol EukGE_WS2 (Affymetrix). Following washing and staining, microarray chips were scanned twice at 3 m resolution using a Hewlett-Packard confocal scanner and hybridisation intensities for each of the genes/transcripts were collected from scanned images. Analysis of microarray data; Gene expression data were initially analysed with the GeneChip® expression analysis software (Affymetrix Microarray Suite, version 5.0). The fluorescence intensity of the genes/transcripts was measured for each probe array and, to minimise discrepancies due to non-biological variations, normalised by global scaling to 1000. Further data analyses were performed using GeneSpring software (version 6.0; Silicon Genetics, Redwood City, CA). Per gene normalisation was applied in which the expression signal of each gene in C. parvum-infected cells (raw data) was normalised to the median of its measurements in the mock-infected samples (control); and the ratio of expression levels between mock and infected samples was calculated as the mean value of normalised signal vs. control signal among three replicates. To identify genes with significantly altered expression, a series of statistical analyses (filtering) were performed: cut-off values for ratio of expression levels 1.80 and 0.55 were used to filter genes with expression level fold changes greater than ±1.8 in all three independent samples. Genes with fold change variations >1.5 across the three samples were excluded. Furthermore, a statistical group comparison using Student's t-test/ANOVA was conducted to compare mean expression levels between mock-infected and infected samples, and the genes with significant differential expression (P<0.05) were selected. Identified differentially expressed genes were then annotated using GeneSpring's Build Simplified Ontology constructor which hierarchically groups genes into meaningful biological categories (gene lists) based on the Gene Ontology Consortium Classifications. Various lists of regulated genes were created by cross-referencing annotated gene lists and applying assorted statistical and visualisation methods.
