Subject #1: a 7cm terminal ileum was obtained from right hemi-colectomy specimen. The mucosal layers were dissected from the underlying muscular layers. The tissue was sectioned into 3 mm2 segments.
1 x 106 oocysts per sample of C. parvum, C. hominis or sterile excystation solution were inoculated onto the apical surface of the explants. The explant tissues were kept in vitro for 24 hours at 37°C in a chamber containing 95% O2 and 5% CO2 and then collected in RNA later (RNase inhibitor solution) and stored frozen. Control tissue from prior to infection was placed immediately in RNA later. Explant samples were disrupted and homogenized mechanically by sonication and total RNA was extracted with the RNAeasy Mini Kit (Qiagen) per manufacturer’s protocol. To extract the RNA from cultured cells we disrupt cells using QIAsheredder colums and the RNAeasy plus extraction kit (Qiagen). The quality of the RNA obtained was confirmed by capillary electrophoresis on an Agilent 2100 Bioanalyzer. Subsequently, gene expression profiles were probed by microarray analysis using the Affymetrix Human Genome U133 Plus 2.0 Array (33,000 human genes). Data were background adjusted, normalized and summarized by the RMA method. The Bioconductor package was used for this analysis. The result was a data set of expression value, with each probe set having a single expression value per chip, and the data was in log 2 scale. This data was then filtered by the present (“P”) or absent (“A”) calls which were calculated by Affymetrix’ software GCOS 2.0 (Affymetrix, Santa Clara, CA). Probe sets that had “A” calls on all chips were excluded in further analysis. The data were then imported into SAS 9.0 (SAS Institute Inc., Cary, NC). The Type III test p-values were adjusted by the Benjamini-Hochberg method to control FDR, resulting in 2370 probe sets.
Growth protocol
Ileal tissue was obtained from 3 individuals undergoing surgical procedures for unrelated non-infectious conditions. Laboratory personnel collected ileal tissue in the operating room at time of surgery. The tissue was immediately transported to the laboratory in CMRL Medium-1066. The mucosal layers were removed mechanically and the mucosa from each patient was divided into four 3 mm2 pieces. Each piece was placed luminal side up, onto polycarbonate coated insert cups. The inserts were then placed into culture wells containing CMRL Medium-1066 supplemented with 5 g/L D-glucose, 0.3 mM Glutamax, 0.3 g/L L-methionine, 0.3 g/L tricine, 5% heat inactivated FBS, 100,000 units/L penicillin, 100,000 mcg/L streptomycin, and 250 mcg/L Amphotericin B. A small amount of culture medium was also added to the apical surface of each insert cup to keep the explants hydrated. The explants were incubated for at least 3 hours in a chamber containing 95% O2 and 5% CO2 prior to ex vivo infection.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted with the RNAeasy Mini Kit (Qiagen) per manufacturer’s protocol. Total RNA was extracted with the RNAeasy Mini Kit (Qiagen) per manufacturer’s protocol.
Label
Biotin
Label protocol
Affymetrix One Cycle Target and Labeling Protocol, exactly as described in Affymetrix Expression Analysis Technical Manual
Hybridization protocol
Affymetrix One Cycle Target and Labeling Protocol Hybridization and Staining, as described in Expression Analysis Technical Manual with following exception(s): Hybridization volume increased to 250.0µL. Fluidics Script: EukGE ws2v5; Array: HGU133 Plus 2.0; Date Scanned: 07/28/05.
Scan protocol
Affymetrix standard protocol
Description
Human ileal explant cultures infected with cryptosporidium
