Six- to 8-week-old athymic nu/nu mice (Harlan, Indianapolis, IN) were inoculated subcutaneously with five million HCT116 human colon cancer cells. The tumor was harvested once the tumor volumes reached ~500 mm3.
Extracted molecule
total RNA
Extraction protocol
Vegetative bacteria or spores were collected by centrifugation and homogenized by vortexing in RNAwiz buffer (RiboPureTM-Bacteria kit, Ambion, Austin, TX) containing 250 µL of Zirconia beads (Biospec, Bartlesville, OK) for 10 minutes at 4°C, followed by a 5-min break. This cycle was repeated 12 times to ensure disruption of the spores (as assessed by phase contrast microscopy and RNA yield). RNA was purified with the RiboPureTM-Bacteria kit (Ambion) following the manufacturer’s instructions. RNA was prepared from infected or uninfected tumors by homogenizing (50 strokes) the dissected tumors in a glass homogenizer containing a 10-fold volume of the RNAwiz buffer on ice. The homogenate was aliquoted into 0.5-mL RNase-free tubes. The samples were then processed exactly as described above, using a 12-cycle homogenization with Zirconia beads.
Label
biotin
Label protocol
total RNA was reverse transcribed with SuperScript II Reverse Transcriptase (Invitrogen). The first-strand cDNA was fragmented by DNase I (Promega, Madison WI) and labeled with biotinylated ddATP through a terminal transferase end-labeling reaction.
Hybridization protocol
Hybridization with the end-labeled samples was carried out at 45°C for 16 hours in 100 mM MES, 1 M NaCl, 20 mM EDTA, 0.01% Tween 20 and 10.5% glycerol.
Scan protocol
After washing, the arrays were stained with Cy3-Streptavidin, followed by scanning with an Axon 4000B scanner.
Description
The features were extracted by using the NimbleScan software.
Data processing
Data was analyzed with the Bioconductor project using quantile normalization (see:Bolstad B, Irizarry R, Astrand M, Speed T (2003) A comparison of normalization methods for high density oligonucleotide array data based on bias and variance. Bioinformatics 19 (2), p185-193) and gene calls generated using the RMA algorithm (Robust multichip average described in Biostatistics 2003 Apr;4(2):249-64 ).
