refer to Series supplementary file for sample characteristics
Treatment protocol
none
Extracted molecule
total RNA
Extraction protocol
The human investigations were performed after approval by the University of South Florida Institutional Review Board and in accord with an assurance filed with and approved by the Department of Health and Human Services. A waiver of informed consent was filed. All tumors and normal tissues were meticulously harvested under an identical, rigorous procurement protocol whereby tissues were macrodissected for purity and snap frozen in liquid nitrogen within 20 min of surgical extirpation. Human colorectal tumor samples (n=100) were microdissected (80% tumor cells) by frozen section guidance. Normal mucosal samples (n=5) were harvested from zones greater than 10 cm away from the gross tumor specimen and subjected to the same procedures as were the tumor samples. Quality control steps: RNA quality was assessed by using an Agilent Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA).
Label
biotin
Label protocol
Standard protocal fro 2 microgram total RNA using oligo dT primers was used.
Hybridization protocol
The protocol and conditions used during hybridization, blocking and washing: Create a hybridization cocktail for a single probe array that contains 0.05 ug/uL fragmented cRNA, 50 pM Control Oligonucleotide B2 (Affymetrix), 20X Eukaryotic Hybridization Controls (1.5 pM bioB, 5 pM bioC, 25 pM bioD, 100 pM cre) (Affymetrix), 0.1 mg/mL Herring Sperm DNA (Promega), 0.5 mg/mL Acetylated BSA (Invitrogen), and 1X Hybridization Buffer. Heat hybridization cocktail to 99°C for 5 minutes, to 45°C for 5 minutes, and spin at maximum speed in a microcentrifuge for 5 minutes. Fill probe array with 200 uL of 1X Hybridization Buffer. Incubate at 45°C for 10 minutes in the GeneChip Hybridization Oven 640 (Affymetrix) rotating at 60 rpm. Remove 1X Hybridization Buffer and fill probe array with 200 uL of the hybridization cocktail. Incubate at 45°C for 16 hrs in the Hybridization Oven rotating at 60 rpm.
Scan protocol
The image file was captured on an Affymetrix Gene Chip Scanner 3000
Description
Sample type: Human colorectal carcinomas and normal adult human colon tissue.
Data processing
Processed with Microarray suite 5.0 (Affymetrix) to generate .CEL files that were subject to RMA normalization (Irizarry et al 2003) using Bioconductor.org software. Standard Affymetrix internal control genes, overall percent present call genes and signal to noise estimates were used to check the quality of the assay per chip. Quality by the signals of the 3' probe set to the 5' probe set of the internal control genes, GAPDH and B-actin, with acceptable 3' to 5' ratios between1-3 were used. Prokaryotic Spike controls were used to determine the hybridization of target RNA to the array occurred properly. GeneSpring 7.2 (Agilent technologies Inc. Palo Alto, California) was used to normalization, Clustering and filtering. The Raw CEL files were processed using the RMA (Robust Multichip Average) from Bioconductor.org. Then, depending on the biological question to be addressed, the samples were referenced either to the median gene expression value across all the samples or to the expression levels of normal control adult colon samples.
