All of the cell lines were obtained from the ATCC (American Type Culture Collection) and cultured following their recommendations, except p53HCT116, a derivative of HCT116 with a homozygous disruption of TP53 (Bunz et al., 1998), which was kindly provided by Dr. Curtis C. Harris of the National Cancer Institute, NIH.
Extracted molecule
total RNA
Extraction protocol
DNA and RNA was extracted from the cell lines following standard procedures (http://www.riedlab.nci.nih.gov/protocols. asp). Nucleic acid quantification was determined using the Nanodrop ND-1000 UV-VIS spectrophotometer (Nanodrop, Rockland, DE) and RNA quality was assessed using the Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA). Normal colon RNA isolated postmortem from five different donors without a history of colorectal cancer was purchased from Ambion (Applied Biosystems, Foster City, CA).
Label
Cy3
Label protocol
One mg each of cell line or normal human colon RNA (Ambion, Austin, TX) were amplified and labeled with Cy3, respectively, using a T7 RNA Polymerase (Low RNA Input Fluorescent Linear Amplification Kit, Agilent) according to the manufacturer’s protocols
Hybridization protocol
Cy3 labeled cRNA was hybridized on 44K or 4x44K oligonucleotide-based Whole Human Genome Microarray (Agilent) according to the manufacturer’s protocol version 4.0.
Scan protocol
Microarrays were washed and processed using an Agilent G2565BA scanner.
Description
Gene expression
Data processing
Raw data were log2 transformed and normalized to 75 percentile according to Agilent protocol.
