Total RNA extracted using Trizol following manufacturer's instructions
Label
Cy5
Label protocol
Two ug of total RNA was amplified and labeled using Ambion's Amino Allyl MessageAmp™ aRNA kit and protocol (Ambion Inc., Austin, TX). For each microarray experiment, 2.0-μg aliquots of aRNA were labeled with Cy5 (Amersham Biosciences, Buckinghamshire, UK). The labeled aRNAs were mixed with equal amounts of Cy3-labeled reference aRNA. To the mixture of labeled reference and sample RNA, 20 μg human COT-1 DNA (Invitrogen, Carlsbad, CA), 8 μg yeast tRNA (Invitrogen) and 20 μg polyadenylic acid (Sigma-Aldrich, St. Louis, MO) were added.
Channel 2
Source name
Reference material consisting of pooled RNAs isolated from five colorectal cancer cell lines (HCT116, LS411N, SW480, HCT15, Caco2) and five normal rectum samples.
Total RNA extracted using Trizol following manufacturer's instructions
Label
Cy3
Label protocol
Two ug of total RNA was amplified and labeled using Ambion's Amino Allyl MessageAmp™ aRNA kit and protocol (Ambion Inc., Austin, TX). For each microarray experiment, 2.0-μg aliquots of aRNA were labeled with Cy5 (Amersham Biosciences, Buckinghamshire, UK). The labeled aRNAs were mixed with equal amounts of Cy3-labeled reference aRNA. To the mixture of labeled reference and sample RNA, 20 μg human COT-1 DNA (Invitrogen, Carlsbad, CA), 8 μg yeast tRNA (Invitrogen) and 20 μg polyadenylic acid (Sigma-Aldrich, St. Louis, MO) were added.
Hybridization protocol
Preheated hybridization buffer (25% formamide, 5× SSC, 0.1% SDS) was added and arrays were hybridised overnight 42°C to human 35K oligo microarrays
Scan protocol
Scanned on an Agilent G2565BA scanner.
Description
Expanded descriptions of Channel 1 'Characteristics': TEM: transanal endoscopic microsurgical resection. TME: total mesorectal excision AA: pure adenomas(in paper A/A), AC: adenoma fraction from cases with a carcinoma focus(in paper A/C), CA: tumor fractions consisting of a mixture of adenoma and carcinoma tissue(in paper AC/C), CC: carcinomas without lymph node metastasis(in paper C/C), CC(N): carcinomas with lymph node metastasis(in paper C/C(N+)). The stages (T1N0) represent the TNM classification of colorectal tumors (International Union Against Cancer (UICC)).
Data processing
Images were quantified using Genepix 5.1 Software. The Limma (linear models for microarray data) package of Bioconductor (http://www.bioconductor.org) was used for importing the data, normalizing the arrays and identifying differentially expressed genes. Control spots and spots with more than 10% saturation, a diameter smaller than 60 μm or signal intensity less than 20 counts above background were excluded from the analysis. Data were corrected for local background (method normexp) and normalized within arrays by print-tip loess normalization and between arrays by quantile normalization.
