ARRAY: H210_15 PATIENT: D9 HISTOLOGY: Normal MUTATION: APC
Extracted molecule
total RNA
Extraction protocol
As described at Cardoso J, Molenaar L, de Menezes RX, van Leerdam M, Rosenberg C, Moslein G, Sampson J, Morreau H, Boer JM, Fodde R: Chromosomal instability in MYH- and APC-mutant adenomatous polyps, Cancer Res 2006, 66:2514-2519.
Label
Cy5
Label protocol
Each total RNA microdissected sample that passed the quality controls was linearly amplified with two rounds of amplification using the MessageAmp kit (Ambion, Huntingdon, UK), according to manufacturer’s protocol. One µg aliquots of target and reference aRNA were labeled with Cy5-dUTP and Cy3-dUTP respectively (Amersham Biosciences, Amersham, UK) by reverse transcription using the CyScribe First Strand cDNA Labeling kit (Amersham Biosciences, Amersham, UK).
Hybridization protocol
Each labeling reaction was purified with the CyScribe GFX purification kit (Amersham Biosciences, Amersham, UK). Subsequently, both labeled cDNAs were hybridized on a human 18K cDNA microarray encompassing 19,200 spots (representing 18,432 independent cDNAs) produced and obtained from The Netherlands Cancer Institute Microarray Facility (Amsterdam, The Netherlands), according to manyfacturer´s protocol.
Scan protocol
Sixteen-bit fluorescent images from the expression arrays were acquired with an Agilent DNA Microarray Scanner (Agilent Technologies, Inc., Palo Alto, CA).
Description
D9_44N
Data processing
The resulting TIFF images were analyzed with the software GenePix Pro 4.0 (Axon Instruments, Union City, CA) and a unique .gpr file was generated for each sample. Each .gpr file was directly loaded into the R environment using the marray package to extract the background-corrected Cy3 and Cy5 median raw intensity per spot. Intensity data was normalized with the variance stabilization and normalization function implemented in the vsn package (http://www.R-project.org/).
