


Experiment Design:
*	The goal of the experiment : High Resolution Copy Number Analysis of 
Paraffin Embedded Archival Tissue Using SNP BeadArrays 
*	A brief description of the experiment:
 High density SNP microarrays provide insight into the genomic events that 
occur in diseases like cancer by their capability to measure both LOH and 
genomic copy numbers. Where currently available methods are restricted to 
the use of fresh frozen tissue, we now describe the design and validation of 
copy number measurements using the Illumina BeadArray platform and its 
application to formalin fixed, paraffin embedded (FFPE) tissue. In fresh 
frozen tissue, using a set of colorectal tumors with numerous chromosomal 
aberrations, our method measures copy number patterns that are 
comparable to values from established platforms, like Affymetrix GeneChip 
and BAC array-CGH. Moreover, paired comparisons of fresh frozen and 
FFPE tissue showed nearly identical patterns of genomic changes. We 
conclude that this method enables the use of paraffin embedded material for 
research into both LOH and numerical chromosomal abnormalities.
*	Keywords
 deletion, gain, human, normalization_testing_design, LOH
*	Experimental factors - 
SNP / copy number detection method, conservation method
*	Experimental design :
Tissue was processed from 4 individuals with a colorectal tumor
From each individual DNA was isolated from blood, frozen tumor material, and 
FFPE material.
The DNA from frozen tumor material was hybridized to Illumina BeadArray, 
Affymetrix GeneChip and BAC array
The DNA from blood and FFPE tumor material was hybridized to Illumina 
BeadArray 
*	Quality control steps taken: 
Technical replicates were hybridized to different platforms
*	Links to the publication, any supplemental websites or database accession 
numbers.

Samples used, extract preparation and labelling: 
*	The origin of each biological sample 
T44: 57 year old, male, right-sided Dukes B carcinoma 
T106: 61 year old, female, Dukes C rectal carcinoma
T108: 52 year old, male, Dukes C rectal carcinoma
T514: 74 year old, female, rectal adenoma
*	Manipulation of biological samples and protocols used 
Patients diagnosed with a colorectal tumor. The tumor was surgically removed 
either endoscopically or abdominally. 
*	Technical protocols for preparing the hybridization extract and labeling.
From fresh frozen tissue, twenty 30-µm-thick sections were cut from each tumor. 
DNA was isolated with the Genomic Wizard kit, according to the manufacturers' 
protocol (Promega, Madison, WI). Leukocyte DNA was obtained by salting out 
precipitation as described previously (Miller et al., 1988). DNA from FFPE tissue 
was extracted with the Chelex extraction method (De Jong et al., 2004). Briefly, 
three tissue punches (diameter 0.6 mm) were obtained by a tissue microarrayer 
(Beecher Instruments, Sun Prairie, WI), both from paraffin blocks containing 
tumor and blocks from the same patient that did not contain tumor tissue. DNA 
was isolated with Chelex and proteinase K. FFPE DNA was subsequently cleaned 
up using the Genomic Wizard kit (Promega). DNA concentrations were measured 
with the PicoGreen method (Invitrogen-Molecular Probes, Carlsbad, CA), and 
DNA quality was checked on a 1% agarose gel.
*	External controls (spikes), if used.
      
Hybridization procedures and parameters:  
*	The protocol and conditions used for hybridization, blocking and washing, 
including any post-processing steps such as staining.
For Illumina BeadArrays and Affymetrix GeneChips, the standard manufacturers' 
protocols were used
BAC array slides were produced at the LUMC department of Molecular Cell 
Biology. This platform contains 3700 probes spotted in triplicate and uses 1 mb 
spaced BACs distributed by the Welcome Trust Sanger Institute (Hinxton, UK). 
Genomic DNAs from test and reference samples were labelled using Cy3- and 
Cy5-dCTP (GE-Healthcare, Amersham, Roosendaal, The Netherlands) in an 
overnight random prime labelling reaction (BioPrime Random Prime labelling 
Kit, Invitrogen, Breda, The Netherlands). Labelled test and reference samples 
were pooled and precipitated in the presence of Cot1 DNA (Invitrogen, Breda, 
The Netherlands). DNA pellet was dissolved in hybridisation mixture. Sample 
was hybridized for 20 hours followed post-hybridisation washes and drying of 
slides by using an automated hybridisation station (Tecan Benelux BVBA, 
Giesen, The Netherlands). Scanned with a GenePix 4100A scanner (Axon 
Instruments, Westburg BV, Leusden, The Netherlands)(Knijnenburg et al., 2005)  


      Reference List
      
De Jong,A.E., van Puijenbroek,M., Hendriks,Y., Tops,C., Wijnen,J., Ausems,M.G., 
Meijers-Heijboer,H., Wagner,A., Van Os,T.A., Brocker-Vriends,A.H., Vasen,H.F., and 
Morreau,H. (2004). Microsatellite Instability, Immunohistochemistry, and Additional 
PMS2 Staining in Suspected Hereditary Nonpolyposis Colorectal Cancer. Clin. Cancer 
Res. 10, 972-980.
Knijnenburg,J., Szuhai,K., Giltay,J., Molenaar,L., Sloos,W., Poot,M., Tanke,H.J., and 
Rosenberg,C. (2005). Insights from genomic microarrays into structural chromosome 
rearrangements. Am. J. Med. Genet. A 132, 36-40.
Miller,S.A., Dykes,D.D., and Polesky,H.F. (1988). A simple salting out procedure for 
extracting DNA from human nucleated cells. Nucleic Acids Res. 16, 1215.


