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Nucleic Acids Research Advance Access published online on October 29, 2009
Nucleic Acids Research, doi:10.1093/nar/gkp881
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© The Author(s) 2009. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples

Galen Hostetter1,*, Su Young Kim1, Stephanie Savage1, Gerald C. Gooden1, Michael Barrett1, Jian Zhang1, Lalitamba Alla1, April Watanabe1, Janine Einspahr2, Anil Prasad2, Brian J. Nickoloff3, John Carpten1, Jeffrey Trent1, David Alberts2 and Michael Bittner1

1Translational Genomics Research Institute, 445 N. 5th Street, Phoenix, AZ 85004, 2Arizona Cancer Center, University of Arizona, Tucson, AZ 85724 and 3Department of Pathology, Loyola University Medical Center, Maywood, IL, USA

*To whom correspondence should be addressed. Tel: +1 602 343 8810; Fax: +1 602 343 8840; Email: ghostetter{at}tgen.org

Received January 16, 2009. Revised September 29, 2009. Accepted October 2, 2009.

Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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