title: Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors.
abstract: Identification of prognostic and predictive genomic markers requires long-term clinical follow-up of patients. Extraction of high-quality DNA from archived formalin-fixed, paraffin-embedded material is essential for such studies. Of particular importance is a robust reproducible method of whole genome amplification for small tissue samples. This is especially true for high-resolution analytical approaches because different genomic regions and sequences may amplify differentially. We have tested a number of protocols for DNA amplification for array-based comparative genomic hybridization (CGH), in which relative copy number of the entire genome is measured at 1 to 2 mb resolution. Both random-primed amplification and degenerate oligonucleotide-primed amplification approaches were tested using varying amounts of fresh and paraffin-extracted normal and breast tumor input DNAs. We found that random-primed amplification was clearly superior to degenerate oligonucleotide-primed amplification for array-based CGH. The best quality and reproducibility strongly depended on accurate determination of the amount of input DNA using a quantitative polymerase chain reaction-based method. Reproducible and high-quality results were attained using 50 ng of input DNA, and some samples yielded quality results with as little as 5 ng input DNA. We conclude that random-primed amplification of DNA isolated from paraffin sections is a robust and reproducible approach for array-based CGH analysis of archival tumor samples.
authors.affiliation: Cancer Center, Box 0808, University of California San Francisco, San Francisco, CA 94143-0808, USA.
authors.forename: Sandy
authors.initials: S
authors.lastname: Devries
authors.forename: Sarah
authors.initials: S
authors.lastname: Nyante
authors.forename: Jim
authors.initials: J
authors.lastname: Korkola
authors.forename: Richard
authors.initials: R
authors.lastname: Segraves
authors.forename: Kentaro
authors.initials: K
authors.lastname: Nakao
authors.forename: Dan
authors.initials: D
authors.lastname: Moore
authors.forename: Hanik
authors.initials: H
authors.lastname: Bae
authors.forename: Monica
authors.initials: M
authors.lastname: Wilhelm
authors.forename: Shelley
authors.initials: S
authors.lastname: Hwang
authors.forename: Frederic
authors.initials: F
authors.lastname: Waldman
mesh_terms: D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics; D004273:DNA, Neoplasm / Q000032:analysis / Q000302:isolation & purification*; D005260:Female; D005404:Fixatives; D005557:Formaldehyde / Q000737:chemistry; D006801:Humans; D021141:Nucleic Acid Amplification Techniques; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding; D011379:Prognosis; D015203:Reproducibility of Results
chemical_list: D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde