Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors.
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.
Devries
Sandy
S
Cancer Center, Box 0808, University of California San Francisco, San Francisco, CA 94143-0808, USA.
Nyante
Sarah
S
Korkola
Jim
J
Segraves
Richard
R
Nakao
Kentaro
K
Moore
Dan
D
Bae
Hanik
H
Wilhelm
Monica
M
Hwang
Shelley
S
Waldman
Frederic
F
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
D004273:DNA, Neoplasm; D005404:Fixatives; D005557:Formaldehyde