Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors

J Mol Diagn. 2005 Feb;7(1):65-71. doi: 10.1016/S1525-1578(10)60010-4.

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Breast Neoplasms / diagnosis*
  • Breast Neoplasms / genetics
  • DNA, Neoplasm / analysis
  • DNA, Neoplasm / isolation & purification*
  • Female
  • Fixatives
  • Formaldehyde / chemistry
  • Humans
  • Nucleic Acid Amplification Techniques*
  • Oligonucleotide Array Sequence Analysis / methods*
  • Paraffin Embedding
  • Prognosis
  • Reproducibility of Results

Substances

  • DNA, Neoplasm
  • Fixatives
  • Formaldehyde