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 randomprimed amplification was clearly superior to degenerate oligonucleotide-primed amplification for arraybased 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 highquality 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 randomprimed amplification of DNA isolated from paraffin sections is a robust and reproducible approach for array-based CGH analysis of archival tumor samples.
(J Mol Diagn 2005, 7:65-71)Genomic analysis of tumor DNA allows identification of alterations in sequence and copy number for individualized diagnostic, prognostic, and therapeutic decision making. This is especially relevant as personalized treatments for cancer patients based on specific genomic alterations become clinically available. Although DNA extracted from freshly acquired samples is optimum for these analyses, it is not always feasible to freeze away such samples given the constraints of clinical practice. Thus, having optimized protocols for extraction of DNA from formalin blocks is a necessary adjunct to recently developed clinical testing for tumor DNA alterations.One of the most useful approaches for analysis of DNA copy number alterations over the entire genome uses an array-based comparative genomic hybridization (CGH) analysis of DNA clones at 1-mb resolution.1-3 Current protocols for array CGH commonly require g quantities of high-quality DNA.4 Such material is generally not available from paraffin sections of formalin-fixed material, especially when tumors are small or require microdissection to remove contaminating normal or necrotic elements.A number of protocols have been reported for extraction and genomic amplification of archival material for application to standard (chromosome-based) or even array-based CGH, with varying quality of the resultant analyses. Degenerate oligonucleotide-primed (DOP) polymerase chain reaction (PCR) 5,6 and genomic representation amplification 7,8 have been tested using s...