Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.
Array-based comparative genomic hybridisation (aCGH) has diverse applications in cancer gene discovery and translational research. Currently, aCGH is performed primarily using high molecular weight DNA samples and its application to formalin-fixed and paraffin-embedded (FFPE) tissues remains to be established. To explore how aCGH can be reliably applied to archival FFPE tissues and whether it is possible to apply aCGH to small numbers of cells microdissected from FFPE tissue sections, we have systematically performed aCGH on 15 pairs of matched frozen and FFPE astrocytic tumour tissues using a well-established in-house human 1 Mb BAC/PAC genomic array. By spiking tumour DNA with normal DNA, we demonstrated that at least 70% of tumour DNA was required for reliable aCGH analysis. Using aCGH data from frozen tissue as a reference, it was found that only FFPE astrocytic tumour tissues that supported PCR amplification of >300 bp DNA fragment provided high quality, reproducible aCGH data. The presence of necrosis in a tissue specimen had an adverse effect on the quality of aCGH, while fixation in formalin for up to 96 h of fresh tissue did not appear to affect the quality of the result. As little as 10-20 ng DNA from frozen or FFPE tissues could be readily used for aCGH analysis following whole genome amplification (WGA). Furthermore, as few as 2000 microdissected cells from haematoxylin-stained slides of archival FFPE tissues could be successfully used for aCGH investigations when WGA was used. By careful assessment of DNA integrity and review of histology, to exclude necrosis and select specimens with a high proportion of tumour cells, it is feasible to preselect archival FFPE tissues adequate for aCGH analysis. With the help of microdissection and WGA, it is also possible to apply aCGH to histologically defined lesions, such as carcinoma in situ.
Johnson
Nicola A
NA
Division of Molecular Histopathology, Department of Pathology, University of Cambridge, Cambridge, Cambridgeshire, UK.
Hamoudi
Rifat A
RA
Ichimura
Koichi
K
Liu
Lu
L
Pearson
Danita M
DM
Collins
V Peter
VP
Du
Ming-Qing
MQ
D001254:Astrocytoma / Q000378:metabolism* / Q000473:pathology; D018070:Biological Specimen Banks; D014408:Biomarkers, Tumor / Q000378:metabolism*; D004273:DNA, Neoplasm / Q000032:analysis*; D005557:Formaldehyde; D006801:Humans; D042282:Microdissection; D009336:Necrosis; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016612:Paraffin Embedding; D015203:Reproducibility of Results; D013997:Time Factors; D014021:Tissue Preservation
D014408:Biomarkers, Tumor; D004273:DNA, Neoplasm; D005557:Formaldehyde