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[1606] Is Formalin-Fixed Tissue Useful for Oligo-Array CGH Studies? A Comparison with Molecular Fixative

D Gugic, M Nassiri, V Vincek, M Nadji, AR Morales. University of Miami Miller School of Medicine, Miami, FL

Background: Array-based comparative genomic hybridization (a-CGH) is a promising tool for clinical genomic studies. The impact of pre-analytical sample preparation methods on the quality of results, however, has not been fully evaluated.
Design: Parallel sections of normal male human skin biopsy samples (n=3) were collected fresh, fixed in formalin, and in Molecular Fixative (Sakura Finetek). Genomic DNA was isolated after 8-24 hrs from the samples and subjected to amplification and labeling. Labeled samples were then co-hybridized with normal reference female DNA to Agilent oligonucleotide-based a-CGH 44k slides. Pre-analytical parameters such as DNA yield, quality of genomic DNA and labeling efficacy were evaluated. Also, microarray analytical variables, including the feature signal intensity, data distribution dynamic range, signal to noise ratio, and background intensity levels were used to assess the quality of data.
Results: The DNA yield and the quality of genomic DNA (as evaluated by spectrophotometery and gel electrophoresis) were similar in Fresh and Molecular Fixative samples. Also, the labeling efficacy of dye incorporation was comparable between the two types of samples, as were the scan parameters and stem plot analysis of a-CGH result. Formalin-fixed samples, on the other hand, showed various errors such as oversaturation, non-uniformity in replicates and decreased signal to noise ratio. In short, a-CGH results of formalin samples were not interpretable.
Conclusions: The DNA extracts of formalin-fixed material is not practical for oligonucleotide-based a-CGH studies. On the other hand, Molecular Fixative preserves tissue DNA in a manner similar to fresh state with no discernable analytical differences. Spectrophotometric and gel-based evaluation of DNA quality are not good indicators of suitability of DNA for a-CGH studies.
Category: Techniques

Monday, March 26, 2007

Poster # 215, Monday Afternoon