Preservation of skin DNA for oligonucleotide array CGH studies: a feasibility study.
Array-based comparative genomic hybridization (a-CGH) is a promising tool for clinical genomic studies. However, pre-analytical sample preparation methods have not been fully evaluated for this purpose. Parallel sections of normal male human skin biopsy samples were collected and immediately immersed in saline, formalin and a molecular fixative for 8, 12 and 24 h. Genomic DNA was isolated 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-analytic 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 assessed for data quality. DNA yield and quality of genomic DNA--as evaluated by spectrophotometry and gel electrophoresis--were similar for fresh and molecular fixative-exposed samples. In addition, labeling efficacy of dye incorporation was not drastically different. There was no difference between fresh and molecular fixative material comparing 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. Overall, the a-CGH result of formalin samples was not interpretable. DNA extracted from formalin-fixed tissue samples is not suitable for oligonucleotide-based a-CGH studies. On the other hand, the molecular fixative preserves tissue DNA similar to its fresh state with no discernable analytical differences.
Nassiri
Mehdi
M
Department of Pathology, University of Miami, Miller School of Medicine, Miami, FL 33136, USA. mnassiri@med.miami.edu
Gugic
Dijana
D
Olczyk
Joseph
J
Ramos
Sharon
S
Vincek
Vladimir
V
D004247:DNA / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D008297:Male; D009693:Nucleic Acid Hybridization / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012867:Skin / Q000737:chemistry*; D014021:Tissue Preservation
D004247:DNA