Preservation of skin DNA for oligonucleotide array CGH studies: a feasibility study

Abstract

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 Wxative for 8, 12 and 24 h. Genomic DNA was isolated from the samples and subjected to ampliWcation 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 eYcacy 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 Wxative-exposed samples. In addition, labeling eYcacy of dye incorporation was not drastically diVerent. There was no diVerence between fresh and molecular Wxative material comparing scan parameters and stem plot analysis of a-CGH result. Formalin-Wxed 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-Wxed tissue samples is not suitable for oligonucleotide-based a-CGH studies. On the other hand, the molecular Wxative preserves tissue DNA similar to its fresh state with no discernable analytical diVerences.

Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples

“…More recently, aCGH, utilizing tens to hundreds of thousands of long 60-mer oligonucleotide probes, provides intragenic resolution at ranges of 6.5–35 kb with sufficient sensitivity to detect intragenic single copy changes ( 17 ). Early reports of application of FFPE samples to the aCGH platform include the comparison of BAC and aCGH analysis of prostate tumors ( 18 ) and failure by multiple parameters on skin biopsy split samples that assessed effects of various fixatives ( 19 ). Sample preparation methods in utilizing FFPE materials are more uneven and difficult to apply to genome-wide assays.…”

Preservation of biomolecules in breast cancer tissue by a formalin-free histology system

“…It is not surprising, therefore, that alternative formalin-free tissue handling methodologies have been introduced in recent years. We have previously reported our experience with a new, simple and practical, yet standardized, tissue fixation and processing method that preserves histomorphology and protects macromolecules at ambient temperature [ 4 - 9 ]. This method is easily applicable to both clinical and research settings.…”

The role of the pathology department in the preanalytical phase of molecular analyses

“…Regarding a-chorionic gonadotrophin, early studies suggested that DNA samples obtained from FFPE tissues were uninterpretable because of various errors such as oversaturation and nonuniformity in replicates. 71 Recent advances have surmounted these impediments and even compared these new methods to suggest optimal a-chorionic gonadotrophin platforms. 72 …”