Southern blot analysis of DNA extracted from formalin-fixed pathology specimens.

Author(s): Dubeau L, Chandler LA, Gralow JR, Nichols PW, Jones PA

Publication: Cancer Res, 1986, Vol. 46, Page 2964-9

PubMed ID: 3009004 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of fixative, fixation duration, and tissue type on DNA analysis by Southern blot.

   Summary of Findings:

   The authors report that intact DNA was unobtainable from specimens exposed to picric acid (Bouin's fixative) or mercuric chloride (Zenker's fixative or B5). Further, when the specimen was autolyzed or improperly fixed, DNA was not recoverable, and when specimens were necrotic, DNA was degraded. However, the authors report that if tissue was properly fixed and not necrotic, the ultraviolet spectra of DNA obtained from formalin-fixed specimens were similar to those from snap-frozen specimens. The length of incubation needed to solubilize the DNA was dependent on the stromal density with spleen requiring only 2 days for 80% of the DNA to be solubilized versus 6 days for a uterine leiomyoma. With fixation times longer than 12 h, the percentage of spoolable DNA decreased. The unspoolable DNA was found to be largely unable to penetrate agarose gels or to be degraded and consequently was unsuitable for Southern blot analysis. In formalin-fixed specimens, some DNA remained undigestable, perhaps due to denaturation during the embedding process. DNA from formalin-fixed specimens also had reduced mobility when compared to snap-frozen specimens. The authors conclude that DNA suitable for Southern blot analysis can be obtained from properly formalin fixed-specimens.

   Biospecimens

   • Tissue - Spleen
   • Tissue - Ovary
   • Tissue - Bladder
   • Tissue - Uterus

   Preservative Types

   • Formalin
   • Other Preservative
   • Frozen

   Diagnoses:

   • Not specified
   • Neoplastic - Carcinoma
   • Neoplastic - Germ Cell
   • Neoplastic - Benign

   Platform:

   Analyte	Technology Platform
   DNA	Southern blot
   Morphology	Light microscopy
   DNA	Electrophoresis
   DNA	Spectrophotometry
   DNA	Dot blot or slot blot

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	B-5 fixative Bouin's fixative Formalin (buffered) Snap frozen Zenker's fluid
   Biospecimen Preservation	Time in fixative	12 h 36 h 60 h 110 h
   Biospecimen Acquisition	Biospecimen location	Spleen Uterus Ovary Bladder

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2. Study Purpose

   The purpose of this study was to determine the optimal solubilization time for efficient removal of degraded DNA during DNA extraction from formalin-fixed specimens.

   Summary of Findings:

   Low molecular weight DNA (degraded DNA) was efficiently solubilized during an initial 24 h incubation. A second solubilization step (24-60 h) did not result in increased solubilization of low molecular weight DNA. Based on these findings the authors recommend an initial 6-12 h solubilization step (24 h in tissue with a high stromal density) be performed and the supernatant discarded prior to solubilization of formalin-fixed DNA for southern blotting.

   Biospecimens

   • Tissue - Breast

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte purification	0-24 h solubilization 24-60 h solubilization

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