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Optimization of DNA extraction from formalin-fixed tissue and its clinical application in Duchenne muscular dystrophy.

Author(s): Forsthoefel K F, Papp A C, Snyder P J, Prior T W

Publication: Am J Clin Pathol, 1992, Vol. 98, Page 98

PubMed ID: 1615933 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to optimize DNA extraction and PCR amplification from a variety of tissues, including postmortem spleen, liver, and cerebellum, duodenal biopsy tissue, and surgical lymph node tissue.

   Summary of Findings:

   The authors report that lysis at 37 degrees C for 24 hours or 54 degrees C for 4 hours yielded predominantly low-molecular weight DNA unsuitable for PCR. Lysis at 54 degrees C for 24 hours resulted in high-molecular weight DNA. Purification of each of these lysates resulted in successful PCR only for the samples incubated at 54 degrees C for 24 hours, with the exception of liver specimens, which required an additional 24 hours in lysate. DNA from unpurified lysates was not successfully amplified by PCR; the authors report that a simple salting-out purification step was sufficient to purify extracted DNA. Reduction of lysis volume increased the DNA yield from small specimens, whereas employing less than one microgram of DNA prevented inhibition of the PCR reaction for larger specimens. Differences between surgical/biopsy and postmortem tissue were not addressed.

   Biospecimens

   • Tissue - Spleen
   • Tissue - Brain
   • Tissue - Liver
   • Tissue - Small Bowel
   • Tissue - Lymph Node

   Preservative Types

   • Formalin

   Diagnoses:

   • Autopsy
   • Muscular Dystrophy

   Platform:

   Analyte	Technology Platform
   DNA	Spectrophotometry
   DNA	PCR
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein digestion	Lysis at 37 degrees C for 24 hours Lysis at 54 degrees C for 4 hours Lysis at 54 degrees C for 24 hours
   Biospecimen Acquisition	Biospecimen location	Duodenum Lymph node Spleen Liver Cerebellum
   Analyte Extraction and Purification	Analyte purification	No purification of extracted DNA Salting out of extracted DNA
   PCR Specific	Template/input amount	< 1 ug > 1 ug

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