Use of neuropathological tissue for molecular genetic studies: parameters affecting DNA extraction and polymerase chain reaction.

Author(s): Kösel S, Graeber MB

Publication: Acta Neuropathol, 1994, Vol. 88, Page 19-25

PubMed ID: 7941968 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to optimize a DNA extraction protocol from FFPE brain specimens, targeting the variables of proteinase K concentration and duration, and incubation temperature.

   Summary of Findings:

   DNA yield was maximized with a 16 hour incubation at 50 degrees C with 4 mg/ml of proteinase K.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Formalin

   Diagnoses:

   • Lewy Body Disease
   • Autopsy

   Platform:

   Analyte	Technology Platform
   DNA	Electrophoresis
   DNA	Fluorometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein digestion	Proteinase K (4 mg/ml) 37 degrees C 50 degrees C Proteinase K (0.5 mg/ml) Proteinase K (1 mg/ml)
   Analyte Extraction and Purification	Incubation duration/condition	4 h 16 h

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2. Study Purpose

   The purpose of this study was to compare yields and PCR applicability of DNA extracted from brain specimens that were fixed in unbuffered formalin for different durations (3 days - 26 years) and those fixed in unbuffered formalin and paraffin embedded.

   Summary of Findings:

   DNA yield was adversely impacted by the duration of unbuffered formalin fixation, with fixation for 1 year or longer exhibiting the most profound effects. While FFPE brain specimens produced a significantly greater DNA yield compared to case-matched specimens stored in unbuffered formalin, PCR results were superior in specimens stored in unbuffered formalin. In general, PCR success was independent of DNA yield, but adversely affected by prolonged fixation duration (17 days or longer) and paraffin embedding.

   Biospecimens

   • Tissue - Brain

   Preservative Types

   • Formalin

   Diagnoses:

   • Lewy Body Disease
   • Autopsy

   Platform:

   Analyte	Technology Platform
   DNA	Fluorometry
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Formalin (buffered) Formalin (unbuffered)
   Biospecimen Preservation	Time in fixative	3 d 7 d 14 d 1-5 yr 18 yr 26 yr
   Biospecimen Preservation	Embedding medium	Paraffin Not embedded

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