The importance of fixation procedures on DNA template and its suitability for solution-phase polymerase chain reaction and PCR in situ hybridization.

Author(s): O'Leary JJ, Browne G, Landers RJ, Crowley M, Healy IB, Street JT, Pollock AM, Murphy J, Johnson MI, Lewis FA

Publication: Histochem J, 1994, Vol. 26, Page 337-46

PubMed ID: 8040006 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine if the type of fixative, fixation temperature (0-37 degrees C) or duration (6 h-1 wk) impact successful PCR analysis of DNA extracted from paraffin-embedded specimens. The efficacy of two different DNA extraction methods was also evaluated.

   Summary of Findings:

   For specimens fixed for 24 h at room temperature, DNA extraction by specimen boiling in HPLC water for 20 min resulted in low molecular weight DNA, while incubation in proteinase K for 5 days yielded comparatively high molecular weight DNA regardless of the type of fixative used. The efficacy of PCR amplification of a short beta-globin amplicon (110 bp) was comparable among the two extraction methods although results were fixative-specific, as boiling was more successful for formalin-fixed specimens and proteinase K was favorable with Carnoy's fixed specimens. Specimens preserved with fixatives containing mercuric-chloride (buffered formaldehyde sublimate, Zenker's, Helly's) failed to generate a PCR product, while Bouin's and glutaraldehyde produced inconsistent results. Short fixation times (6-48 h) at elevated temperatures (21-37 degrees C) were favorable for successful PCR amplification.

   Biospecimens

   • Tissue - Tonsil

   Preservative Types

   • Formalin
   • Other Preservative

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Protein digestion	0.1 mg/ml proteinase K, 5 days, 37 degrees C Boiling in HPLC water for 20 min
   Biospecimen Preservation	Time in fixative	6 h 24 h 48 h 72 h 1 wk
   Biospecimen Preservation	Temperature of fixation/preservation	0-4 degrees C 21 degrees C (room temperature) 37 degrees C
   Biospecimen Preservation	Type of fixation/preservation	B-5 fixative Bouin's fixative Carnoy's solution Formalin (buffered) Glutaraldehyde Helly's fluid Zenker's fluid

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