PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

Author(s): Greer CE, Peterson SL, Kiviat NB, Manos MM

Publication: Am J Clin Pathol, 1991, Vol. 95, Page 117-24

PubMed ID: 1846996 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 type, fixation duration, and DNA purification on amplification of different size fragments of the beta-globin gene.

   Summary of Findings:

   Fixation in NBF or acetone allowed for amplification of a 1327 bp product, regardless of fixation duration. When 1 ul, instead of 10 ul, of template DNA from formalin-fixed specimens was used, the 1327 bp product was reduced for all fixation durations, and the 989 bp product was reduced when fixation was for 24 h. Amplification of the 1327 bp product was not successful when specimens were fixed in Methacarn or formalin-alcohol-acetic acid for more than 1 h, or in Clarke's fixative, alcoholic formalin, paraformaldehyde, or Zamboni's solution for more than 4 h. Fixation in Carnoy's solution prevented amplification of products longer than 989 bp, and when fixation was for 24 h, products longer than 268 bp were not amplified. Amplification of products longer than 268 bp was not possible from Zenker's-fixed specimens. Furthermore, when fixation in Zenker's solution was for more than 4 h, amplification of only the 110 bp product was successful. When specimens were fixed in Bouin's fixative, the only successful amplification was the 110 bp product from a specimen fixed for 1 h, regardless of input quantity. The authors report that further purification of the template with phenol-chloroform extraction did not improve amplification. By electrophoresis, only specimens fixed in Zamboni's solution showed decreased fragment size with longer fixation.

   Biospecimens

   • Tissue - Cervix

   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)
   Biospecimen Preservation	Type of fixation/preservation	Bouin's fixative Acetone Formalin (buffered) Methacarn Zamboni's solution Zenker's fluid Carnoy's solution Clarke's fluid Paraformaldehyde Acetic formalin alcohol
   Biospecimen Preservation	Time in fixative	1 h 4 h 24 h
   PCR Specific	Length of gene fragment	110 bp 268 bp 536 bp 989 bp 1327 bp
   PCR Specific	Targeted nucleic acid	Beta globin
   Analyte Extraction and Purification	Analyte isolation method	Proteinase K alone Proteinase K followed by phenol-chloroform extraction

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