Sample preparation and PCR amplification from paraffin-embedded tissues.

Author(s): Greer CE, Wheeler CM, Manos MM

Publication: PCR Methods Appl, 1994, Vol. 3, Page S113-22

PubMed ID: 7920232 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to review the effects of fixative type, and fixation duration on maximum PCR target length.

   Summary of Findings:

   A 1327 bp fragment was amplifiable in specimens fixed in formalin for up to 1 day, acetone for 1-8 days, Clarke's fixative for 4 h, ethanol for 24 h to 30 days, acetic formalin alcohol for 1 h, methacarn for 1 h, paraformaldehyde for 1 h, OmniFix for 24-72 h, and Zamboni's solution for 4 h, but in specimens fixed in alcoholic formalin for 24 h or Carnoy's solution for 4 h, the maximum product was 989 bp. Fixation in Zenker's fluid only allowed for amplification of 268 bp when fixed for 4 h or less, and fixation in Bouin's fixative only allowed for amplification of the 110 bp product when fixed for 1 h or less.

   Biospecimens

   • Tissue - Cervix

   Preservative Types

   • Formalin
   • Other Preservative
   • Ethanol

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Acetone Alcoholic formalin Acetic formalin alcohol Bouin's fixative Carnoy's solution Clarke's fluid Ethanol Methacarn Omnifix Paraformaldehyde Zamboni's solution Zenker's fluid
   Biospecimen Preservation	Time in fixative	1 h 4 h 24 h 72 h 8 days 30 days
   PCR Specific	Length of gene fragment	110 bp 268 bp 989 bp 1327 bp
   PCR Specific	Targeted nucleic acid	Beta globin

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2. Study Purpose

   The purpose of this study was to determine the effects of storage duration and target length on the amplification of beta-globin from formalin-fixed paraffin-embedded (FFPE) cervical carcinomas.

   Summary of Findings:

   Amplification of a 268 bp fragment of beta globin was successful 90% of the time in specimens stored for less than 16 y but only 45% of the time in specimens stored for 20 years. A 536 bp fragment was amplified in 60% of 5 y old specimens and none of the specimens which were 20 y old. The effect of specimen age was most dramatic when amplification of a 989 bp fragment was attempted and could not be amplified in any specimens stored for 5 y or longer.

   Biospecimens

   • Tissue - Cervix

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	1-20 years
   PCR Specific	Targeted nucleic acid	Beta globin
   PCR Specific	Length of gene fragment	268 bp 536 bp 989 bp

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