Extraction and amplification of DNA from formalin-fixed, paraffin-embedded tissues.

Author(s): Wu L, Patten N, Yamashiro CT, Chui B

Publication: Appl Immunohistochem Mol Morphol, 2002, Vol. 10, Page 269-74

PubMed ID: 12373156 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to optimize and validate a heat-mediated method for DNA extraction from archival formalin-fixed paraffin-embedded (FFPE) specimens used in conjunction with commercially available DNA extraction kits in several tissue types.

   Summary of Findings:

   Genomic DNA extracted directly from FFPE specimens (sans deparaffinization) with an 98 degrees C incubation in lysis buffer prior to proteinase K digestion yielded a significantly greater PCR efficiency compared to extraction without the heating step; the extent of improvement was dependent on the length of the generated amplicon (ranging from a 3- to an 8-fold increase). Comparable results were achieved with the HighPure DNA preparation kit (Roche Molecular Biochemicals) and the QIAamp DNA mini kit (QIAGEN Ltd.). Similar results were obtained with multiplex PCR (specifically a robust improvement for longer amplicons), tissue types, and specimens stored as paraffin blocks for varying durations (6 months to 8 years).

   Biospecimens

   • Tissue - Colorectal
   • Tissue - Breast
   • Tissue - Kidney
   • Tissue - Spleen
   • Tissue - Small Bowel
   • Tissue - Pancreas
   • Tissue - Lung
   • Tissue - Liver
   • Tissue - Ovary
   • Tissue - Thyroid Gland
   • Tissue - Tonsil

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	Real-time qPCR
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Temperature of heat-induced retrieval	98 degrees C, 15 min Room temperature
   Analyte Extraction and Purification	Analyte isolation method	QIAamp DNA mini kit HighPure DNA preparation kit
   Storage	Storage duration	6 months 1 yr 8 yr
   Biospecimen Acquisition	Biospecimen location	Thyroid Breast Colon Kidney Spleen Tonsil Pancreas Lung Jejunum Ileum Liver Ovary
   PCR Specific	Length of gene fragment	90 bp 368 bp

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