Novel enzyme immunoassay and optimized DNA extraction for the detection of polymerase-chain-reaction-amplified viral DNA from paraffin-embedded tissue.

Author(s): Merkelbach S, Gehlen J, Handt S, Füzesi L

Publication: Am J Pathol, 1997, Vol. 150, Page 1537-46

PubMed ID: 9137080 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to evaluate four DNA extraction methods from formalin-fixed, paraffin-embedded (FFPE) tissue for amplification of viral sequences using poylmerase chain reaction (PCR). The tissues examined included liver, spleen, gastrointestinal tract, lymph, nasopharyngeal, and head and neck.

   Summary of Findings:

   The authors identified the QIAamp method of DNA extraction, which promotes DNA binding to a silica column, as the optimal method of DNA extraction from archived FFPE specimens. DNA extracted by the QIAamp method resulted in high yield and DNA quality, yielding fragments ranging in size from 100 to 25,000 kb. While yields using the InstaGene method were half those obtained with QIAamp, DNA integrity was comparable to QIAamp extracted samples. The age of archived FFPE tissue blocks did not influence DNA quality. Surgical- and biopsy-sized specimens extracted by the QIAamp method resulted in successful PCR amplification of fragments ranging in size from 110 to 435 bp.

   Biospecimens

   • Tissue - Liver
   • Tissue - Lymph Node
   • Tissue - Nasopharynx
   • Tissue - Spleen
   • Tissue - Head and Neck
   • Tissue - Esophagus
   • Tissue - Colorectal
   • Tissue - Stomach
   • Tissue - Small Bowel

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Lymphoma
   • Neoplastic - Carcinoma
   • Cytomegalovirus
   • Mononucleosis

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	Electrophoresis

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Simple boiling method InstaGene (BioRad) ExWax DNA extraction kit (Oncor) QIAamp (Qiagen)
   Storage	Storage duration	1 week - 5 years
   PCR Specific	Length of gene fragment	110 bp 240 bp 435 bp
   Biospecimen Aliquots and Components	Aliquot size/volume	Surgical specimen (20 mm X 20 mm) Biopsy specimen (2 mm X 2 mm)
   Biospecimen Acquisition	Method of tissue acquisition	Surgical resection Biopsy

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