Rapid techniques for DNA extraction from routinely processed archival tissue for use in PCR.

Author(s): Sepp R, Szabó I, Uda H, Sakamoto H

Publication: J Clin Pathol, 1994, Vol. 47, Page 318-23

PubMed ID: 8027368 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to compare the utility of four rapid DNA extraction techniques among FFPE specimens (fixed for 24-48 h) for use in the PCR application. The tissue types evaluated included skin, soft tissue, tonsils, esophagus, duodenum, colon, pancreas, lung, kidney, urinary bladder, meninges, ovary, cervix, normal placenta, and endometrium, as well as non-Hodgkin's lymphoma. The amplified targets were portions of the p53, HPV, and IgH genes.

   Summary of Findings:

   PCR amplification of larger surgical specimens was successful in all cases, producing a single product of predicted size and equal intensity for all DNA extraction methods examined. For smaller biopsy-sized specimens, DNA from 17-33% or 50-80% of specimens extracted using the boiling water method failed to produce a product or produced a product of reduced intensity, respectively. Single products of equivalent intensity were observed among biopsy-sized specimens extracted by Chelex-boiling, proteinase k digestion, or a combination of the two methods. The largest product size achieved (983 bp) was in large surgical specimens that underwent DNA extraction by proteinase K digestion or proteinase K digestion followed by Chelex boiling.

   Biospecimens

   • Tissue - Cervix
   • Tissue - Skin
   • Tissue - Tonsil
   • Tissue - Esophagus
   • Tissue - Small Bowel
   • Tissue - Colorectal
   • Tissue - Pancreas
   • Tissue - Lung
   • Tissue - Kidney
   • Tissue - Bladder
   • Tissue - Brain
   • Tissue - Ovary
   • Tissue - Placenta
   • Tissue - Endometrium
   • Tissue - Lymph Node
   • Tissue - Other

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Boiling in distilled water Boiling in 5% Chelex-100 Proteinase K Proteinase K and boiling in 5% Chelex-100
   Biospecimen Aliquots and Components	Aliquot size/volume	16 mm X 18 mm (Surgical specimen) 1.7 mm X 2.0 mm (Biopsy specimen)
   PCR Specific	Length of gene fragment	80-120 bp 408 bp 647 bp 983 bp
   PCR Specific	Targeted nucleic acid	p53 HPV16 IgH
   Biospecimen Acquisition	Method of tissue acquisition	Biopsy Surgical resection

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2. Study Purpose

   The purpose of this study was to determine if DNA sample storage for 3 months at -20 degrees C affects PCR amplification efficiency in FFPE specimens extracted by different methods.

   Summary of Findings:

   After prolonged DNA sample storage (3 months at -20 degrees C) PCR efficiency was dependent on product size and extraction method. PCR generated products of 408 bp yielded less intense amplicons after DNA sample storage, while successful generation of a 647 bp PCR product was achieved only in samples extracted by proteinase K or proteinase K-Chelex boiling methods.

   Biospecimens

   • Tissue - Cervix

   Preservative Types

   • Formalin

   Diagnoses:

   • Neoplastic - Carcinoma
   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   PCR Specific	Length of gene fragment	80-166 bp 408 bp 647 bp
   Analyte Extraction and Purification	Analyte isolation method	Boiling in distilled water Boiling in 5% Chelex-100 Proteinase K digestion Proteinase K followed by boiling in 5% Chelex-100
   PCR Specific	Targeted nucleic acid	p53 HPV16 IgH
   Storage	Storage duration	3 months

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