Comparative analysis of four methods to extract DNA from paraffin-embedded tissues: effect on downstream molecular applications.

Author(s): Huijsmans CJ, Damen J, van der Linden JC, Savelkoul PH, Hermans MH

Publication: BMC Res Notes, 2010, Vol. 3, Page 239

PubMed ID: 20840759 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 protein digestion and DNA extraction method on PCR and SNP detection from FFPE breast, prostate, lung and colon tissues.

   Summary of Findings:

   Amplification of spiked PhHV was significantly inhibited when DNA was extracted using the Gentra kit, but not when DNA was extracted by the other three methods. The lowest CT in the TaqMan SNP genotyping assay were obtained when DNA was extracted using the QIAmp or EasyMag kits after proteinase K pretreatment and when the PCR mastermix was homemade. The 600 bp multiplex PCR product was amplifiable from 3 of 4 tissues when a proteinase K pretreatment step was used along with the QIAmp or EasyMag kit, and in all 4 tissues when proteinase K pretreatment was combined with heat extraction. When there was no proteinase K pretreatment, the DNA yield was significantly lower.

   Biospecimens

   • Tissue - Breast
   • Tissue - Colorectal
   • Tissue - Lung
   • Tissue - Prostate

   Preservative Types

   • Formalin

   Diagnoses:

   • Not specified

   Platform:

   Analyte	Technology Platform
   DNA	PCR
   DNA	SNP assay

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Heat-treatment QIAamp DNA-blood-mini-kit NucliSens EasyMAG Gentra Capture-Column-kit
   Analyte Extraction and Purification	Protein digestion	Proteinase k None
   PCR Specific	Length of gene fragment	200 bp 400 bp 600 bp
   SNP assay Specific	Reaction solution	Homemade mastermix ABI mastermix

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