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New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women’s health study
  1. B Thyagarajan1,
  2. K E Anderson1,
  3. F Kong1,
  4. F R Selk2,
  5. C F Lynch2,
  6. M D Gross3
  1. 1University of Minnesota, Division of Epidemiology, Suite 300, West Bank Office Building, Minneapolis MN-55454, USA
  2. 2Department of Epidemiology, University of Iowa, Iowa City, IA 52242, USA
  3. 3Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN-55455, USA
  1. Correspondence to:
 Myron D Gross
 Department of Laboratory Medicine and Pathology, University of Minnesota, MMC 609, 420 Delaware Street SE, Minneapolis, MN-55455, USA; grossepi.umn.edu

Abstract

Background: The use of paraffin wax embedded tissue samples as a source of DNA for genotype analysis has been limited because of difficulties in DNA extraction and single nucleotide polymorphism (SNP) analysis.

Aims: To test the feasibility of applying the combination of a commonly used DNA isolation procedure, PureGene, and a high throughput SNP analysis method, the polymerase chain reaction (PCR)-INVADER assay, to genotype several types of paraffin wax embedded breast tissues.

Methods: Twenty formalin fixed, paraffin wax blocks were obtained from five participants in the Iowa women’s health study. Each participant provided several types of tissue including normal lymph node, normal nipple/areola tissue, inflammatory/fibrotic breast tissue, or normal breast tissue, and tumour tissue.

Results: Good quality DNA (260/280 ratio >1.6) was obtained from all tissues. Normal lymph nodes yielded the largest amount of DNA (97.1 μg). DNA obtained from the samples was tested for a germline C1183T polymorphism in the MnSOD gene by three methods—PCR-RFLP (restriction fragment length polymorphism), INVADER assay, and PCR-INVADER assay. Of the 20 samples, PCR-RFLP genotyped 16, the PCR-INVADER assay 18, and the INVADER assay two. This methodology was then used to analyse five additional genotypes and confirmed the general applicability of the method.

Conclusions: This study demonstrated the feasibility of (1) using several paraffin wax embedded breast tissues as a source of DNA for germline genetic analysis, with lymph nodes providing the highest yield, and (2) using the combination of a common extraction method with a high throughput SNP analysis method, the PCR–INVADER assay.

  • MnSOD, manganese superoxide dismutase
  • PCR, polymerase chain reaction
  • RFLP, restriction fragment length polymorphism
  • SNP, single nucleotide polymorphism
  • Dna genotyping
  • paraffin wax embedded tissue blocks
  • single nucleotide polymorphisms

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