New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer: the Iowa women's health study.
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.


OBJECTIVE
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 mug). 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.
Thyagarajan
B
B
University of Minnesota, Division of Epidemiology, Suite 300, West Bank Office Building, Minneapolis, MN 55454, USA.
Anderson
K E
KE
Kong
F
F
Selk
F R
FR
Lynch
C F
CF
Gross
M D
MD
D001943:Breast Neoplasms / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification*; D005240:Feasibility Studies; D005260:Female; D005838:Genotype; D006801:Humans; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D020641:Polymorphism, Single Nucleotide; D013482:Superoxide Dismutase / Q000235:genetics
D004273:DNA, Neoplasm; D013482:Superoxide Dismutase