TY - JOUR
T1 - New approaches for genotyping paraffin wax embedded breast tissue from patients with cancer
T2 - The Iowa women's health study
AU - Thyagarajan, B.
AU - Anderson, Kristin E.
AU - Kong, F.
AU - Selk, F. R.
AU - Lynch, C. F.
AU - Gross, Myron D.
PY - 2005/9
Y1 - 2005/9
N2 - 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.
AB - 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.
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U2 - 10.1136/jcp.2004.023374
DO - 10.1136/jcp.2004.023374
M3 - Article
C2 - 16126877
AN - SCOPUS:24644456556
SN - 0021-9746
VL - 58
SP - 955
EP - 961
JO - Journal of Clinical Pathology
JF - Journal of Clinical Pathology
IS - 9
ER -