Detection of (NAD(P)H:Quinone oxidoreductase-1, EC 1.6.99.2) 609C-->T and 465C-->T polymorphisms in formalin-fixed, paraffin-embedded human tumour tissue using PCR-RFLP.

Roger M. Phillips, Saurajyoti Basu, John E. Brown, G. Michael Flannigan, Paul M. Loadman, Sandie W. Martin, Brian Naylor, Rajiv Puri, Tariq Shah

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

NQO1 is a cytosolic flavoprotein that plays a dual role in the detoxification of potentially carcinogenic compounds and the bioreductive activation of quinone based anticancer drugs. Two polymorphic variants of NQO1 exist (NQO1*2 and NQO1*3) which cause significant phenotypic reductions in NQO1 protein content and activity. Current methods for detecting NQO1 polymorphisms commonly use PCR-RFLP techniques and have exclusively used DNA isolated from fresh tissues. This study describes a method that is suitable for analysing NQO1 polymorphisms in genomic DNA isolated from formalin-fixed paraffin-embedded tissue. The method utilises two rounds of PCR amplification using a nested primer strategy that generates specific PCR products followed by RFLP analysis using either Hinf1 (for NQO1*2) or Msp1 (for NQO1*3). Whilst existing methods proved unsatisfactory (low product yield and poor specificity), the nested primer strategy produced good quality PCR products suitable for RFLP analysis and genotyping of NQO1*2 and NQO1*3 in archival tissue samples. The ability to utilise the vast archives of human tissue held by pathology laboratories would be of considerable benefit as retrospective studies comparing NQO1 genotype status, patient history and treatment outcomes could be conducted.

LanguageEnglish
Pages1005-1010
Number of pages6
JournalInternational Journal of Oncology
Volume24
Issue number4
DOIs
Publication statusPublished - Apr 2004
Externally publishedYes

Fingerprint

Restriction Fragment Length Polymorphisms
NAD
Paraffin
Formaldehyde
Oxidoreductases
Polymerase Chain Reaction
Neoplasms
Flavoproteins
DNA
Retrospective Studies
Genotype
Pathology
benzoquinone
Pharmaceutical Preparations
Proteins

Cite this

Phillips, Roger M. ; Basu, Saurajyoti ; Brown, John E. ; Flannigan, G. Michael ; Loadman, Paul M. ; Martin, Sandie W. ; Naylor, Brian ; Puri, Rajiv ; Shah, Tariq. / Detection of (NAD(P)H:Quinone oxidoreductase-1, EC 1.6.99.2) 609C-->T and 465C-->T polymorphisms in formalin-fixed, paraffin-embedded human tumour tissue using PCR-RFLP. In: International Journal of Oncology. 2004 ; Vol. 24, No. 4. pp. 1005-1010.
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abstract = "NQO1 is a cytosolic flavoprotein that plays a dual role in the detoxification of potentially carcinogenic compounds and the bioreductive activation of quinone based anticancer drugs. Two polymorphic variants of NQO1 exist (NQO1*2 and NQO1*3) which cause significant phenotypic reductions in NQO1 protein content and activity. Current methods for detecting NQO1 polymorphisms commonly use PCR-RFLP techniques and have exclusively used DNA isolated from fresh tissues. This study describes a method that is suitable for analysing NQO1 polymorphisms in genomic DNA isolated from formalin-fixed paraffin-embedded tissue. The method utilises two rounds of PCR amplification using a nested primer strategy that generates specific PCR products followed by RFLP analysis using either Hinf1 (for NQO1*2) or Msp1 (for NQO1*3). Whilst existing methods proved unsatisfactory (low product yield and poor specificity), the nested primer strategy produced good quality PCR products suitable for RFLP analysis and genotyping of NQO1*2 and NQO1*3 in archival tissue samples. The ability to utilise the vast archives of human tissue held by pathology laboratories would be of considerable benefit as retrospective studies comparing NQO1 genotype status, patient history and treatment outcomes could be conducted.",
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Detection of (NAD(P)H:Quinone oxidoreductase-1, EC 1.6.99.2) 609C-->T and 465C-->T polymorphisms in formalin-fixed, paraffin-embedded human tumour tissue using PCR-RFLP. / Phillips, Roger M.; Basu, Saurajyoti; Brown, John E.; Flannigan, G. Michael; Loadman, Paul M.; Martin, Sandie W.; Naylor, Brian; Puri, Rajiv; Shah, Tariq.

In: International Journal of Oncology, Vol. 24, No. 4, 04.2004, p. 1005-1010.

Research output: Contribution to journalArticle

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