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.
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.
Phillips
Roger M
RM
Cancer Research Unit, Tom Connors Cancer Research Centre, University of Bradford, UK. r.m.phillips@bradford.ac.uk
Basu
Saurajyoti
S
Brown
John E
JE
Flannigan
G Michael
GM
Loadman
Paul M
PM
Martin
Sandie W
SW
Naylor
Brian
B
Puri
Rajiv
R
Shah
Tariq
T
D002295:Carcinoma, Transitional Cell / Q000201:enzymology / Q000235:genetics*; D005557:Formaldehyde; D005838:Genotype; D006801:Humans; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics*; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic; D012150:Polymorphism, Restriction Fragment Length; D014183:Transplantation, Heterologous / Q000473:pathology; D001749:Urinary Bladder Neoplasms / Q000201:enzymology / Q000235:genetics*
D005557:Formaldehyde; D016660:NAD(P)H Dehydrogenase (Quinone); C447479:NQO1 protein, human