gms | German Medical Science

Background: Genetic susceptibility to environmental or occupational diseases is believed to play an important role in the inter-individual different risk of carcinomas. Especially the Cytochrome P450 oxidase system is involved in the metabolism of carcinogens. Therefore genetic polymorphism within the Cytochrome P450 enzyme, via changes of enzyme activity, could be associated with a different susceptibility to cancer.

Nevertheless the determination of the phenotype is insufficient for a real characterisation of the enzyme activity.

To demonstrate considerable inter-individual variability of Cytochrome P450 oxidase activity due to exogenous (tobacco smoke) or endogenous (genetic polymorphism) influence, a sensitive quantitative determination of enzyme activity is necessary.

Therefore we introduce an approach for analysis of gene expression using real-time quantitative PCR and ∆∆CT-method.

Methods and results: Peripheral blood samples from 20 Caucasians were collected and total RNA was isolated by Trizol®-method. After DNAse treatment total RNA again was precipitated. For further analysis cDNA was reverse transcribed and the absence of genomic DNA was confirmed by ACTB PCR and gel electrophoresis. A Real-time RT PCR Assay to quantify the mRNA expression of the Cytochrome Cyp 1A1, Cyp 1A2, Cyp 1A3 and Cyp 1B1 as well as the housekeeping genes ACTB, HPRT, B2M and GAPDH was established using Light Cycler (Fa. Roche).

Conclusion: With the established method it will be possible to evaluate the influence of gene polymorphisms as well as exogenous events on the gene expression. Thus relevant polymorphism can be identified. In connection with the knowledge of the operating factors it will be possible to estimate the individual risk of illness.