**3.3 Our method for gene polymorphism analysis**

The workers' blood samples are harvested in tubes containing either heparin or K2-EDTA from the medical staff of the company following workers' informed consent and according to the ethical guidelines of our research institute. All procedures performed in this study involving human participants [18] are in accordance with the ethical standards of our institutional committee and with the local health unit. Urine samples may be immediately frozen at −20°C after harvesting, and saliva and buccal cells may be stored at RT, while the blood samples may be stored up to 24 hours at a temperature between 4 and 10°C or immediately frozen for the genetic analysis. Blood genomic DNA is isolated by using a DNA blood kit, checked for the quality by agarose gel electrophoresis, quantified by a nanophotometer and stored at 4°C or −20°C. Although there are many novel and reliable techniques used to assess the gene polymorphisms such as Taqman assay, amplification refractory mutation system mass spectrometry (PCR-ARMS), confronting two-primer pair (CTPP-PCR), high-resolution melting and different types of mini-sequencing, our choice is oriented towards the traditional method based on polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) (**Figure 2**). We have tried also the fast and less expensive CTPP-PCR protocol developed previously in which the resulting genotype is obtained by a traditional PCR carried out with two couples of primers, avoiding to proceed for the enzymatic digestion [11]. However in our hands such protocol turned out

**Figure 2.** *Methodological procedure for gene polymorphism analysis by PCR-RFLP.*

to be unreliable. In particular we applied this alternative method to detect two polymorphisms of one gene involved in response to oxidative stress and ototoxicity (NRF2 -617C/A and -653A/G), but the data were not completely satisfactory since CTPP-PCR produced contradictory results, particularly for the heterozygosis classification, requiring another orthogonal technique for confirming the data [20]. In the following section, we propose a list of the gene polymorphisms that we usually evaluate in the biological monitoring of the occupational exposure. They have been grouped on the basis of the enzyme function, i.e. detoxification, oxidative stress and DNA repair. The majority of them has been analyzed and reported in our previous published papers as biomarkers of susceptibility to the exposure of several organic compounds including styrene, toluene, ethylbenzene, benzene as well as biomarkers of genotoxic damage and of oxidative stress [14–17, 21]. **Table 1** shows a list of the analyzed susceptibility biomarkers together with the PCR-RFLP protocols which have been used by our group with some modifications.
