Qiaamp dna ffpe tissue isolation kit

Genomic DNA was isolated from formalin-fixed paraffin-embedded tissue samples (FFPE) obtained by biopsy/resection using the QIAamp^® DNA FFPE Tissue isolation kit (Qiagen, United Kingdom). Ethylenediaminetetraacetic acid (EDTA) peripheral blood was drawn from healthy controls by venipuncture and further used for leukocyte isolation using BloodPrep Chemistry for ABI PRISM™ 6100 Nucleic Acid PrepStation (Applied Biosystems, CA, United States). The concordance between germline and somatic DNA in variations of pharmacogenetic genes is around 100%, according to a recent large-scale literature research. Spectrophotometric analysis was used to evaluate the concentration and purity of the extracted DNA samples (Nanodrop, Shimadzu). For MTHFR genotyping, restriction fragment length polymorphism analysis (PCR-RFLP) was employed as previously described (Jakovljevic et al., 2012 (link); Cavic et al., 2014 (link); 2016 (link)). The analysis was performed by gel electrophoresis on a chip using the Agilent DNA 1000 Kit on the Agilent 2100 Bioanalyzer. To ensure adequate genotyping, a previously established heterozygote sample was used as a method of control, and genotyping was carried out blind to case-control status. 10% of samples, chosen at random, underwent Sanger sequencing analysis to verify the accuracy of the findings.