Detection of IL28B SNP DNA from Buccal Epithelial Cells, Small Amounts of Serum, and Dried Blood Spots

Background & Aims: Point mutations in the coding region of the interleukin 28 gene (rs12979860) have recently been identified for predicting the outcome of treatment of hepatitis C virus infection. This polymorphism detection was based on whole blood DNA extraction. Alternatively, DNA for genetic diagnosis has been derived from buccal epithelial cells (BEC), dried blood spots (DBS), and genomic DNA from serum. The aim of the study was to investigate the reliability and accuracy of alternative routes of testing for single nucleotide polymorphism allele rs12979860CC. Methods: Blood, plasma, and sera samples from 200 patients were extracted (400 mL). Buccal smears were tested using an FTA card. To simulate postal delay, we tested the influence of storage at ambient temperature on the different sources of DNA at five time points (baseline, 48 h, 6 days, 9 days, and 12 days) Results: There was 100 % concordance between blood, plasma, sera, and BEC, validating the use of DNA extracted from BEC collected on cytology brushes for genetic testing. Genetic variations in HPTR1 gene were detected using smear technique in blood smear (3620 copies) as well as in buccal smears (5870 copies). These results are similar to those for whole blood diluted at 1/10. A minimum of 0.04 mL, 4 mL, and 40 mL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma. No significant variation between each time point was observed for the different sources of DNA. IL28B SNPs analysis at these different time points showed the same results using the four sources of DNA

Quantity of DNA obtained by different preparation methods and quantification of HPTR-1 gene in blood, sera, and plasma.

<p>Blood, sera, and plasma were successively diluted at 10, 100, 1000, and 10000 in PBS. 400 µL of each type of sample were extracted with MagnaPur and eluted in 100 µL of elution buffer. A minimum of 0.04 µL, 4 µL, and 40 µL was necessary to obtain exploitable results respectively for whole blood, sera, and plasma.</p

Study Design.

<p>Study Design.</p

IL28 SNP according to sampling types (buccal smear, whole blood, plasma, and DBS).

<p>IL28 SNP according to sampling types (buccal smear, whole blood, plasma, and DBS).</p

Threshold for DNA detection among different sampling types.

<p>Threshold for DNA detection among different sampling types.</p