Optimizing DNA Extraction from Frozen Blood Samples for Studying Telomere Length

Abstract

Background: Optimizing DNA extraction for the preservation of telomere length – as a biomarker for ageing and vari-ous diseases – is highly important. Long-term stored blood samples are considered essential resources for genetic studies. In this study, different lysis buffers were used along with CTAB extraction buffer to inves-tigate the quality of extracted DNA and the effect of its accompanying inhibitors on quantitative PCR (q-PCR) in telomere studies. Materials and Methods: DNA extraction was performed using six RBC lysis buffers and CTAB-based extraction buffer. DNA integrity was evaluated by gel electrophoresis, quantity by absorbance at 260 nm and its purity with expected values of 1.8 and 2-2.2 for the ratios of A260/A280 and A260/A230. The quantitative effect of each buffer in q-PCR and the repeatability of the results were assessed by calculating the reaction efficiency, coefficient of determination (R2), and percentage of coefficient of variation (%CV). Results: Buffer number 5 (10 mM Tris-HCl, pH 7.6, 50 mM NaCl) yielded the highest amount of DNA extraction (324.93 ng/ml, %CV 11.53). All the extracted DNA samples were pure, as indicated by the acceptable A260/A280 and A260/A230ratios (p>0.05). Gel analysis revealed that the extracted DNA of all the buffers ex-cept one was intact. The DNA molecule extracted with buffer number 1 (155 mM NH4Cl, 10 mM KHCO3, and 5 mM EDTA) showed the best performance in q-PCR for HBG gene (efficiency=0.126, R2=0.97) and telomere (efficiency=0.99, R2=0.99). Conclusion: The DNA molecule extracted from frozen blood samples by buffer number 1 showed the least q-PCR inhibition for telomere study.

Keywords

• telomere
• dna extraction
• hemoglobin
• blood
• buffer