Volume 27, Issue 1 (Iranian South Medical Journal 2024)
Iran South Med J 2024, 27(1): 37-52 |
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Sobhanparast S, Soleymani J, Aftabi Y, Chaparzadeh N. Optimizing DNA Extraction from Frozen Blood Samples for Studying Telomere Length. Iran South Med J 2024; 27 (1) :37-52
URL: http://ismj.bpums.ac.ir/article-1-1984-en.html
URL: http://ismj.bpums.ac.ir/article-1-1984-en.html
1- Department of Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran
Tuberculosis & Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2- Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3- Tuberculosis & Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4- Department of Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran ,Nchapar@azaruniv.ac.ir
Tuberculosis & Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
2- Pharmaceutical Analysis Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
3- Tuberculosis & Lung Disease Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
4- Department of Biology, School of Basic Sciences, Azarbaijan Shahid Madani University, Tabriz, Iran ,
Abstract: (135 Views)
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.
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.
Type of Study: Original |
Subject:
Biochemistry. Cell Biology and Genetics
Received: 2024/06/2 | Accepted: 2024/09/15 | Published: 2024/10/28
Received: 2024/06/2 | Accepted: 2024/09/15 | Published: 2024/10/28
References
1. Shammas MA. Telomeres, lifestyle, cancer, and aging. Curr Opin Clin Nutr Metab Care 2011; 14(1): 28–34. [DOI]
2. Denham J, Marques FZ, Charchar FJ. Leukocyte telomere length variation due to DNA extraction method. BMC Res Notes 2014; 7: 877. [DOI]
3. Di Pietro F, Ortenzi F, Tilio M, et al. Genomic DNA extraction from whole blood stored from 15- to 30-years at -20 °C by rapid phenol-chloroform protocol: A useful tool for genetic epidemiology studies. Mol Cell Probes 2011; 25(1): 44–8. [DOI]
4. Lin J, Smith DL, Esteves K, et al. Telomere length measurement by qPCR – Summary of critical factors and recommendations for assay design. Psychoneuroendocrinology 2019; 99: 271–8. [DOI]
5. Goldberg S. Mechanical/physical methods of cell distribution and tissue homogenization. Methods Mol Biol 2014; 1295: 1–20. [DOI]
6. Posch A. Sample preparation guidelines for two-dimensional electrophoresis. Arch Physiol Biochem 2014; 120(5): 192–197. [DOI]
7. Akane A, Matsubara K, Nakamura H, et al. Identification of the heme compound copurified with deoxyribonucleic acid (DNA) from bloodstains, a major inhibitor of polymerase chain reaction (PCR) amplification. J Forensic Sci 1994; 39(2): 362–372. [DOI]
8. de Franchis R, Cross NCP, Foulkes NS, et al. A potent inhibitor of Taq polymerase copurifies with human genomic DNA. Nucleic Acids Res 1988; 16(21): 10355. [DOI]
9. Sidstedt M, Hedman J, Romsos EL, et al. Inhibition mechanisms of hemoglobin, immunoglobulin G, and whole blood in digital and real-time PCR. Anal Bioanal Chem 2018; 410(10): 2569–2583. [DOI]
10. Lucena-Aguilar G, Sánchez-López AM, BarberánAceituno C, et al. DNA source selection for downstream applications based on dna quality indicators analysis. Biopreserv Biobank 2016; 14(4): 264–270. [DOI]
11. Boardman LA, Skinner HG, Litzelman K. elomere length varies by DNA extraction method: Implications for epidemiologic research - Response. Cancer Epidemiol Biomarkers Prev 2014; 23(6): 1131. [DOI]
12. Cunningham JM, Johnson RA, Litzelman K, et al. Telomere length varies by DNA extraction method: Implications for epidemiologic research. Cancer Epidemiol Biomarkers Prev 2013; 22(11): 2047–2054. [DOI]
13. Martin-Ruiz CM, Baird D, Roger L, et al. Reproducibility of telomere length assessment: An international collaborative study. Int J Epidemiol 2015; 44(5): 1749–1754. [DOI]
14. Joglekar MV, Satoor SN, Wong WKM, et al. An optimised step-by-step protocol for measuring relative telomere length. Methods Protoc 2020; 3(2): 27. [DOI]
15. Cawthon RM. Telomere measurement by quantitative PCR. Nucleic Acids Res 2002; 30(10): e47. [DOI]
16. Guha P, Das A, Dutta S, et al. A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples. J Clin Lab Anal 2018; 32(1): e22181. [DOI]
17. Chacon-Cortes D, Haupt LM, Lea RA, et al. Comparison of genomic DNA extraction techniques from whole blood samples: A time, cost and quality evaluation study. Mol Biol Rep 2012; 39(5): 5961–5966. [DOI]
18. Brown WE, Hu JC, Athanasiou KA. Ammonium-chloride-potassium lysing buffer treatment of fully differentiated cells increases cell purity and resulting neotissue functional properties. Tissue Eng - Part C Methods 2016; 22(9): 895–903. [DOI]
19. Galyuk EN, Lando DY, Egorova VP, et al. Na2CO3 influence on DNA double helix stability: Strong anion destabilizing effect. J Biomol Struct Dyn 2003; 20(6): 801–809. [DOI]
20. Borges A, Rosa MS, Recchia GH, et al. CTAB methods for DNA extraction of sweetpotato for microsatellite analysis. Sci Agric 2009; 66(4): 529–534. [DOI]
21. Song Y, Fahs A, Feldman C, et al. A reliable and effective method of DNA isolation from old human blood paper cards. Springerplus 2013; 2: 616. [DOI]
22. Terry CF, Harris N, Parkes HC. Detection of genetically modified crops and their derivatives: Critical steps in sample preparation and extraction. J AOAC Int. 2002; 85(3): 768–774. [DOI]
23. Budelier K, Schorr J. Purification of DNA by Anion‐ Exchange Chromatography. Curr Protoc Mol Biol 2001: Chapter 2: Unit2. 1B. [DOI]
24. Carpi FM, Di Pietro F, Vincenzetti S, et al. Human DNA extraction methods: Patents and applications. Recent Patents DNA Gene Seq 2011; 5(1): 1–7. [DOI]
25. Demeke T, Jenkins GR. Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits. Anal Bioanal Chem 2010; 396(6): 1977–1990. [DOI]
26. Thomas JC, Khoury R, Neeley CK, et al. A fast CTAB method of human DNA isolation for polymerase chain reaction Applications. Biochem Educ 1997; 25(4): 233–235. [DOI]
27. Shokrzadeh M, Mohammadpour A. Evaluation of a modified salt-out method for DNA extraction from whole blood lymphocytes: A simple and economical method for gene polymorphism. Pharm Biomed Res 2018; 4(2): 28–32. [DOI]
28. Ebadi Z, Ghaffarian S. Association of WDR79 (rs2287497C>T) Poly-morphism with Breast Cancer Susceptibility in Northwest of Iran. Iran South Med J 2023; 26(1): 1-13. [DOI]
29. Aronhime S, Calcagno C, Jajamovich GH, et al. DCEMRI of the liver: Effect of linear and nonlinear conversions on hepatic perfusion quantification and reproducibility. J Magn Reson Imaging 2014; 40(1): 90–8. [DOI]
30. Liu P, Zhu Z, Zeng C, et al. Specific absorption spectra of hemoglobin at different PO2 levels: potential noninvasive method to detect PO2 in tissues. J Biomed Opt 2012; 17(12): 125002. [DOI]
31. Al-Soud WA, Rådström P. Purification and characterization of PCR-inhibitory components in blood cells. J Clin Microbiol 2001; 39(2): 485–493. [DOI]
32. Zhou L, Lei Q, Guo J, et al. Long-term whole blood DNA preservation by cost-efficient cryosilicification. Nat Commun 2022; 13: 6265. [DOI]
33. Kuffel A, Gray A, Daeid NN. Impact of metal ions on PCR inhibition and RT-PCR efficiency. Int J Legal Med 2021; 135(1): 63–72. [DOI]
34. Heikrujam J, Kishor R, Behari Mazumder P. the chemistry behind plant dna isolation protocols. In: Boldura OM, Baltă C, Awwad NS eds. Biochemical analysis tools - Methods for bio-molecules studies. IntechOpen, 2020. [DOI]
35. Lahiri DK, Schnabel B. DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality. Biochem Genet 1993; 31(7): 321–328. [DOI]
36. Opel KL, Chung D, McCord BR. A study of PCR inhibition mechanisms using real time PCR. J Forensic Sci 2010; 55(1): 25–33. [DOI]
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