Donmuş Çözünmüş Kanın Farklı Değerlerde Santrifüjünün DNA İzolasyonu Üzerine Etkileri

Year 2023, , 154 - 163, 30.04.2023

Mevlut Arslan

https://doi.org/10.53433/yyufbed.1130525

Abstract

Kandan DNA izolasyonu, nDNA ve mtDNA’yı elde etmek için yaygın kullanılan bir uygulamadır. Donmuş çözünmüş kanın (DÇK) santrifüjü sonrası elde edilen çökeltiden DNA izolasyonun gerçekleştirilebileceği daha önce gösterilmişti ve bu ön uygulama DNA izolasyonu üzerine olumlu sonuçlara sahipti. Fakat, bu ön uygulama için hangi santrifüj değerlerinin kullanılabileceği ve bunların etkileri bilinmemektedir. Bu çalışmanın amacı belirtilen ön uygulama için uygun santrifüj değerlerini belirlemek ve bu santrifüj değerlerinin izole edilen DNA üzerine ektilerini göstermekti. Bu amaçla, DÇK’nin farklı değerlerde santrifüjüyle elde edilen çökelti ve süpernatantdan DNA izolasyonları gerçekleştirildi. Sonra, izole edilen DNA örneklerinden spektrofotometrik, jel elektroforezi ve gerçek-zamanlı PCR analizleri gerçekleştirildi. Sonuç olarak, DÇK’nin, 5.000×g’de 2 dakika ya da üzeri değerde santrifüjü genetik materyali tamamen çöktürmeyi sağladı. Bu durum ayrıca yüksek miktarda DNA elde edilmesine imkan sağladı. Belirtilen santrifüj seviyelerinde elde edilen çökeltiden izole edilen DNA örneklerinde mtDNA/nDNA oranı değişmedi fakat, DNA bütünlüğü azaldı. Sonuç olarak, DÇK’nin 5.000×g’de 2 dakika ya da üzerinde santrifüjü, DNA izolasyonundan önce DÇK’de bulunan genetik materyali çöktürmek ve yıkamak için kullanılabilir.

Keywords

DNA izolasyonu, Donmuş-çözünmüş kan, Kan, Santrifüj

Supporting Institution

Van Yüzüncü Yıl Üniveristesi, Bilimsel Araştırma Projeleri Koodinasyon Birimi

Project Number

THD-2019-8401

Effects of Centrifugation at Different Levels of Freeze-Thawed Blood on DNA Isolation

Abstract

DNA isolation from blood is a commonly used application to obtain nDNA and mtDNA. It was previously shown that DNA isolation could be performed from the pellet obtained after centrifugation of freeze-thawed blood (FTB), and this pretreatment had constructive results on DNA isolation. However, which centrifugation levels can be used for this pretreatment, and their effects are unknown. The aim of the study was to determine appropriate centrifugation levels for this pretreatment and show their effects on isolated DNA. For this purpose, DNA isolations were carried out from pellet and supernatant obtained by centrifugation at different levels of FTB. Then, spectrophotometric, gel electrophoresis, and real-time PCR analyses were performed in the isolated DNA samples. As a result, centrifugation of FTB at 5,000×g for 2 min or over let genetic material to pellet completely. This also caused to obtain high amount of DNA. mtDNA/nDNA ratios did not change in the isolated DNA samples from pellets obtained by defined centrifugation levels, but the DNA integrity decreased. To conclude, centrifugation of FTB at 5,000×g for 2 min or over can be used to harvest and wash genetic material found in FTB before DNA isolations.

Keywords

Blood, Centrifuge, DNA isolation, Freeze-thawed blood

Project Number

THD-2019-8401

References

• Arslan, M. (2022). Effects of centrifugation and washing of freeze-thawed blood on isolated DNA characteristics. Turkish Journal of Veterinary and Animal Sciences, 46(1), 130-138. doi:10.3906/vet-2106-94
• Arslan, M., Tezcan, E., Camcı, H., & Avcı, M. K. (2021). Effect of DNA concentration on band intensity and resolution in agarose gel electrophoresis. Van Health Sciences Journal, 14(3), 326-333. doi:10.52976/vansaglik.969547
• Bulla, A., De Witt, B., Ammerlaan, W., Betsou, F., & Lescuyer, P. (2016). Blood DNA yield but not integrity or methylation is impacted after long-term storage. Biopreservation and Biobanking, 14(1), 29-38. doi:10.1089/bio.2015.0045
• Cartozzo, C., Singh, B., Boone, E., & Simmons, T. (2018). Evaluation of DNA Extraction Methods from Waterlogged Bones: A Pilot Study. Journal of Forensic Sciences, 63(6), 1830-1835. doi:10.1111/1556-4029.13792
• Chacon-Cortetes, D., & Griffith, L.R. (2014). Methods for extracting genomic DNA from whole blood samples: Current perspectives. Journal of Biorepository Science for Applied Medicine, 2014(2), 1-9. doi:10.2147/bsam.S46573
• Cottle, C., Porter, A. P., Lipat, A., Turner-Lyles, C., Nguyen, J., Moll, G., & Chinnadurai, R. (2022). Impact of cryopreservation and freeze-thawing on therapeutic properties of mesenchymal stromal/stem cells and other common cellular therapeutics. Current Stem Cell Reports, 8(2), 72-92. doi:10.1007/s40778-022-00212-1
• Craig, J. M., Vena, N., Ramkissoon, S., Idbaih, A., Fouse, S. D., Ozek, M., Sav, A., Hill, D. A., Margraf, L. R., Eberhart, C. G., Kieran, M. W., Norden, A. D., Wen, P. Y., Loda, M., Santagata, S., Ligon, K. L., & Ligon, A. H. (2012). DNA fragmentation simulation method (FSM) and fragment size matching improve aCGH performance of FFPE tissues. PloS One, 7(6), e38881. doi:10.1371/journal.pone.0038881
• Dagur, P. K., & McCoy, J. P. Jr. (2015). Collection, storage, and preparation of human blood cells. Current Protocols in Cytometry, 73(5), 1-16. doi:10.1002/0471142956.cy0501s73
• Dahm, R. (2005). Friedrich Miescher and the discovery of DNA. Developmental Biology, 278(2), 274-288. doi:10.1016/j.ydbio.2004.11.028
• Evans, S. O., Jameson, M. B., Cursons, R. T. M., Peters, L. M., Bird, S., & Jacobson, G. M. (2016). Development of a qPCR method to measure mitochondrial and genomic DNA damage with application to chemotherapy-induced DNA damage and cryopreserved cells. Biology, 5(4), 39. doi:10.3390/biology5040039
• Fuentes-Pardo, A. P., & Ruzzante, D. E. (2017). Whole-genome sequencing approaches for conservation biology: Advantages, limitations and practical recommendations. Molecular Ecology, 26(20), 5369-5406. doi:10.1111/mec.14264
• Gao, X., Jia, M., Zhang, Y., Breitling, L. P., & Brenner, H. (2015). DNA methylation changes of whole blood cells in response to active smoking exposure in adults: A systematic review of DNA methylation studies. Clinical Epigenetics, 7, 113. doi:10.1186/s13148-015-0148-3
• Gautam, A., (2022). Phenol-Chloroform DNA Isolation Method. In A. Gautam (Ed.), DNA and RNA Isolation Techniques for Non-Experts (pp. 33-39). Cham, Springer International Publishing. doi:10.1007/978-3-030-94230-4_3
• Ghatak, S., Muthukumaran, R. B., & Nachimuthu, S. K. (2013). A simple method of genomic DNA extraction from human samples for PCR-RFLP analysis. Journal of Biomolecular Techniques, 24(4), 224-231. doi:10.7171/jbt.13-2404-001
• Green, M.R. & Sambrook, J. (2018). Isolation and quantification of DNA. Cold Spring Harbor Protocols, 2018(6), pdb. top093336. doi:10.1101/pdb.top093336
• Heard, B. E. (1955). The histological appearances of some normal tissues at low temperatures. British Journal of Surgery, 42(174), 430-437. doi:10.1002/bjs.18004217416
• Hjorthaug, H. S., Gervin, K., Mowinckel, P., & Munthe-Kaas, M. C. (2018). Exploring the influence from whole blood DNA extraction methods on Infinium 450K DNA methylation. PloS One, 13(12), e0208699. doi:10.1371/journal.pone.0208699
• Kaya, Z., Almalı, N., Sahin, E. S., Duran, S., Görgisen, G., & Ates, C. (2022). Association of insulin-like growth factor binding protein-7 promoter methylation with esophageal cancer in peripheral blood. Molecular Biology Reports, 49, 3423-3431. doi:10.1007/s11033-022-07173-y
• Lahiri, D. K., & Schnabel, B. (1993). DNA isolation by a rapid method from human blood samples: Effects of MgCl2, EDTA, storage time, and temperature on DNA yield and quality. Biochemical Genetics, 31, 321-328. doi:10.1007/bf02401826
• Lee, S. V., & Bahaman, A. R. (2012). Discriminatory Power of Agarose Gel Electrophoresis in DNA Fragments Analysis. In S. Magdeldin (Ed.), Gel Electrophoresis Principles and Basics (pp. 41-56). Crotia: IntechOpen. Lippi, G. (2012). Interference studies: Focus on blood cell lysates preparation and testing. Clinical laboratory, 58(3-4), 351–355.
• Livak, K. J., & Schmittgen, T. D. (2001). Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods, 25(4), 402-408. doi:10.1006/meth.2001.1262
• Lu, H. Y., Zhao, G. L., & Fu, M. F. (2016). Polymorphisms in the vascular endothelial growth factor (VEGF) gene associated with asthma. Genetics and Molecular Research, 15(2), gmr.15027880. doi:10.4238/gmr.15027880
• Lucena-Aguilar, G., Sánchez-López, A. M., Barberán-Aceituno, C., Carrillo-Ávila, J. A., López-Guerrero, J. A., & Aguilar-Quesada, R. (2016). DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreservation and Biobanking, 14(4), 264–270. doi:10.1089/bio.2015.0064
• McGann, L. E., Yang, H., & Walterson, M. (1988). Manifestations of cell damage after freezing and thawing. Cryobiology, 25(3), 178-185. doi:10.1016/0011-2240(88)90024-7
• Olins, A. L., & Olins, D. E. (1974). Spheroid chromatin units (v bodies). Science, 183(4122), 330-332. doi:10.1126/science.183.4122.330
• Rzehak, P., Saffery, R., Reischl, E., Covic, M., Wahl, S., Grote, V., Xhonneux, A., Langhendries, J. P., Ferre, N., Closa-Monasterolo, R., Verduci, E., Riva, E., Socha, P., Gruszfeld, D., & Koletzko, B. (2016). Maternal smoking during pregnancy and DNA-Methylation in children at age 5.5 years: Epigenome-wide-analysis in the European childhood obesity project (CHOP)-study. PloS One, 11(5), e0155554. doi:10.1371/journal.pone.0155554
• R. (2017). R Core Team, R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna.
• Sloviter, H. A. (1962). Mechanism of hæmolysis caused by freezing and its prevention. Nature, 193, 884-885. doi:10.1038/193884a0
• Steponkus, P. L., & Lynch, D. V. (1989). Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation. Journal of Bioenergetics and Biomembranes, 21(1), 21-41. doi:10.1007/bf00762210
• Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and protein extraction: The past and the present. Journal of Biomedicine & Biotechnology, 2009, 574398. doi:10.1155/2009/574398
• Tansey, W. P. (2006). Freeze-thaw lysis for extraction of proteins from mammalian cells. CSH Protocols, 2006(7), pdb.prot4614. doi:10.1101/pdb.prot4614
• Trusal, L. R., Guzman, A. W., & Baker, C. J. (1984). Characterization of freeze-thaw induced ultrastructural damage to endothelial cells in vitro. In Vitro, 20(4), 353–364. doi:10.1007/bf02618599
• Tweedie, J. W., & Stowell, K. M. (2005). Quantification of DNA by agarose gel electrophoresis and analysis of the topoisomers of plasmid and M13 DNA following treatment with a restriction endonuclease or DNA topoisomerase I. Biochemistry and Molecular Biology Education, 33(1), 28-33. doi:10.1002/bmb.2005.494033010410
• Visvikis, S., Schlenck, A., & Maurice, M. (1998). DNA extraction and stability for epidemiological studies. Clinical Chemistry and Laboratory Medicine, 36(8), 551-555. doi:10.1515/CCLM.1998.094
• Wang, F., Wang, L., Briggs, C., Sicinska, E., Gaston, S. M., Mamon, H., Kulke, M. H., Zamponi, R., Loda, M., Maher, E., Ogino, S., Fuchs, C. S., Li, J., Hader, C., & Makrigiorgos, G. M. (2007). DNA degradation test predicts success in whole-genome amplification from diverse clinical samples. The Journal of Molecular Diagnostics: JMD, 9(4), 441–451. doi:10.2353/jmoldx.2007.070004