Effects of centrifugation and washing of freeze-thawed blood on isolated DNA characteristics

DNA isolations from the whole blood are commonly performed to obtain DNA for molecular research and diagnostics. Generally, blood samples are taken into anticoagulant tubes and stored in deep freezers until DNA isolation. In fresh blood, pretreatments or leukocytes preparations can be performed and suggested for advanced DNA isolation. However, similar applications in freeze-thawed blood (FTB) have not been shown yet. In the study, centrifugation and washing of FTB were applied as pretreatment before DNA isolations, and their effects on isolated DNA characteristics including DNA integrity, quality, quantity, mitochondrial (mt), and nuclear (n) DNA levels were investigated. Microscopic and flow cytometric analyses were used to check leukocyte integrity in FTB. Spectrophotometric analysis was carried out to determine DNA quality and quantity in the isolated DNA samples. Real-time PCR analyses were used to check mtDNA/nDNA ratio and DNA integrity at the quantitative level. Cell integrity analyses showed that most of the leukocytes were intact in FTB. Therefore, centrifugation enabled intact leukocytes and nuclear pellets in FTB to be harvested and washed and could be applied as pretreatment before DNA isolations. PBS and water washing of FTB led to obtaining high-quality DNA without changing the nDNA/mtDNA ratio and DNA integrity. TE washing of FTB increased DNA quality and enriched nDNA level about 2-fold without changing DNA integrity. Centrifugation and harvesting of a higher volume of FTB increased isolated DNA yield and quality but decreased DNA integrity and nDNA level. To conclude, the pretreatments of FTB had the advantage to obtain DNA with high-quality and high-quantity and can be used before DNA isolation, but they may affect mtDNA/nDNA ratios and DNA integrity levels. The relevant pre-treatment used in the present study can be used and improved for desired DNA isolation from FTB samples.

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1. Chacon-Cortetes D, Griffith LR. 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
2. Guha P, Das A, Dutta S, Chaudhuri TK. A rapid and efficient DNA extraction protocol from fresh and frozen human blood samples. Journal of Clinical Laboratory Analysis 2018; 32(1): e22181. doi: 10.1002/jcla.22181
3. Ferro P, Ortega-Pinazo J, Martínez B, Jiménez Á, GómezZumaquero JM et al. On the use of buffy or whole blood for obtaining DNA of high quality and functionality: What is the best option? Biopreserv Biobank 2019; 17 (6): 577-582. doi: 10.1089/bio.2019.0024
4. Dean L. Blood groups and red cell antigens. USA: National Center for Biotechnology Information; 2005.
5. Mao X, Huang TJ, Ho C-M. The lab on a chip approach for molecular diagnostics. In: Grody WW, Nakamura RM, Strom CM, Kiechle FL (editors). Molecular Diagnostics. San Diego: Academic Press; 2010. p. 21-34.
6. Garraud O, Cognasse F. Are platelets cells? and if yes, are they immune cells? Frontiers in Immunology 2015; 6: 70. doi: 10.3389/fimmu.2015.00070
7. Al-Soud WA, Jonsson LJ, Radstrom P. Identification and characterization of immunoglobulin G in blood as a major inhibitor of diagnostic PCR. Journal of Clinical Microbiology 2000; 38(1): 345-350. doi: 10.1128/JCM.38.1.345-350.2000
8. Sengar DP, Jerome FN, Douglas RJ. A simple method for separation of buffy coat from peripheral blood of chickens. Canadian Journal of Comparative Medicine 1968; 32(4): 593- 597.
9. Mychaleckyj JC, Farber EA, Chmielewski J, Artale J, Light LS et al. Buffy coat specimens remain viable as a DNA source for highly multiplexed genome-wide genetic tests after long term storage. Journal of Translational Medicine 2011; 9: 91. doi: 10.1186/1479-5876-9-91
10. Boyd MA, Tennant SM, Melendez JH, Toema D, Galen JE et al. Adaptation of red blood cell lysis represents a fundamental breakthrough that improves the sensitivity of Salmonella detection in blood. Journal of Applied Microbiology 2015; 118 (5): 1199-1209. doi: 10.1111/jam.12769
11. Kamangu EN. Comparison of the quality of the DNA extracted from different media at the laboratory of molecular biology of the faculty of medicine of UNIKIN. International Journal of Molecular Biology: Open Access 2019; 4 (1): 27-28. doi: 10.15406/ijmboa.2019.04.00094
12. Foley C, O’Farrelly C, Meade KG. Technical note: Comparative analyses of the quality and yield of genomic DNA from invasive and noninvasive, automated and manual extraction methods. Journal of Dairy Science 2011; 94 (6): 3159-3165. doi: 10.3168/ jds.2010-3987
13. Tansey WP. Freeze-thaw lysis for extraction of proteins from mammalian cells. Cold Spring Harbor Protocols 2006; 2006 (7): pdb.prot4614. doi:10.1101/pdb.prot4614
14. McGann LE, Yang H, Walterson M. Manifestations of cell damage after freezing and thawing. Cryobiology 1988; 25(3): 178-185. doi: 10.1016/0011-2240(88)90024-7
15. Dagur PK, McCoy JP, Jr. Collection, storage, and preparation of human blood cells. Current Protocols in Cytometry 2015; 73: 5.1.1-5.1.16. doi: 10.1002/0471142956.cy0501s73
16. Lippi G. Interference studies: focus on blood cell lysates preparation and testing. Clinical laboratory 2012; 58 (3-4): 351-355.
17. Lahiri DK, Nurnberger JI, Jr. A rapid non-enzymatic method for the preparation of HMW DNA from blood for RFLP studies. Nucleic Acids Research 1991; 19 (19): 5444. doi: 10.1093/nar/19.19.5444
18. Parzer S, Mannhalter C. A rapid method for the isolation of genomic DNA from citrated whole blood. Biochemical Journal 1991; 273(Pt 1)(Pt 1): 229-231. doi: 10.1042/bj2730229
19. Jiang L, Jiang J, Yang J, Liu X, Wang J et al. Genome-wide detection of copy number variations using high-density SNP genotyping platforms in Holsteins. BMC Genomics 2013; 14: 131. doi: 10.1186/1471-2164-14-131
20. Bae JS, Cheong HS, Kim LH, NamGung S, Park TJ et al. Identification of copy number variations and common deletion polymorphisms in cattle. BMC Genomics 2010; 11: 232. doi: 10.1186/1471-2164-11-232
21. Silva VHd, Regitano LCdA, Geistlinger L, Pértille F, Giachetto PF et al. Genome-wide detection of CNVs and their association with meat tenderness in Nelore Cattle. PloS one 2016; 11 (6): e0157711. doi: 10.1371/journal.pone.0157711
22. Gould MP, Bosworth CM, McMahon S, Grandhi S, Grimerg BT et al. PCR-free enrichment of mitochondrial DNA from human blood and cell lines for high quality next-generation DNA sequencing. PloS one 2015; 10 (10): e0139253. doi: 10.1371/journal.pone.0139253
23. Ye J, Coulouris G, Zaretskaya I, Cutcutache I, Rozen S et al. Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 2012; 13: 134. doi: 10.1186/1471-2105-13-134
24. Arslan M. A new primer designing for PCR-RFLP analysis of A and B genetic variants of bovine kappa-casein. Harran Üniversitesi Veteriner Fakültesi Dergisi 2020; 9(1): 6-11. doi: 10.31196/huvfd.651821
25. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method. Methods 2001; 25 (4): 402-408. doi: 10.1006/ meth.2001.1262
26. Evans SO, Jameson MB, Cursons RTM, Peters LM, Bird S et al. Development of a qPCR method to measure mitochondrial and genomic DNA damage with application to chemotherapyinduced DNA damage and cryopreserved cells. Biology 2016; 5 (4): 39. doi: 10.3390/biology5040039
27. R Core Team. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria; 2017.
28. Lucena-Aguilar G, Sánchez-López AM, Barberán-Aceituno C, Carrillo-Ávila JA, López-Guerrero JA et al. DNA source selection for downstream applications based on DNA quality indicators analysis. Biopreservation and Biobanking 2016; 14 (4): 264-270. doi: 10.1089/bio.2015.0064
29. Steponkus PL, Lynch DV. Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation. Journal of Bioenergetics and Biomembranes 1989; 21(1): 21- 41. doi: 10.1007/bf00762210
30. Sherman JK. Comparison of in vitro and in situ ultrastructural cryoinjury and cryoprotection of mitochondria. Cryobiology 1972; 9 (2): 112-122. doi: 10.1016/0011-2240(72)90018-1
31. Tsvetkov T, Tsonev L, Minkov I. A quantitative evaluation of the extent of inner mitochondrial membrane destruction after freezing-thawing based on functional studies. Cryobiology 1986; 23 (5): 433-439. doi: 10.1016/0011-2240(86)90028-3
32. Rowe AW, Lenny LL. Cryopreservation of granulocytes for transfusion: studies on human granulocyte isolation, the effect of glycerol on lysosomes, kinetics of glycerol uptake and cryopreservation with dimethyl sulfoxide and glycerol. Cryobiology 1980; 17 (3): 198-212. doi: 10.1016/0011- 2240(80)90027-9
33. Sawant PL, Desai ID, Tappel AL. Digestive capacity of purified lysosomes. Biochimica et Biophysica Acta 1964; 85: 93-102. doi: 10.1016/0926-6569(64)90170-1
34. Volger H, Heber U, Berzborn RJ. Loss of function of biomembranes and solubilization of membrane proteins during freezing. Biochimica et Biophysica Acta 1978; 511 (3): 455-469. doi: 10.1016/0005-2736(78)90281-x
35. Trusal LR, Guzman AW, Baker CJ. Characterization of freezethaw induced ultrastructural damage to endothelial cells in vitro. In Vitro 1984; 20 (4): 353-364. doi: 10.1007/bf02618599
36. McGann LE, Yang HY, Walterson M. Manifestations of cell damage after freezing and thawing. Cryobiology 1988; 25 (3): 178-185. doi: 10.1016/0011-2240(88)90024-7
37. Yang H, Arnaud F, McGann LE. Cryoinjury in human granulocytes and cytoplasts. Cryobiology 1992; 29 (4): 500- 510. doi: 10.1016/0011-2240(92)90053-5
38. Olins AL, Olins DE. Spheroid chromatin units (v bodies). Science 1974; 183 (4122): 330-332. doi: 10.1126/ science.183.4122.330
39. Belmont AS, Bruce K. Visualization of G1 chromosomes: a folded, twisted, supercoiled chromonema model of interphase chromatid structure. Journal of Cell Biology 1994; 127 (2): 287- 302. doi: 10.1083/jcb.127.2.287
40. Hübner MR, Eckersley-Maslin MA, Spector DL. Chromatin organization and transcriptional regulation. Current Opinion in Genetics and Development 2013; 23 (2): 89-95. doi: 10.1016/j.gde.2012.11.006
41. Koshy L, Anju AL, Harikrishnan S, Kutty VR, Jissa VT et al. Evaluating genomic DNA extraction methods from human whole blood using endpoint and real-time PCR assays. Molecular Biology Reports 2017; 44 (1): 97-108. doi: 10.1007/ s11033-016-4085-9
42. Chacon-Cortes D, Haupt LM, Lea RA, Griffiths LR. Comparison of genomic DNA extraction techniques from whole blood samples: a time, cost and quality evaluation study. Molecular Biology Reports 2012; 39 (5): 5961-5966. doi: 10.1007/s11033-011-1408-8
43. Psifidi A, Dovas CI, Bramis G, Lazou T, Russel CL et al. Comparison of eleven methods for genomic DNA extraction suitable for large-scale whole-genome genotyping and longterm DNA banking using blood samples. PloS one 2015; 10 (1): e0115960. doi: 10.1371/journal.pone.0115960
44. Stulnig TM, Amberger A. Exposing contaminating phenol in nucleic acid preparations. Biotechniques 1994; 16 (3): 402-404.
45. Ahmad S, Ghosh A, Nair DL, Seshadri M. Simultaneous extraction of nuclear and mitochondrial DNA from human blood. Genes and Genetic Systems 2007; 82 (5): 429-432. doi: 10.1266/ggs.82.429
46. Filograna R, Mennuni M, Alsina D, Larsson N-G. Mitochondrial DNA copy number in human disease: the more the better? FEBS Letters 2021; 595 (8): 976-1002. doi: 10.1002/1873-3468.14021
47. Weikard R, Kuehn C. Different mitochondrial DNA copy number in liver and mammary gland of lactating cows with divergent genetic background for milk production. Molecular Biology Reports 2018; 45 (5): 1209-1218. doi: 10.1007/s11033- 018-4273-x
48. Darr CR, Moraes LE, Connon RE, Love CC, Teague S et al. The relationship between mitochondrial DNA copy number and stallion sperm function. Theriogenology 2017; 94: 94-99. doi: 10.1016/j.theriogenology.2017.02.015
49. Zhang X, Wang T, Ji J, Wang H, Zhu X et al. The distinct spatiotemporal distribution and effect of feed restriction on mtDNA copy number in broilers. Scientific Reports 2020; 10 (1): 3240. doi: 10.1038/s41598-020-60123-1
50. Wai T, Ao A, Zhang X, Cyr D, Dufort D et al. The role of mitochondrial DNA copy number in mammalian fertility. Biology of Reproduction 2010; 83 (1): 52-62. doi: 10.1095/ biolreprod.109.080887
51. Heard BE. The histological appearances of some normal tissues at low temperatures. British Journal of Surgery 1955; 42 (174): 430-437. doi: 10.1002/bjs.18004217416
52. 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. Biochemical Genetics 1993; 31 (7): 321-328. doi: 10.1007/BF00553174
53. Noda M, Ma Y, Yoshikawa Y, Imanaka T, Mori T et al. A singlemolecule assessment of the protective effect of DMSO against DNA double-strand breaks induced by photo-and gamma-rayirradiation, and freezing. Scientific Reports 2017; 7 (1): 8557. doi: 10.1038/s41598-017-08894-y