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Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma

  • Technological Innovations
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Abstract

Purpose

Methylated cell-free fetal DNA (cffDNA) in maternal plasma can potentially be used as a biomarker for accurate noninvasive prenatal testing (NIPT) of fetal disorders. Recovery and purification of cffDNA are key steps for downstream applications. In this study, we aimed to developed and evaluated different aspects of an optimized method and compared its efficiency with common methods used for extraction of methylated cffDNA.

Methods

Single factor experiments, Plackett-Burman (PB) design, and response surface methodology (RSM) were conducted for conventional Triton/Heat/Phenol (cTHP) method optimization. The total cell-free DNA (cfDNA) was extracted from pooled maternal plasma using the optimized method called the Triton/Heat/Phenol/Glycogen (THPG), cTHP method, a column-based kit, and a magnetic bead-based kit. In the next step, methylated cfDNA from the extracted total cfDNA was enriched using a methylated DNA immunoprecipitation (MeDIP) kit. Real-time quantitative polymerase chain reaction was performed on the RASSF1 gene and hyper region to determine the genomic equivalents per milliliter (GEq/ml) values of the methylated cfDNA and cffDNA, respectively.

Results

The optimum values of the significant factors affecting cfDNA extraction from 200 μl of plasma were 3% SDS, 1% Triton X-100, 0.9 μg/μl glycogen, and 0.3 M sodium acetate. The GEq/ml values of methylated cffDNA extracted using the THPG method were significantly higher than for the tested extraction methods (p < 0.001).

Conclusions

Our results indicate that the THPG method is more efficient than the other tested methods for extraction of low copy number methylated cffDNA from a small volume of maternal plasma.

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References

  1. Allyse M, Minear MA, Berson E, Sridhar S, Rote M, Hung A, et al. Non-invasive prenatal testing: a review of international implementation and challenges. Int J Women's Health. 2015;7:113–26.

    Article  CAS  Google Scholar 

  2. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A. 2008;105:16266–71.

    Article  PubMed  PubMed Central  Google Scholar 

  3. Chandrasekharan S, Minear MA, Hung A, Allyse M. Noninvasive prenatal testing goes global. Sci Transl Med. 2014;6:231fs15.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Bischoff FZ, Lewis DE, Simpson JL. Cell-free fetal DNA in maternal blood: kinetics, source and structure. Hum Reprod Update. 2005;11:59–67.

    Article  CAS  PubMed  Google Scholar 

  5. Wright CF, Wei Y, Higgins JP, Sagoo GS. Non-invasive prenatal diagnostic test accuracy for fetal sex using cell-free DNA a review and meta-analysis. BMC Res Notes. 2012;5:476.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Tounta G, Kolialexi A, Papantoniou N, Tsangaris GT, Kanavakis E, Mavrou A. Non-invasive prenatal diagnosis using cell-free fetal nucleic acids in maternal plasma: progress overview beyond predictive and personalized diagnosis. EPMA J. 2011;2:163–71.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Yang H, Xu HB, Liu TT, He XL. Systematic review of noninvasive prenatal diagnosis for abnormal chromosome genetic diseases using free fetal DNA in maternal plasma. Genet Mol Res. 2015;14:10603–8.

    Article  CAS  PubMed  Google Scholar 

  8. Chiu RW, Lo YM. Clinical applications of maternal plasma fetal DNA analysis: translating the fruits of 15 years of research. Clin Chem Lab Med. 2013;51:197–204.

    CAS  PubMed  Google Scholar 

  9. Canick JA, Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE. The impact of maternal plasma DNA fetal fraction on next generation sequencing tests for common fetal aneuploidies. Prenat Diagn. 2013;33:667–74.

    Article  CAS  PubMed  Google Scholar 

  10. Old RW, Crea F, Puszyk W, Hulten MA. Candidate epigenetic biomarkers for non-invasive prenatal diagnosis of Down syndrome. Reprod BioMed Online. 2007;15:227–35.

    Article  CAS  PubMed  Google Scholar 

  11. Ioannides M, Papageorgiou EA, Keravnou A, Tsaliki E, Spyrou C, Hadjidaniel M, et al. Inter-individual methylation variability in differentially methylated regions between maternal whole blood and first trimester CVS. Mol Cytogenet. 2014;7:73.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Papageorgiou EA, Fiegler H, Rakyan V, Beck S, Hulten M, Lamnissou K, et al. Sites of differential DNA methylation between placenta and peripheral blood: molecular markers for noninvasive prenatal diagnosis of aneuploidies. Am J Pathol. 2009;174:1609–18.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Xiang Y, Zhang J, Li Q, Zhou X, Wang T, Xu M, et al. DNA methylome profiling of maternal peripheral blood and placentas reveal potential fetal DNA markers for non-invasive prenatal testing. Mol Hum Reprod. 2014;20:875–84.

    Article  CAS  PubMed  Google Scholar 

  14. Repiská G, Sedláčková T, Szemes T, Celec P, Minárik G. Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma. Clin Chem Lab Med. 2013;51:1185–9.

    Article  CAS  PubMed  Google Scholar 

  15. Lim JH, Park SY, Kim SY, Kim DJ, Kim MJ, Yang JH, et al. Effective method for extraction of cell-free DNA from maternal plasma for non-invasive first-trimester fetal gender determination: a preliminary study. J Genet Med. 2010;7:53–8.

    Article  Google Scholar 

  16. Keshavarz Z, Moezzi L, Ranjbaran R, Aboualizadeh F, Behzad-Behbahani A, Abdullahi M, et al. Evaluation of a modified DNA extraction method for isolation of cell-free fetal DNA from maternal serum. Avicenna J Med Biotechnol. 2015;7:85–8.

    PubMed  PubMed Central  Google Scholar 

  17. Jorgez CJ, Dang DD, Simpson JL, Lewis DE, Bischoff FZ. Quantity versus quality: optimal methods for cell-free DNA isolation from plasma of pregnant women. Genet Med. 2006;8:615–9.

    Article  CAS  PubMed  Google Scholar 

  18. Xue X, Teare MD, Holen I, Zhu YM, Woll PJ. Optimizing the yield and utility of circulating cell-free DNA from plasma and serum. Clin Chim Acta. 2009;404:100–4.

    Article  CAS  PubMed  Google Scholar 

  19. Heydari R, Zahiri HS, Noghabi KA, Rad VV, Khaniki GB. A statistical approach to the optimization of cold-adapted amylase production by Exiguobacterium sp. SH3. Starch. 2012;64:955–63.

    Article  CAS  Google Scholar 

  20. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, et al. Presence of fetal DNA in maternal plasma and serum. Lancet. 1997;350:485–7.

    Article  CAS  PubMed  Google Scholar 

  21. Lim JH, Park SY, Kim SY, Kim DJ, Choi JE, Kim MH, et al. Effective detection of fetal sex using circulating fetal DNA in first-trimester maternal plasma. FASEB J. 2012;26:250–8.

    Article  CAS  PubMed  Google Scholar 

  22. Chan KC, Zhang J, Hui AB, Wong N, Lau TK, Leung TN, et al. Size distributions of maternal and fetal DNA in maternal plasma. Clin Chem. 2004;50:88–92.

    Article  CAS  PubMed  Google Scholar 

  23. Legler TJ, Liu Z, Mavrou A, Finning K, Hromadnikova I, Galbiati S, et al. Workshop report on the extraction of foetal DNA from maternal plasma. Prenat Diagn. 2007;27:824–9.

    Article  CAS  PubMed  Google Scholar 

  24. Lim JH, Lee BY, Kim JW, Han YJ, Chung JH, Kim MH, et al. Evaluation of extraction methods for methylated cell-free fetal DNA from maternal plasma. J Assist Reprod Genet. 2018;35:637–41.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Warton K, Graham L-J, Yuwono N, Samimi G. Comparison of 4 commercial kits for the extraction of circulating DNA from plasma. Cancer Gene Ther. 2018;228-229:143–50.

    Article  CAS  Google Scholar 

  26. Fregel R, González A, Cabrera VM. Improved ethanol precipitation of DNA. Electrophoresis. 2010;31:1350–2.

    Article  CAS  PubMed  Google Scholar 

  27. Lever MA, Torti A, Eickenbusch P, Michaud AB, Šantl-Temkiv T, Jørgensen BB. A modular method for the extraction of DNA and RNA, and the separation of DNA pools from diverse environmental sample types. Front Microbiol. 2015;6:476.

    Article  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Author notes

  1. Mostafa Akbariqomi and Reza Heidari contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Medicine, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mostafa Akbariqomi, Reza Heidari, Nafiseh Sadat Sanikhani, Fatemeh Mahmoudian & Gholamreza Tavoosidana

  2. Department of Gynecology & Obstetric, Shohada Tajrish Educational Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Soraya Saleh Gargari

  3. Department of Medical Genetics, Faculty of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Mir Davood Omrani

  4. Department of Genetics, Faculty of Basic Science, Shahrekord University, Shahrekord, Iran

    Garshasb Rigi

  5. Research Institute of Biotechnology, Shahrekord University, Shahrekord, Iran

    Garshasb Rigi

  6. Nanobiotechnology Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

    Hamid Kooshki

  7. Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran

    Mohammad Ali Mazlomi

Authors
  1. Mostafa Akbariqomi
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  2. Reza Heidari
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  4. Mir Davood Omrani
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  9. Mohammad Ali Mazlomi
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  10. Gholamreza Tavoosidana
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Corresponding author

Correspondence to Gholamreza Tavoosidana.

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The study was approved by the ethical committee of the Tehran University of Medical Sciences, Iran (IR.TUMS.REC.1394.1156). All participant provided written informed consent prior to venipuncture.

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Akbariqomi, M., Heidari, R., Gargari, S.S. et al. Evaluation and statistical optimization of a method for methylated cell-free fetal DNA extraction from maternal plasma. J Assist Reprod Genet 36, 1029–1038 (2019). https://doi.org/10.1007/s10815-019-01425-w

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  • DOI: https://doi.org/10.1007/s10815-019-01425-w

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