Abstract
Background: Circulating nucleic acids acquired non-invasively have been confirmed as useful biomarkers in cancer and prenatal medicine. The most important molecules in the field of circulating nucleic acids research are circulating DNA and miRNA. In this study, the possibility of co-isolation of total circulating DNA, cell-free fetal DNA and miRNA from the plasma of pregnant women was tested, and the yields of co-isolated circulating nucleic acids using two commercial kits and three protocols were compared.
Methods: Cell-free fetal DNA and miRNA from the plasma of pregnant women carrying male fetuses were co-isolated with the miRCURY™ RNA Isolation Kit according to the original protocol and the QIAamp Circulating Nucleic Acid Kit (CNA kit) according to the manufacturer’s protocol for DNA isolation and miRNA isolation. For comparison of DNA isolation, the AR and DYS14 gene-based assays were used for the detection and quantification of total circulating and cell-free fetal DNA. For miRNA detection and quantification, the miR-16 and miR-451 assays were used.
Results: Two different protocols for isolation using the CNA kit did not significantly differ in the yields of isolated tcDNA and cffDNA; however, the amount of isolated cffDNA using the miRCURY™ RNA Isolation Kit was significantly less (p<0.05, F=4.776). There was a statistically significant difference for miRNA isolation (p<0.0001, F=859 for miR-16 and p<0.0001, F=854.4 for miR-451), with the highest amount of isolated miRNA obtained using the miRCURY™ RNA Isolation Kit.
Conclusions: All three methods used in our study were successful in the co-isolation of tcDNA, cffDNA and miRNA from the same sample. The best combined results were obtained with the miRCURY™ RNA Isolation Kit.
Acknowledgments
This study was supported by funding from the Slovak Research and Development Agency (VMSP-II-0030-09, APVV-0720-10) and Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic (VEGA-1/0285/11).
Conflict of interest statement
Authors’ conflict of interest disclosure: The authors state that there are no conflicts of interest regarding the publication of this article. Research funding played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
References
1. Huang X, Yuan T, Tschannen M, Sun Z, Jacob H, Du M, et al. Characterization of human plasma-derived exosomal RNAs by deep sequencing. BMC Genomics 2013;14:319.10.1186/1471-2164-14-319Search in Google Scholar PubMed PubMed Central
2. Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, et al. Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci USA 2008;105:10513–8.10.1073/pnas.0804549105Search in Google Scholar PubMed PubMed Central
3. Gibbings DJ, Ciaudo C, Erhardt M, Voinnet O. Multivesicular bodies associate with components of miRNA effector complexes and modulate miRNA activity. Nat Cell Biol 2009;11:1143–9.10.1038/ncb1929Search in Google Scholar PubMed
4. Pinzani P, Salvianti F, Pazzagli M, Orlando C. Circulating nucleic acids in cancer and pregnancy. Methods 2010;50:302–7.10.1016/j.ymeth.2010.02.004Search in Google Scholar PubMed
5. Sozzi G, Conte D, Leon M, Ciricione R, Roz L, Ratcliffe C, et al. Quantification of free circulating DNA as a diagnostic marker in lung cancer. J Clin Oncol 2003;21:3902–8.10.1200/JCO.2003.02.006Search in Google Scholar PubMed
6. Chan KC, Jiang P, Zheng YW, Liao GJ, Sun H, Wong J, et al. Cancer genome scanning in plasma: detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. Clin Chem 2013;59:211–24.10.1373/clinchem.2012.196014Search in Google Scholar PubMed
7. Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, et al. Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med 2013;368:1199–209.10.1056/NEJMoa1213261Search in Google Scholar PubMed
8. Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 2013;497:108–12.10.1038/nature12065Search in Google Scholar PubMed
9. Lo YM, Leung TN, Tein MS, Sargent IL, Zhang J, Lau TK, et al. Quantitative abnormalities of fetal DNA in maternal serum in preeclampsia. Clin Chem 1999;45:184–8.10.1093/clinchem/45.2.184Search in Google Scholar
10. Zhong XY, Laivuori H, Livingston JC, Ylikorkala O, Sibai BM, Holzgreve W, et al. Elevation of both maternal and fetal extracellular circulating deoxyribonucleic acid concentrations in the plasma of pregnant women with preeclampsia. Am J Obstet Gynecol 2001;184:414–9.10.1067/mob.2001.109594Search in Google Scholar PubMed
11. Zhong XY, Burk MR, Troeger C, Jackson LR, Holzgreve W, Hahn S. Fetal DNA in maternal plasma is elevated in pregnancies with aneuploid fetuses. Prenat Diagn 2000;20:795–8.10.1002/1097-0223(200010)20:10<795::AID-PD897>3.0.CO;2-PSearch in Google Scholar
12. Lo YM, Patel P, Wainscoat JS, Sampietro M, Gillmer MD, Fleming KA. Prenatal sex determination by DNA amplification from maternal peripheral blood. Lancet 1989;2:1363–5.Search in Google Scholar
13. Fan HC, Quake SR. Detection of aneuploidy with digital polymerase chain reaction. Anal Chem 2007;79:7576–9.10.1021/ac0709394Search in Google Scholar
14. Lo YM, Chan KC, Sun H, Chen EZ, Jiang P, Lun FM, et al. Maternal plasma DNA sequencing reveals the genome-wide genetic and mutational profile of the fetus. Sci Transl Med 2010;2:61ra91.10.1126/scitranslmed.3001720Search in Google Scholar
15. Bushati N, Cohen SM. microRNA functions. Annu Rev Cell Dev Biol 2007;23:175–205.10.1146/annurev.cellbio.23.090506.123406Search in Google Scholar
16. Enquobahrie DA, Abetew DF, Sorensen TK, Willoughby D, Chidambaram K, Williams MA. Placental microRNA expression in pregnancies complicated by preeclampsia. Am J Obstet Gynecol 2011;204:178 e12–21.10.1016/j.ajog.2010.09.004Search in Google Scholar
17. Gunel T, Zeybek YG, Akcakaya P, Kalelioglu I, Benian A, Ermis H, et al. Serum microRNA expression in pregnancies with preeclampsia. Genet Mol Res 2011;10:4034–40.10.4238/2011.November.8.5Search in Google Scholar
18. Camps C, Buffa FM, Colella S, Moore J, Sotiriou C, Sheldon H, et al. hsa-miR-210 Is induced by hypoxia and is an independent prognostic factor in breast cancer. Clin Cancer Res 2008;14:1340–8.10.1158/1078-0432.CCR-07-1755Search in Google Scholar
19. van der Meer D, Uchimoto ML, Williams G. Simultaneous analysis of micro-RNA and DNA for determining the body fluid origin of DNA profiles. J Forensic Sci 2013;58:967–71.10.1111/1556-4029.12160Search in Google Scholar
20. Repiska G, Sedlackova T, Szemes T, Celec P, Minarik G. Selection of the optimal manual method of cell free fetal DNA isolation from maternal plasma. Clin Chem Lab Med 2013;51:1185–9.10.1515/cclm-2012-0418Search in Google Scholar
21. McAlexander MA, Phillips MJ, Witwer KW. Comparison of methods for miRNA extraction from plasma and quantitative recovery of RNA from cerebrospinal fluid. Front Genet 2013;4:83.10.3389/fgene.2013.00083Search in Google Scholar
22. Lo YM, Tein MS, Lau TK, Haines CJ, Leung TN, Poon PM, et al. Quantitative analysis of fetal DNA in maternal plasma and serum: implications for noninvasive prenatal diagnosis. Am J Hum Genet 1998;62:768–75.10.1086/301800Search in Google Scholar PubMed PubMed Central
23. Blondal T, Jensby Nielsen S, Baker A, Andreasen D, Mouritzen P, Wrang Teilum M, et al. Assessing sample and miRNA profile quality in serum and plasma or other biofluids. Methods 2013;59:S1–6.10.1016/j.ymeth.2012.09.015Search in Google Scholar PubMed
24. Kirschner MB, Kao SC, Edelman JJ, Armstrong NJ, Vallely MP, van Zandwijk N, et al. Haemolysis during sample preparation alters microRNA content of plasma. PLoS One 2011;6:e24145.10.1371/journal.pone.0024145Search in Google Scholar PubMed PubMed Central
25. McDonald JS, Milosevic D, Reddi HV, Grebe SK, Algeciras-Schimnich A. Analysis of circulating microRNA: preanalytical and analytical challenges. Clin Chem 2011;57:833–40.10.1373/clinchem.2010.157198Search in Google Scholar PubMed
26. Song J, Bai Z, Han W, Zhang J, Meng H, Bi J, et al. Identification of suitable reference genes for qPCR analysis of serum microRNA in gastric cancer patients. Dig Dis Sci 2012;57:897–904.10.1007/s10620-011-1981-7Search in Google Scholar PubMed
27. Sourvinou IS, Markou A, Lianidou ES. Quantification of circulating miRNAs in plasma: effect of preanalytical and analytical parameters on their isolation and stability. J Mol Diagn 2013;15:827–34.10.1016/j.jmoldx.2013.07.005Search in Google Scholar PubMed
28. Hanson EK, Lubenow H, Ballantyne J. Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs. Anal Biochem 2009;387:303–14.10.1016/j.ab.2009.01.037Search in Google Scholar PubMed
29. Zubakov D, Boersma AW, Choi Y, van Kuijk PF, Wiemer EA, Kayser M. MicroRNA markers for forensic body fluid identification obtained from microarray screening and quantitative RT-PCR confirmation. Int J Legal Med 2010;124:217–26.10.1007/s00414-009-0402-3Search in Google Scholar PubMed PubMed Central
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