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.
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.
Authors: Louise C Laurent; Asim B Abdel-Mageed; P David Adelson; Jorge Arango; Leonora Balaj; Xandra Breakefield; Elizabeth Carlson; Bob S Carter; Blanca Majem; Clark C Chen; Emanuele Cocucci; Kirsty Danielson; Amanda Courtright; Saumya Das; Zakaria Y Abd Elmageed; Daniel Enderle; Alan Ezrin; Marc Ferrer; Jane Freedman; David Galas; Roopali Gandhi; Matthew J Huentelman; Kendall Van Keuren-Jensen; Yashar Kalani; Yong Kim; Anna M Krichevsky; Charles Lai; Madhu Lal-Nag; Clara D Laurent; Trevor Leonardo; Feng Li; Ivana Malenica; Debasis Mondal; Parham Nejad; Tushar Patel; Robert L Raffai; Renee Rubio; Johan Skog; Robert Spetzler; Jie Sun; Kahraman Tanriverdi; Kasey Vickers; Liang Wang; Yaoyu Wang; Zhiyun Wei; Howard L Weiner; David Wong; Irene K Yan; Ashish Yeri; Stephen Gould Journal: J Extracell Vesicles Date: 2015-08-28