There has been a recent surge of interest in analysis of nucleic acids recovered from cell-free biological fluids. Research studies have shown the feasibility of detecting genetic variants associated with recurrence of cancer after treatment, in cell-free DNA (cfDNA) from plasma and urine, at earlier time points compared to conventional imaging tests, and numerous RNA-based biomarkers have been reported in plasma. To increase our understanding of the characteristics of cfDNA and cfRNA extracted from bodily fluids, we carried out studies to compare yields and size distribution of nucleic acids extracted from plasma and urine from healthy donors.
cfDNA was extracted using magnetic-bead-based kits and analyzed on an Agilent® 2100 HS® DNA chip to determine concentration and size distribution. Urine cfDNA was converted into whole-genome libraries and used for paired-end sequencing on an Illumina® MiSeq® sequencer, and analyzed to determine the mapping characteristics (genomic coverage and % fetal DNA). Plasma RNA was extracted and used to generate small RNA sequencing libraries, which were sequenced and mapped to small RNA databases.
Differences in yield and size distribution of cfDNA were observed for different biofluids, for different donors, and for sequential urine samples collected from the same donor. Some urine cfDNAs included very low molecular weight DNA, likely comprising transrenal DNA. The proportion of very high molecular weight cfDNA (larger than 1 kb) obtained from urine and plasma could not be attributed to differences in sample collection or preanalytical processing. The processing steps used to produce whole-genome libraries from plasma cfDNA containing large amounts of very high MW DNA enriches for library products consistent with nucleosome-protected cfDNA, with depletion of products derived from high MW DNA. Variation in percentage of fetal DNA was observed in sequentially collected maternal urine samples. Yields of RNA from platelet-poor plasma were generally much lower compared to yields of RNA from platelet-rich plasma from the same donor. High yields of cfDNA consistent with nucleosome-protected DNA could be recovered from platelet-poor plasma that had low yields of RNA. Further experiments are needed to assess the extent of differences in genomic mapping characteristics between cfDNA comprising smaller and larger.