Circulating Nucleic Acids in Blood of Healthy Male and Female Donors

The analysis of circulating nucleic acids (cirNAs) in blood provides helpful information for medical diagnostics (1). Thus, there has been intensive investigation of the factors influencing nucleic acid concentrations in plasma and serum, such as changes in DNA concentrations in serum and plasma during storage of blood samples (2) and the influence of blood processing on cell-free DNA quantification (3)(4)(5). Recent observations demonstrated that cirNAs are found not only free in blood plasma but also bound to the cell surface (6). Physiologic factors determining cirNA concentrations in blood remain to be elucidated. In the present study, we compared the concentration of cell-surface–bound and cell-free cirNAs in blood samples from 15 healthy men and 20 healthy women. Venous blood (8 mL) was collected into 13-mL tubes (cat. no. 55.459; Sarstedt) containing 2 mL of sterile phosphate-buffered saline (PBS) containing 50 mmol/L EDTA. The samples were stored at 4 °C and treated within 4 h after blood collection. Plasma was collected after low-speed centrifugation (400g for 20 min) followed by high-speed centrifugation (16 000g for 5 min). Blood cells were fractionated into leukocytes and erythrocytes, and extracellular nucleic acids were eluted from the cell surface with PBS containing 5 mmol/L EDTA (PBS/EDTA) and trypsin solutions as described previously (6). Briefly, cells were washed with 9 volumes of PBS/EDTA) and centrifuged (400g for 10 min). Cells were resuspended and incubated for 5 min at room temperature with 2.5 g/L trypsin with subsequent inactivation of the enzyme by trypsin inhibitor. Cells were then pelleted by centrifugation, and the supernatant was collected as trypsin eluate. RNA and DNA were isolated (7) and quantified by a fluorescence-based method (8). Plasma (1 mL), PBS/EDTA eluate (2 mL), and trypsin eluate (1 mL) were used for cirNA quantification. Detection limits for RNA and DNA calculated to the initial blood volume were 8 ng/mL in plasma, 20 ng/mL in trypsin eluate, and 40 ng/mL in PBS/EDTA eluate. Statistical tests were carried out with STATISTICA 6.0 software.