Concentrations of Circulating RNA from Healthy Donors and Cancer Patients Estimated by Different Methods

Abstract

Circulating RNA (cirRNA) was isolated from plasma and cell surface-bound fractions of blood of healthy women and breast cancer patients. RNA samples were DNase treated and quantified by a SYBR Green II assay. Concentrations of RNA sequences of GAPDH, Ki-67 mRNA, and 18S rRNA were measured by real-time quantitative PCR (RT-qPCR) after reverse transcription with random hexamer primers. The obtained data spread over three orders of magnitude for GAPDH and Ki-67 mRNA signals and two orders of magnitude for the copy number of 18S rRNA in blood fractions in both groups. In blood of healthy donors, no correlation was found between the copy number of GAPDH, Ki-67 mRNA, and 18S rRNA and RNA concentrations measured by the SYBR Green II assay. Within the group of breast cancer patients, the concentration GAPDH and Ki-67 mRNA correlated with the concentration of total RNA only in the cell surface-bound fraction; whereas the concentration of 18S rRNA correlated with total RNA in both, the cell surface-bound fraction and blood. The copy number of Ki-67 mRNA correlated with copy numbers of GAPDH mRNA in all fractions of cirRNA of healthy donors and breast cancer patients. A correlation between copy numbers of Ki-67 mRNA and 18S rRNA was found only in cell surface-bound fraction of breast cancer patients. The data described here demonstrate the necessity of searching for more suitable RNA markers in order to estimate total cirRNA concentrations by RT-qPCR, although mRNA of GAPDH could be used for normalization of the level of cancer-specific mRNA among patients.

Extracellular nucleic acids

“…(37) Using highly sensitive and reliable fluorescence-based method, we determined the mean concentration of RNA in blood plasma of healthy individuals as 2.5 ng/ml. (38) Kopreski and co-authors reported in 1999 that exRNA circulate in blood of healthy people and diseased patients with sufficient integrity as to allow reverse transcriptionpolymerase chain reaction (RT-PCR) amplification (Table 1). This was a surprising finding, because ribonuclease is present in both plasma and serum.…”

“…(37) The spectrum of mRNAs whose presence was demonstrated in plasma and other body fluids, like urine and milk, extends from genes overexpressed in a variety of different tumors, (39) to fetal genes in the blood plasma of pregnant women, (40) and housekeeping genes, delectable in the plasma of healthy persons. (37,38) RT-PCR detectable fragments of ribosomal 18S rRNA were found in the extracellular RNA pool circulating in blood plasma of healthy subjects and cancer patients. (38) exRNA secreted by primary and cultivated human cells into the culture medium were shown to contain ribosomal 28S rRNA fragments.…”

“…(37,38) RT-PCR detectable fragments of ribosomal 18S rRNA were found in the extracellular RNA pool circulating in blood plasma of healthy subjects and cancer patients. (38) exRNA secreted by primary and cultivated human cells into the culture medium were shown to contain ribosomal 28S rRNA fragments. (21) To explain the circulating exRNA stability, it was suggested that exRNA is included within lipoprotein vesicles that are either actively secreted or released during programmed cell death, possibly as apoptotic bodies.…”

“…Cancer patients demonstrated increased amounts of the free exDNA in plasma and only negligible amounts of cell-surface-bound exDNA compared with healthy women and patients with benign breast tumors. (38) It is known that cancer tumor invasion is accompanied by increased level of proteases in tissues. These enzymes can damage the cell-surface nucleic-acid-binding proteins and prevent DNA binding to the cells.…”

The Biology of CNAPS

“…Leukocyte breakdown could release a range of RNAs into the blood so possibly accounting for the RNA amount already determined [2].…”