Incubation of human blood fractions leads to changes in apparent miRNA abundance

A basic investigation on the presence and composition of miRNA species and their reaction to in vitro incubation and stimulation (borosilicate glass beads), in plasma, platelet-rich plasma (PRP), red blood cells (RBC), peripheral blood mononuclear cells (PBMC) and polymorphonuclear (PMN) cells was performed. 19 specific miRNAs were compared in control samples (0 hours), incubated 24 hour samples, and incubated 24 hour samples with glass bead stimulation for each blood fraction.

All 19 miRNAs were expressed in all blood fractions albeit at different levels for different miRNAs. Incubation resulted in significant changes in the abundance of miR-21, miR-155, Let-7c and Let 7f in plasma, miR-21, miR-23a and miR-150 in RBC and miR-15b, miR-126, miR155 and Let-7g in PBMC, while no change was seen in PRP and PMN.

Interestingly, in the samples incubated with glass beads, no miRNAs were significantly affected in plasma, RBC, PBMC and PMN, while expression of miR-25, miR15a, miR-126 and miR223 was significantly changed in PRP. Thus, PRP, as the only blood fraction depended on stimulation to change its miRNA profile upon incubation.

For the other fractions, stimulation either leveled out the changes induced by incubation alone (plasma, RBC and PBMC) or no effect was seen in either case. This study confirms the presence of miRNA in anucleate cells like RBC and platelets. The up-regulation of four specific miRNAs in isolated plasma upon incubation was surprising; one possible explanation is that miRNA complexes in plasma may become more accessible for cDNA synthesis and qPCR upon incubation.

Accessibility could be a new point of regulation for soluble miRNAs.