Differential stability of cell-free circulating microRNAs: implications for their utilization as biomarkers.
BACKGROUND
MicroRNAs circulating in the blood, stabilized by complexation with proteins and/or additionally by encapsulation in lipid vesicles, are currently being evaluated as biomarkers. The consequences of their differential association with lipids/vesicles for their stability and use as biomarkers are largely unexplored and are subject of the present study.


METHODS
The levels of a set of selected microRNAs were determined by quantitative reverse-transcription PCR after extraction from sera or vesicle- and non-vesicle fractions prepared from sera. The stability of these microRNAs after incubation with RNase A or RNase inhibitor, an inhibitor of RNase A family enzymes was studied.


RESULTS
The levels of microRNA-1 and microRNA-122, but not those of microRNA-16, microRNA-21 and microRNA-142-3p, declined significantly during a 5-h incubation of the sera. RNase inhibitor prevented the loss of microRNAs in serum as well as the degradation of microRNA-122, a microRNA not expressed in blood cells, in whole blood. Stabilization of microRNA-122 was also achieved by hemolysis. Prolonged incubation of the sera led to enrichment of vesicle-associated relative to non-vesicle-associated microRNAs. Vesicle-associated microRNAs were more resistant to RNase A treatment than the respective microRNAs not associated with vesicles.


CONCLUSIONS
Serum microRNAs showed differential stability upon prolonged incubation. RNase inhibitor might be useful to robustly preserve the pattern of cell-free circulating microRNAs. In the case of microRNAs not expressed in blood cells this can also be achieved by hemolysis. Vesicle-associated microRNAs appeared to be more stable than those not associated with vesicles, which might be useful to disclose additional biomarker properties of miRNAs.
Department of Medicine I, University Hospital Frankfurt, Frankfurt, Germany.
Verena
V
Köberle
Thomas
T
Pleli
Christian
C
Schmithals
Eduardo
E
Augusto Alonso
Jörg
J
Haupenthal
Halvard
H
Bönig
Jan
J
Peveling-Oberhag
Ricardo M
RM
Biondi
Stefan
S
Zeuzem
Bernd
B
Kronenberger
Oliver
O
Waidmann
Albrecht
A
Piiper
D014408:Biomarkers, Tumor / Q000097:blood*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004912:Erythrocytes / Q000378:metabolism*; D064368:Healthy Volunteers; D006801:Humans; D035683:MicroRNAs / Q000097:blood* / Q000737:chemistry*; D020871:RNA Stability; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012259:Ribonuclease, Pancreatic / Q000037:antagonists & inhibitors; D044967:Serum / Q000378:metabolism*
D014408:Biomarkers, Tumor; D004791:Enzyme Inhibitors; D035683:MicroRNAs; D012259:Ribonuclease, Pancreatic