Improving Accuracy of Urinary miRNA Quantification in Heparinized Patients Using Heparinase I Digestion.
miRNAs have emerged as promising biomarkers because of their association with cell stress and diseases and their easy detection and stability in many body fluids. Because of the sensitivity, the method of choice to detect miRNAs is quantitative RT-PCR (RT-qPCR). Therapeutics, in particular circulating anticoagulants, are notorious for their inhibitory effect on RT-qPCR-based measurements. The effect of heparin contamination on inhibition of RT-qPCR from miRNAs isolated from urine has, however, never been investigated. We obtained urine samples from healthy controls and from heparinized patients undergoing major surgery (live kidney donation or liver transplantation) (n = 27). Samples were spiked with synthetic miRNAs to monitor RNA loss during workup, and levels of endogenous and spiked-in miRNAs were quantified by RT-qPCR. Endogenous miRNAs in urine were protected from degradation, but levels differed substantially within surgery groups. Variability in detection levels of spiked-in miRNAs was low in nonhospitalized controls, but was high in both surgery groups, and the difference in miRNA levels correlated well with the heparin concentration in urinary samples. Treatment of urinary RNA with heparinase I during RT-qPCR strongly reduced this variation in a dose-dependent manner. Heparinase I should therefore be considered as standard step for detection of miRNA in urine from hospitalized individuals.
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, the Netherlands. Electronic address: h.roest@erasmusmc.nl.
Henk P
HP
Roest
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, the Netherlands.
Cornelia J
CJ
Verhoeven
Department of Adult Intensive Care, Erasmus MC-University Medical Center, Rotterdam, the Netherlands.
Jubi E
JE
de Haan
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, the Netherlands.
Jeroen
J
de Jonge
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, the Netherlands.
Jan N M
JN
IJzermans
Department of Surgery, Erasmus MC-University Medical Center, Rotterdam, the Netherlands.
Luc J W
LJ
van der Laan
D000328:Adult; D000925:Anticoagulants / Q000009:adverse effects; D015415:Biomarkers; D016022:Case-Control Studies; D005260:Female; D006493:Heparin / Q000009:adverse effects; D019763:Heparin Lyase; D006801:Humans; D008297:Male; D035683:MicroRNAs / Q000235:genetics* / Q000652:urine; D008875:Middle Aged; D059748:Proteolysis; D020871:RNA Stability; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D015203:Reproducibility of Results
D000925:Anticoagulants; D015415:Biomarkers; D035683:MicroRNAs; D006493:Heparin; D019763:Heparin Lyase