Improving Accuracy of Urinary miRNA Quantification in Heparinized Patients Using Heparinase I Digestion

Abstract

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.

Identification and Validation Model for Informative Liquid Biopsy-Based microRNA Biomarkers: Insights from Germ Cell Tumor In Vitro, In Vivo and Patient-Derived Data

“…When applying the new miR-23a/451a cutoff determined by us in a second validation cohort, we found no significant differences in distribution of Ct values of spike-in, normalizer nor the specific targets miR-371a-3p/miR-375. Importantly, we believe the major reason for the little impact of hemolysis on specific targets in our study is the distinct microRNA isolation procedure: we have performed a magnetic bead capture of microRNAs, which are isolated and purified from the serum contents, eliminating the potentially harmful effects of molecules such as hemoglobin or heparin, which are known to inhibit the PCR reaction [60][61][62][63]. Indeed, we have witnessed this effect in RNA isolated from kidney preservation fluids (described in [54]) containing heparin contamination, a commonly used anticoagulant.…”

Cell‐free microRNAs as early predictors of graft viability during ex vivo normothermic machine perfusion of human donor livers

“…Complement DNA (cDNA) synthesis was performed using the Taqman microRNA Reverse Transcription Kit (Applied Biosystems, Carlsbad, CA, USA) as suggested by the manufacturer with minor modifications as described previously . To eliminate qRT‐PCR inhibition by heparin, the RT reactions were co‐incubated with 6 IU heparinase (New England Biolabs) . One hundred amol of synthetic Cel‐miR‐54 was added to monitor the presence of PCR inhibiting components that co‐eluted with total RNA, and/or incomplete heparin degradation.…”

“…16 To eliminate qRT-PCR inhibition by heparin, the RT reactions were co-incubated with 6 IU heparinase (New England Biolabs). 17 One hundred amol of synthetic Cel-miR-54 was added to monitor the presence of PCR inhibiting components that co-eluted with total RNA, and/or incomplete heparin degradation. cDNA samples were diluted to 100 μL with nucleasefree water and stored at −20°C until further use.…”

Evaluation of RNA isolation methods for microRNA quantification in a range of clinical biofluids.

“…Blood was collected in Vacutainer serum tubes (Becton Dickinson, Breda, The Netherlands), centrifugated at 18 °C for 10 min at 800 g to separate serum. Urine was collected as described previously (34). Cell-free material was stored at -20 °C until further use.…”