Improving Accuracy of Urinary miRNA Quantification in Heparinized Patients Using Heparinase I Digestion Henk P. Roest, Cornelia J. Verhoeven, Jubi E.

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Improving Accuracy of Urinary miRNA Quantification in Heparinized Patients Using Heparinase I Digestion Henk P. Roest, Cornelia J. Verhoeven, Jubi E. de Haan, Jeroen de Jonge, Jan N.M. IJzermans, Luc J.W. van der Laan The Journal of Molecular Diagnostics Volume 18, Issue 6, Pages 825-833 (November 2016) DOI: 10.1016/j.jmoldx.2016.06.006 Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 1 Stability of endogenous miRNAs in urine. Mean stability of endogenous miRNA miR-30a and spiked-in Cel-miR-39 of healthy control samples after increasing incubation times at 37°C. Bars represent SEM. n = 3. The Journal of Molecular Diagnostics 2016 18, 825-833DOI: (10.1016/j.jmoldx.2016.06.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 2 Variation in spiked-in and endogenous miRNA detection. Quantitative RT-PCR results of endogenous miR-30e and miR-92a (A) and spiked-in Cel-miR-39 and miR-238 (B) presented as Cq values in nonheparinized individuals (circles) and heparinized individuals (squares). Bars indicate median with interquartile range. ∗∗∗P < 0.001. The Journal of Molecular Diagnostics 2016 18, 825-833DOI: (10.1016/j.jmoldx.2016.06.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 3 Heparin inhibits quantitative RT-PCR (RT-qPCR) analysis in urinary miRNA samples in a dose-dependent manner and can be neutralized with 6 IU heparinase I. A: RT-qPCR inhibition of 50 amol synthetic Cel-miR-39 at increasing amounts of unfractionated heparin (UH) and nadroparin [low-molecular-weight heparin (LMWH)]. PCR levels without anticoagulants are set at 100%. B: Minimally required amount of heparinase I to reach a plateau of RT-qPCR improvement. Cq levels were converted to miRNA expression levels for Cel-miR-39, miR-30e, and miR-92a. Data are obtained from two LMWH-contaminated urinary RNA samples. Results obtained with 12 IU heparinase I are set to 100%. C: Effect of coincubation with (+) or without (−) 6 IU heparinase I during cDNA synthesis in the nonheparinized group and heparinized group. D: LMWH concentration (IU LMWH/mL) (closed bars) and average RT-PCR inhibition (open bars) in 11 urine samples (1 to 11). E: Correlation analysis between LMWH concentration and the average ΔCq. ΔCq is calculated as the difference in Cq values with or without treatment with 6 IU heparinase I. Values are means ± SD (A and B). n = 15 (C, nonheparinized group); n = 20 (C, heparinized group). ∗∗P < 0.01, ∗∗∗P < 0.001 (C). The Journal of Molecular Diagnostics 2016 18, 825-833DOI: (10.1016/j.jmoldx.2016.06.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 4 Effect of protamine treatment on RT-PCR results. Urine samples of healthy controls were contaminated with 10 IU low-molecular-weight heparin (LMWH), where indicated, and either counteracted with 50 IU protamine sulfate before RNA isolation using the miRNeasy kit (samples A to D) or RNA samples were contaminated with the indicated amount of LWMH and protamine sulfate before RT (samples E to H). Results are presented in Cq. ND, not detected. The Journal of Molecular Diagnostics 2016 18, 825-833DOI: (10.1016/j.jmoldx.2016.06.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions

Figure 5 Kidney biopsy specimens are not affected by the presence of heparin in urine. Paired urine (A) and kidney (B) biopsy specimens were tested by quantitative RT-PCR (RT-qPCR) for the presence of heparin. The y axis indicates the ratio of the RT-qPCR results of every sample with or without the incubation with 6 IU heparinase I. Bars indicate median with interquartile range. The Journal of Molecular Diagnostics 2016 18, 825-833DOI: (10.1016/j.jmoldx.2016.06.006) Copyright © 2016 American Society for Investigative Pathology and the Association for Molecular Pathology Terms and Conditions