H.P. Roest, J.W. Selten, C.J. Verhoeven, R.W.F. de Bruin, J. de Jonge, J.N.M Ijzermans, L.J.W. van der Laan
Chair(s): prof. dr. Herold Metselaar, MDL-arts, Erasmus MC Rotterdam
Thursday 10 march 2016
12:30 - 13:00h at Foyer
Parallel session: Postersessies XI - Opgesplitst in 3 tijdblokken en 3 categoriëen (klinisch, basaal, donatie)
Ischemia-type biliary lesions (ITBL) after liver transplantation strongly increase patient morbidity and impair graft survival. Our earlier study showed that release of hepatocyte- and cholangiocyte-derived miRNAs (HDmiRs and CDmiRs, respectively) into graft preservation solution are predictive for the development of ITBL following transplantation. Recently, concerns are raised that microRNA detection by PCR-analysis can be inhibited by the anti-coagulant heparin. Since graft procurement is performed in the presence of heparin, the PCR detection of HDmiRs and CDmiRs in perfusates might be hampered. The aim of this study was to determine whether heparin in graft perfusates influences miRNA biomarker detection.
Total RNA was isolated from perfusate samples of grafts that developed ITBL (n=22) and non-ITBL (n=35). Specific cDNA for CDmiR-222, CDmiR-296, HDmiR-122, and HDmiR-148a was synthesized in the presence of 6 IU of heparinase I. A synthetic C. elegans miRNA (Cel miR-39) was added as an internal correction for sample variation during the RT step. Relative expression levels were calculated and normalized for Cel miR-39 levels.
Real time PCR results for Cel miR-39 shows a mean Ct level of 20.23+/-1.61, indicative for a strong abrogation of PCR impairment by heparin. Treatment of RNA samples with heparinase I resulted in a considerable increase in detection and, simultaneously, a reduction in standard deviation (3.52 to 1.62) and IQR (3.09 to 1.92) of the Ct levels for all measured miRNAs. Also after heparinase treatment HDmiR/CDmiR ratios were significant different between the ITBL and the non-ITBL group (P values ranging between 0.0011 and 0.013).
This study demonstrates the usefulness of heparinase-treatment of graft perfusate containing traces of heparin in order to optimize miRNA detection. Furthermore, these results confirm that calculations with organ-derived miRNA ratios are useful for analysis. In other situations, the use of heparinase I is encouraged to obtain high enough levels of detection if contamination is suspected.