Background: Delayed graft function (DGF) is a common complication after deceased donor kidney transplantation (KT) which affects short and long-term outcome. Currently available biomarkers in perfusate lack sensitivity in predicting graft outcome.
Our group has already set up a protocol to determine miRNAs in preservation solution during graft perfusion and a panel was selected. The purpose of normalization is to remove technical variation in data which is not related to the biological changes under investigation. Proper normalization is critical for the correct analysis and interpretation of results. New normalization guidelines in miRNAs analysis have been recently established.
The aim of the study was to validate the selected miRNAs panel according to those renewed normalization guidelines as a way of results support.
Material and Methods: A prospective cohort study was conducted on graft dysfunction in KT from ECD. Ethical approval was obtained from Ethics Review Board. As a discovery cohort, a total of 8 samples selected according to immediate and graft function at 1 year were analyzed. miRNAs were previously detected by Real-Time PCR performed using SYBR Green and specific LNA probes for each miRNA of interest (Exiqon) as well as exogenous Spike-in as technical control. Renewed normalization guidelines involved the use of the average of all miRNAs expression. The statistical significant differences were observed by means of GenEX v6 Enterprise software from Exiqon.
Results: Initial normalization was performed using Spike-in (UniSP2) levels. Data analysis by SPSS indicate that miR-486, miR-18a, miR20a, miR363-3p, miR144-3p, miR454-3p, mir223 3p, miR142-5p, miR502-3p, miR144-3p and miR144-5p discriminate between the DGF appearance after transplantation. Moreover, miR130a-3p, miR-30e-5p, miR-324-3p were associated to the diminished renal function after one year (GRF < 30).
A second normalization analysis using GenEX v6 Enterprise software from Exiqon, using the average of all miRNAs expression (Mean Expression Value) as normalization parameter was performed.
This second analysis confirm that 486-5p, 144-3p, 142-5p, 144-5p, are again associated to DGF appearance. Regarding diminished renal function after a year any miRNAs were confirmed by Mean Expression Value.
Conclusions: We have identified some miRNAs in preservation solution that can be associated to DGF after transplantation. Analysis compliance with new normalization international guidelines confirms the significance of a subset of miRNAs previously associated to DGF post-KT.