N laboratory animals. Eight to ten-week old BALB/cJ (Janvier), C57Bl/6J (Janvier), bicm2/m2 (miR155 and WT littermates (miR155 were subjected to cervical heterotopic HTX and sham operations (15,16).RNA isolation and expressionRNA was isolated using the miRVana kit (Ambion, Gent, Belgium). We controlled its integrity and concentration working with Nanodrop (ThermoScientific, Erembodegem, Belgium) and BioAnalyzer (Agilent, Diegem, Belgium). For murine samples, 1 mg of total RNA was reverse transcribed together with the miScript cDNA synthesis kit (Qiagen, Venlo, the Netherlands); for human samples, 0.5 mg of total RNA was reverse transcribed. Real-time quantitative PCR was performed with SYBR green mix (AN3199 web applied Biosystems, Gent, Belgium) on an ABI Prism 7500 (Applied Biosystems). LNA primers (Exiqon, Vedbaek, Denmark) have been used to detection mature miRs. U6 was applied as an endogenous control for miRs; GAPDH was utilised as a housekeeping gene for mRNAs.Human endomyocardial biopsiesHuman material was obtained during sampling for clinical purposes and offered for investigation in line with the Declaration of Helsinki and the ethical committees at UZ Leuven (Leuven, Belgium) and Maastricht University Health-related Center (Maastricht, the Netherlands). Surveillance EMBs included in our analyses have been preferentially taken later than 6 weeks after transplantation to avoid the perioperative phase. Control biopsies consisted of age-matched individuals with unexplained ventricular tachyarrhythmias but with a normal ejection fraction, cardiac morphology, and no systemic or cardiac inflammation or viral persistence. Biopsies were snap-frozen for RNA evaluation and formalin or Bouin fixed for histology.MiR and mRNA expression profilingTotal RNA from human biopsies was subjected to miR expression profiling on NanoStrings’ nCounter platform by PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20082828 the Nucleomics Core on the Flemish Institute of Biotechnology (Merelbeke, Belgium). The gene target list consisted of 654 human miRs, 80 human-associated viral miRs, 6 positive controls, eight adverse controls, and 5 housekeeping genes. Further identification of differentially regulated miRs was performed utilizing the R pipeline offered by the manufacturer. Total RNA from mouse hearts was hybridized to mouse miRNA microarray G4472B (Agilent) determined by miRBase release 12.0, containing 627 mouseHistological and morphometrical analysisMice have been sacrificed three, five, or 7 days soon after HTX. Organs have been removed, rinsed, blotted dry, weighed, and snap frozen. Ahead of weighing, atria were separated in the ventricles. Ventricles have been divided in basal, midventricular, and apical components. The apical portion was split and snap frozen. TheAmerican Journal of Transplantation 2016; 16: 99RNA Mechanisms in Acute Allograft RejectionmiRs, 39 mouse viral miRs, six damaging controls, 6 optimistic controls, and 5 housekeeping genes. Further identification of differentially regulated miRs was performed applying computer software provided by the manufacturer at Hannover Health-related College (MHH, Hannover, Germany). Total RNA from human biopsies and mouse hearts was subjected to mRNA expression profiling on Affymetrix Human PrimeView and Mouse MoGene 1.0 ST v1 microarrays, respectively. The expression information was normalized utilizing the Affymetrix R package and acceptable CustomCDFs in the Microarray Lab (University of Michigan) (19). Differential expression was assessed in R computer software(20) employing the limma package (21). MiRs were regarded as differentially expressed having a p-value of 0.05; mRNAs were deemed diffe.
