4.4. Omics Analysis

One-hundred ng of total RNA from each fraction was labeled and hybridized onto Agilent mouse RNA and miRNA microarrays, Release 18.0 (Agilent Technologies, Santa Clara, CA, USA) following the standard Agilent protocol with RNA and miRNA complete labelling and hybridization kits including Agilent RNA spike-ins. The results were scanned using an Agilent G2565CA microarray scanner. Scanned TIFF image files were processed using Agilent feature extraction software (v10.7.3.1) to extract the raw data. Briefly, samples were shortly separated in a polyacrylamide gel and proteins we re-reduced with dithiothreitol, alkylated with iodoacetamide and digested with trypsin. After extraction, the peptides were finally resuspended in 0.1% trifluoroacetic acid. Approximately 500 ng of peptides were subsequently separated on an Ultimate 3000 rapid separation liquid chromatography system (Thermo Fisher Scientific, Dreieich, Germany) and analyzed on an Orbitrap Elite (Thermo Fisher Scientific, Dreieich, Germany) hybrid mass spectrometer. All omics data were normalized with the quantile method. To find the statistically significant molecules and their GO enrichment, we used the method described previously [62] with the selection threshold θ_DEG = θ_DEM = 4 and significance threshold α_DEG = α_DEM = 0.001. For each mRNA sample, we used four biological replicates; for each miRNA sample we used three biological replicates except two for ECs and KTs.