Analysis and validation of a new extended method for estimating plasma free cortisol including neutrophil elastase and competition from other steroids

The presence of noncoding RNAs, such as microRNAs (miRNAs), in mitochondria has been reported by several studies. The biological roles and functions of these mitochondrial miRNAs ("mitomiRs") have not been sufficiently characterized, but the mitochondrial localization of miRNAs has recently gained significance due to modified mitomiR-populations in certain states of diseases. Here, we describe the isolation and analysis of mitochondrial RNAs from rat liver tissue and HepG2 cells. The principle of the analysis is to prepare mitochondria by differential centrifugation. Cytosolic RNA contamination is eliminated by RNase A treatment followed by Percoll gradient purification and RNA extraction. Small RNA content is verified by capillary electrophoresis. Mitochondrial miRNAs are detected by qPCR following synthesis of cDNA. After qPCR-based mitomiR-profiling, the Normfinder algorithm is applied to identify the suitable reference miRNAs to use as normalizers for mitochondrial input and data analysis. The described procedure depicts a simple way of isolating and quantifying mitomiRs in tissue and cell culture samples.