miR profiling of apoptotic murine neurons and their supernatent

miRNAs were recently discovered to directly activate receptors thereby extending their original role beyond post-transcriptional gene expression regulation. In particular, Here, conducting a systematic screening approach we identified miRNAs that in extracellular form can serve as receptor ligands, and elaborated the consequences of such an interaction in CNS injury. Primary cortical neurons isolated from C57BL/6 mice were first exposed to staurosporine, a potent, non-selective kinase inhibitor, which is widely used to model neuronal apoptosis, for 8 h. While neurons remained morphologically intact within this time period, efficient apoptosis induction through activation of neuronal caspase-3 signaling was verified by western blot. Next, neurons and corresponding supernatant (S/N) were separately harvested and processed for miRNA detection. After enrichment and isolation of the small RNA fractions from neurons and their corresponding S/N, they were analyzed by GeneChip miRNA 4.0 array technology. Microarray data were processed using the robust multi-array average (RMA) method on the R/Bioconductor platform. Most of subsequent analysis was restricted to miRNA probes that were specific for Mus musculus. Replicate samples were taken from neurons and their supernatant after induction of apoptosis. As control, samples were extracted from neurons and their supernatant without induced apoptosis. The abundance levels of miRNA were compared between apoptotic and control neurons as well as between the supernatents of apoptotic and of control neurons, respectively