Purification of mitochondria from skeletal muscle tissue for transcriptomic analyses reveals localization of nuclear-encoded non-coding RNAs [L6 mito]

MiRNAs are non-coding RNAs that regulate gene expression. MiRNAs mostly localise within the cytosol but are also found in the mitochondria where they can regulate the expression of mitochondrial-encoded transcripts. Small RNA library preparation protocols are well described when using total cellular RNA as the template, however, these methods are not directly applicable to total RNA extracted from fractionated cells, such as isolated mitochondria. The aim of this study was to optimise the small RNA library preparation protocol for use with small (<60ng) amounts of total mitochondrial RNA. L6 cells were proliferated to 80% confluence, harvested, and immediately processed for the isolation of mitochondria and extraction of mitochondrial RNA which then underwent a series of dilutions (1.8-120ng). Small RNA libraries were then prepared from a range of mitochondrial RNA inputs with different ratios of adapters (0.1x, 0.3x, 0.5x) and qPCR cycles.