Small RNA Analysis in Cancer Cells

Small RNAs (sRNAs), broadly defined as RNA molecules shorter than ~200 nucleotides in size, include miscellaneous products of transcription and post-transcriptional processing that are variably abundant in cancer cells. While some types of sRNAs, such as microRNA (miRNA), are relatively well-characterized, the biological roles of many others remain unknown. Regardless of functionality, sRNA transcriptomes may reflect the identity of cell types, tissues, and cancers. While deep sequencing remains the best approach for sRNA profiling, there are distinct technical challenges for sRNA extraction, library preparation, sequencing and data analysis. Here, we describe a workflow for the preparation of sRNAs from cancer cells, sRNA libraries for sequencing, and basic bioinformatic analyses of sRNA sequencing data. We describe the preparation and analysis of standard, miRNA-enriched libraries that contain 5'-monophosphorylated RNAs and 3'-hydroxyl ends, as well as a custom protocol for the analysis of sRNAs containing 5'-cap modifications. The chapter should facilitate the analysis of different sRNA classes in cancer cells.