Abstract

Luncheon Technology Spotlight: An Effective NGS Workflow for microRNA Profiling from Liquid Biopsies, Extracellular Vesicles and Small Sample Input - Sample Preservation, RNA Extraction, Amplification and Profiling

Bernard Lam, Senior Research Scientist, Norgen Biotek Corporation

The tremendous advancement in our basic understanding in cancer biology at the molecular level has led to the increased use of molecules such as DNA, RNA and microRNA (miRNA) as a biomarker for diagnosis and prognosis of various diseases. In particular, many research has focused on detecting biomarkers in bodily fluids such as plasma, serum, urine or saliva as they are either readily available as clinical samples or they can be easily obtained using non-invasive approaches. The characteristics of RNA present in bodily fluids are quite different from traditional samples such as cells and tissues. First, many RNA in bodily fluids are either free-circulating or are within extra-cellular vesicles such as exosomes. These RNA are generally small in sizes - either mRNA of less than 1000 nts or miRNAs. More importantly, the RNA present in bodily fluids are usually of very low abundance and this presents challenge for detection and study using many next-generation gene expression technologies which requires a large amount of RNA template, sometime at the microgram level.

Here, we present an effective workflow for studying miRNAs from various bodily fluids. The workflow involves four important modules - (1) Sample Preservation, (2) RNA Extraction, (3) miRNA Amplification, (4) Detection. Using urine RNA as an example, we demonstrated the importance of preservation as a significant amount of miRNA transcripts could be lost during standard freezing storage. For RNA purification, we demonstrated that the use of silicon carbide-resin technology worked more effectively than traditional phenol:chloroform/silica column-based methods in recovering miRNAs from bodily fluids such as plasma and urine. In particular, the silicon carbide technology does not require the use of carrier RNA and it does not have any bias in GC contents of the miRNAs. Even with effective preservation and RNA extraction, many bodily fluids samples may yield very low amount of RNA (sometimes at the nanogr