Abstract

Complete Next-Generation Gene Expression Workflow for microRNAs from Bodily Fluids, Extracellular Vesicles and Small Sample Input - Sample Preservation, RNA Extraction, Amplification and Detection

Bernard Lam, Senior Research Scientist, Norgen Biotek Corporation

The tremendous advancements in our basic understanding of cancer biology at the molecular level have 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, much 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 of the RNAs in bodily fluids are either free-circulating or are within extra-cellular vesicles such as exosomes. These RNAs are generally small in size - either mRNA of less than 1000 nts or miRNAs. More importantly, the RNA present in bodily fluids is usually of very low abundance and this presents challenges for detection and studies using many next-generation gene expression technologies which require a large amount of RNA template, sometimes 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 purified. Even with effective preservation and RNA extraction, many bodily fluid samples may yield very low amounts of RNA (sometimes