Abstract

Lunch Technology Spotlight: Increasing the Sensitivity of Next-Generation Gene Expression Workflow for microRNAs from Bodily Fluids, Extracellular Vesicles and Small Sample Input - Sample Preservation, RNA Extraction and Sequencing Library Preparation

Bernard Lam, Senior Research Scientist, Norgen Biotek Corporation

The use of bodily fluids such as plasma, serum, urine or saliva for non-invasive biomarker discovery has attracted tremendous attention in recent years. However, the characteristics of RNA (and DNA) 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 that is optimized for enhancing the sensitivity for detecting miRNAs from various bodily fluids. The workflow involves the use of a patented silicon carbide nucleic acid purification technology that could enhance the outcome in both sample preparation and library preparation.  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, and does not require the use of carrier RNA. As well, it does not have any bias in GC contents of the miRNAs purified. In addition, we will discuss the adoption of the silicon carbide technology to enhance signals in Next Gen Sequencing library preparation.