(481f) Semi-Preparative Isotachoporesis for Fractionation of RNA From Blood

RNA transcripts circulating in blood or blood plasma may be useful as biomarkers of infection, host response to infection, or numerous other states of health.  RNA-Seq technologies offer the ability for quantitative, unbiased analysis of all circulating RNA within a clinical sample in a single experiment, and development of sample preparation techniques for RNA from blood is an area of active investigation.  Circulating RNA tend to heavily degraded, and are typically <200 nt in length.  This pool includes both "small" RNA (microRNA, etc), as well as degraded mRNA and other larger species.  Both pools can be informative, but are ideally analyzed separately due to disparate requirements for library preparation, sequencing techniques, and bioinformatics analysis.  We have thus developed a technique for small-scale preparative fractionation of RNA into "small" and "large" fractions, utilizing discontinous zone electrophoresis in a "large-channel microfluidics" device sized to process tens to hundreds of nanograms of RNA, acheiving good recovery from samples at the scale of tens of microliters.  Our device operates at a larger scale than typical analytical separations, but at a smaller scale than commercial kits and devices that are mainly geared to fractionating DNA at microgram or larger scale.  We discuss design of the device and effect of operating parameters such as buffer system on performance of the device, and discuss constraints for use in the workflow of preparing sequencing libraries.