An inexpensive semi-automated sample processing pipeline for cell-free RNA

Abstract

Despite advances in automated liquid handling and microfluidics, preparing samples for RNA sequencing at scale generally requires expensive equipment, which is beyond the reach of many academic labs. Manual sample preparation remains a slow, expensive, and error-prone process. Here, we describe a low-cost, semi-automated pipeline to extract cell-free RNA (cfRNA) that like many RNA isolation protocols, can be decomposed into three subparts - RNA extraction, DNA digestion, and RNA cleaning and concentration. RT-qPCR data using a synthetic spike-in confirms comparable RNA quality as compared to manual sample processing, the gold-standard used in our prior work. The semi-automated pipeline also shows striking improvement in sample throughput (+12x), time spent (-11x), cost (-3x), and biohazardous waste produced (-4x) as compared to its manual counterpart. In total, this protocol enables cfRNA extraction from 96 samples simultaneously in 4.5 hours; in practice, this dramatically improves time to results as demonstrated in our recent work [1] where it was used to process 404 samples in 27 hours. Importantly, any lab already has most of the parts required (manual pipette, corresponding tips and kits) to build a semi-automated sample processing pipeline of their own and would only need to purchase or 3D-print a few extra parts ($5.5K total). This pipeline is also generalizable for many nucleic acid extraction applications, thereby increasing the scale of studies, which can be performed in small research labs.