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

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 laboratories. 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 using one of two commercially available, inexpensive and open-source robotic systems: the Opentrons OT1.0 or OT2.0. Like many RNA isolation protocols, ours 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 to the gold standard, manual sample processing. The semi-automated pipeline also shows improvement in sample throughput (+12×), time spent (-11×), cost (-3×) and biohazardous waste produced (-4×) compared with its manual counterpart. This protocol enables cell-free RNA extraction from 96 samples simultaneously in 4.5 h; in practice, this dramatically improves the time to results, as we recently demonstrated. Importantly, any laboratory already has most of the parts required (manual pipette and corresponding tips and kits for RNA isolation, cleaning and concentration) to build a semi-automated sample processing pipeline of their own and would only need to purchase or three-dimensionally print a few extra parts (US$5.5 K-12 K in 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 laboratories.