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 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.
Key points
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A protocol for setting up, calibrating and validating a semi-automated sample processing pipeline for cell-free RNA isolation from clinical samples using the Opentrons 1.0 or 2.0 open-source robotic platform.
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The key benefits of this semi-automated system, as compared with manual extraction, include an increase in sample throughput by 12 fold. When compared with other available protocols for automated RNA extraction, this protocol provides a complementary option for larger sample volumes (>0.5 mL).
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Data availability
Source data are provided with this paper. This file includes raw data to reproduce Fig. 3b–d and Fig. 4. The Supplementary Manual also includes instructions for how to build a system similar to the OT1 from scratch.
Code availability
All software, including the data analysis required to generate Figs. 3 and 4, are available at https://github.com/miramou/cfRNA_pipeline_automation (https://doi.org/10.5821/zenodo.7931552) (ref. 24) and https://github.com/miramou/cfRNA_pipeline (https://doi.org/10.5281/zenodo.7931554) (ref. 25).
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Acknowledgements
We are grateful to both N. Neff and R. Sit for their sequencing expertise. We also thank B. Yu for helping brainstorm and providing indispensable advice around how to best use the Opentrons system. Figures 1 and 2D,E were created with BioRender.com. This work was supported by the Chan Zuckerberg Biohub. M.N.M. is supported by the Stanford Bio-X Bowes Fellowship.
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M.N.M. conceptualized and designed this protocol, and collected and analyzed the data in collaboration with S.R.Q. All authors contributed to the writing and editing of the manuscript.
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S.R.Q. is a founder, consultant and shareholder of Mirvie. M.N.M. is also a shareholder of Mirvie.
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Nature Protocols thanks Eleni Tomazou and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Moufarrej, M. N. et al. Nature 602, 689–694 (2022): https://doi.org/10.1038/s41586-022-04410-z
Moufarrej, M. (2020): https://youtu.be/g6RsSaNvSNA
Supplementary information
Supplementary Information
Supplementary Manual.
Source data
Source Data Figs. 3 and 4
Unprocessed RT–qPCR data for Figs. 3a–c and 4.
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Moufarrej, M.N., Quake, S.R. An inexpensive semi-automated sample processing pipeline for cell-free RNA extraction. Nat Protoc 18, 2772–2793 (2023). https://doi.org/10.1038/s41596-023-00855-2
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DOI: https://doi.org/10.1038/s41596-023-00855-2
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