Department of Bioengineering, Stanford University, Stanford, CA, USA.
The Column Group, San Francisco, CA, USA.
Nat Protoc. 2023 Sep;18(9):2772-2793. doi: 10.1038/s41596-023-00855-2. Epub 2023 Aug 11.
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.
尽管自动化液体处理和微流控技术取得了进步，但大规模制备 RNA 测序样本通常需要昂贵的设备，这超出了许多学术实验室的承受能力。手动样品制备仍然是一个缓慢、昂贵且容易出错的过程。在这里，我们描述了一种低成本、半自动化的方法，使用两种商业上可用的、廉价的开源机器人系统之一（Opentrons OT1.0 或 OT2.0）提取无细胞 RNA。与许多 RNA 分离方案一样，我们的方案可以分解为三个子部分：RNA 提取、DNA 消化以及 RNA 清洗和浓缩。使用合成 RNA 掺入物的 RT-qPCR 数据证实，与手动样品处理的金标准相比，我们的方法具有相当的 RNA 质量。与手动方法相比，半自动化的工作流程还提高了样本通量（提高 12 倍）、用时（减少 11 倍）、成本（降低 3 倍）和生物危害性废物（减少 4 倍）。该方案可在 4.5 小时内同时从 96 个样本中提取无细胞 RNA；实际上，正如我们最近所证明的那样，这极大地缩短了得出结果的时间。重要的是，任何实验室已经拥有构建自己的半自动化样品处理工作流程所需的大部分部件（用于 RNA 分离、清洗和浓缩的手动移液器和相应的吸头和试剂盒），只需购买或三维打印几个额外的部件（总计 5500 美元至 12000 美元）。该工作流程还可推广用于许多核酸提取应用，从而增加可以在小型研究实验室中进行的研究规模。
