Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215, USA.
Anal Chem. 2010 Jun 1;82(11):4344-56. doi: 10.1021/ac100063f.
The life science and healthcare communities have been redefining the importance of ribonucleic acid (RNA) through the study of small molecule RNA (in RNAi/siRNA technologies), micro RNA (in cancer research and stem cell research), and mRNA (gene expression analysis for biologic drug targets). Research in this field increasingly requires efficient and high-throughput isolation techniques for RNA. Currently, several commercial kits are available for isolating RNA from cells. Although the quality and quantity of RNA yielded from these kits is sufficiently good for many purposes, limitations exist in terms of extraction efficiency from small cell populations and the ability to automate the extraction process. Traditionally, automating a process decreases the cost and personnel time while simultaneously increasing the throughput and reproducibility. As the RNA field matures, new methods for automating its extraction, especially from low cell numbers and in high throughput, are needed to achieve these improvements. The technology presented in this article is a step toward this goal. The method is based on a solid-phase extraction technology using a porous polymer monolith (PPM). A novel cell lysis approach and a larger binding surface throughout the PPM extraction column ensure a high yield from small starting samples, increasing sensitivity and reducing indirect costs in cell culture and sample storage. The method ensures a fast and simple procedure for RNA isolation from eukaryotic cells, with a high yield both in terms of quality and quantity. The technique is amenable to automation and streamlined workflow integration, with possible miniaturization of the sample handling process making it suitable for high-throughput applications.
生命科学和医疗保健领域通过研究小分子 RNA（在 RNAi/siRNA 技术中）、微 RNA（在癌症研究和干细胞研究中）和 mRNA（用于生物药物靶点的基因表达分析），重新定义了 RNA 的重要性。该领域的研究越来越需要高效、高通量的 RNA 分离技术。目前，有几种商业试剂盒可用于从细胞中分离 RNA。尽管这些试剂盒从细胞中提取的 RNA 的质量和数量对于许多目的来说已经足够好，但在从小细胞群中提取的效率和自动化提取过程的能力方面仍存在局限性。传统上，自动化一个过程可以降低成本和人员时间，同时提高吞吐量和可重复性。随着 RNA 领域的成熟，需要新的方法来自动化其提取，特别是从低细胞数量和高通量中提取，以实现这些改进。本文介绍的技术是实现这一目标的一步。该方法基于使用多孔聚合物整体（PPM）的固相萃取技术。一种新颖的细胞裂解方法和整个 PPM 提取柱上更大的结合表面确保了从小起始样品中获得高产量，提高了灵敏度并降低了细胞培养和样品存储的间接成本。该方法确保了从真核细胞中快速简单地分离 RNA，无论是在质量还是数量方面都具有很高的产量。该技术适用于自动化和简化的工作流程集成，并且可能通过减小样品处理过程的尺寸使其适用于高通量应用。
