RNA Isolation from Mammalian Cells Using Porous Polymer Monoliths: An Approach for High-Throughput Automation

• Published in: Analytical chemistry (Washington) 2010-06, Vol.82 (11), p.4344-4356
• Main Authors: Chatterjee, Anirban, Mirer, Paul L, Zaldivar Santamaria, Elvira, Klapperich, Catherine, Sharon, Andre, Sauer-Budge, Alexis F
• Format: Article
• Language: English
• Subjects: Air; Analytical chemistry; Animals; Automation; Buffers; Cell Line; Cells; Chemistry; Dogs; Eukaryotes; Exact sciences and technology; Mammals; Polymers; Polymers - chemistry; Porosity; Porous materials; Pressure; Reproducibility of Results; Ribonucleic acid; RNA; RNA - isolation & purification; Solid Phase Extraction - economics; Solid Phase Extraction - instrumentation; Solid Phase Extraction - methods
• ISSN: 0003-2700; 1520-6882
• 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.