Abstract 4426: Automated miRNA purification from plasma, serum or exosomes

Abstract

Abstract Introduction: MicroRNAs (miRNAs) are endogenous small noncoding RNAs 18-24 nucleotides in size that play important roles involving gene regulation associated with cancer, disease control and gene silencing. Because of the impact on disease progression, miRNA research is rapidly shifting towards biomarker discovery. Many of the commercially available miRNA purification methods involve organic extraction during preprocessing. Here, we describe a novel chemistry that enables RNA purification including smaller RNA such as miRNA. This chemistry offers significant advantages with a simple automated workflow, no organic extraction and minimal preprocessing. Method: In this study we show successful purification of small RNA, including miRNA, from 100uL to 500uL liquid samples utilizing a novel chemistry on the Maxwell® RSC instrument, which can process between 1 and 16 samples. Proteinase K and Lysis Buffer C are added to the liquid samples and incubated before being transferred to the Maxwell cartridge for miRNA isolation. Sample types tested include plasma, serum and isolated exosomes. The miRNA were evaluated using RT-qPCR including miR-16, miR-21, and let-7a. Results: The eluates had high levels of small RNA including miRNA at levels comparable to manual competitor methods that require organic extraction. Quantitative PCR showed that the eluates had little or no detectable DNA contamination. Integration of the chemistry onto this automated platform reduced hands-on time while maintaining high quality needed for use in downstream RT-qPCR amplification assays. Summary: This novel chemistry and method enables purification of small RNA from a range of samples types and volumes. The purified RNA has little or no detectable DNA contamination. Phenol extraction is not required, improving safety and simplifying waste disposal. Also, the automation of this workflow decreases manual hands-on time, saving scientist time and reducing the risk of RNase contamination. Citation Format: Michelle Mandrekar, Jami English, Douglas Horejsh, Chris Moreland, Herly Karlen, Marjeta Urh. Automated miRNA purification from plasma, serum or exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4426.