title: Automatic extraction and processing of small RNAs on a multi-well/multi-channel (M&M) chip.
abstract: The study of the regulatory roles in small RNAs can be accelerated by techniques that permit simple, low-cost, and rapid extraction of small RNAs from a small number of cells. In order to ensure highly specific and sensitive detection, the extracted RNAs should be free of the background nucleic acids and present stably in a small volume. To meet these criteria, we designed a multi-well/multi-channel (M&M) chip to carry out automatic and selective isolation of small RNAs via solid-phase extraction (SPE), followed by reverse-transcription (RT) to convert them to the more stable cDNAs in a final volume of 2 μL. Droplets containing buffers for RNA binding, washing, and elution were trapped in microwells, which were connected by one channel, and suspended in mineral oil. The silica magnetic particles (SMPs) for SPE were moved along the channel from well to well, i.e. in between droplets, by a fixed magnet and a translation stage, allowing the nucleic acid fragments to bind to the SMPs, be washed, and then be eluted for RT reaction within 15 minutes. RNAs shorter than 63 nt were selectively enriched from cell lysates, with recovery comparable to that of a commercial kit. Physical separation of the droplets on our M&M chip allowed the usage of multiple channels for parallel processing of multiple samples. It also permitted smooth integration with on-chip RT-PCR, which simultaneously detected the target microRNA, mir-191, expressed in fewer than 10 cancer cells. Our results have demonstrated that the M&M chip device is a valuable and cost-saving platform for studying small RNA expression patterns in a limited number of cells with reasonable sample throughput.
authors.affiliation: Department of Chemistry, University of California, Riverside, CA 92521, USA.
authors.forename: Runtao
authors.initials: R
authors.lastname: Zhong
authors.forename: Kenneth
authors.initials: K
authors.lastname: Flack
authors.forename: Wenwan
authors.initials: W
authors.lastname: Zhong
mesh_terms: D057809:HEK293 Cells / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry; D035683:MicroRNAs / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D012313:RNA / Q000032:analysis / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D052616:Solid Phase Extraction / Q000379:methods
chemical_list: C546177:MIRN191 microRNA, human; D035683:MicroRNAs; D012313:RNA