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.

On-chip miRNA extraction platforms: recent technological advances and implications for next generation point-of-care nucleic acid tests

“…The miRNAs were bound onto silica magnetic particles which were transferred between wells using a magnet. The final elution was prepared within 15min and on-chip RT-PCR was carried out proving similar results to the extraction kit used for comparison while the process was both faster and cheaper 48 . Slouka et al used electrophoresis as a sample pre-treatment method following cell lysis.…”

“…Isotachophoresis which involves the movement of a leading electrolyte (green) and a trailing electrolyte (blue) 50 , f) Magnetic particles for removal of larger nucleic acids in the process of miRNA isolation 48 . This journal is © The Royal Society of Chemistry 20xx…”

“…The final elution was prepared within 15 min and on-chip RT-PCR was carried out proving similar results to the extraction kit used for comparison while the process was both faster and cheaper. 48 Slouka et al used electrophoresis as a sample pretreatment method following cell lysis. This was then followed by concentration of the miRNAs using a cation exchange membrane.…”

“…3 Hybrid methods (off-and on-chip components) of miRNA extraction: A) acoustic waves and seeding particles for EV entrapment, 55 B) electrophoresis in buffer through nanofilter membrane, 57 C) thermo-electric cell lysis, 51,58 D) electrophoretic miRNA purification, 58 E) isotachophoresis which involves the movement of a leading electrolyte (green) and a trailing electrolyte (blue), 50 F) magnetic particles for removal of larger nucleic acids in the process of miRNA isolation. 48 This journal is © The Royal Society of Chemistry 2022 ultrasonication and centrifugation and achieved very high recoveries (94.9-108%). 52 Although these electrophoretic methods seem promising, they still require manual handling at times and are thus not entirely on-chip extraction methods.…”

A 3D mixing-based portable magnetic device for fully automatic immunofluorescence staining of γ-H2AX in UVC-irradiated CD4+ cells

“…25,26 The versatility in sizes, magnetic properties and surface functionalizations, as well as commercial availability of MBs have been promoting their use in lab-on-a-chip devices, 25 thus lowering complexity in chip design and uid control (free of pump and valve), and improving exibility and automation of the assays. 26,27 Researches on MB-based on-chip IF staining have shown the possibilities of magnetic POC IF assays. 11,[28][29][30][31] As for MB-based on-chip IF staining, a front-end MBs mixing step allows efficient capture of target cells, 31 which is determined by the extent of interactions between MBs and cells.…”