Abstract

Enrichment of microRNA Using a Novel Avalanche Photodiode-based Benchtop Cell Sorter

John Tigges, Flow Cytometry Science Center Director, Beth Israel Deaconess Medical Center

In the past few years, extracellular vesicles (EVs) have rapidly become one of the most studied entities. EVs are characterized by their outer lipid layer and internalized cargo such as microRNA (miR). EVs and their cargo have been shown to regulate gene expression and alter cell function in various cell types (Kreimer et al., 2015, Mantel et al., 2013). Isolation and molecular profiling of subsets of EVs (i.e., RNAs, proteins, lipids, metabolites) are critical for understanding the biogenesis of EVs and their potential utility as biomarkers (Pereira et al., 2020). To this end, flow cytometry has been employed for the analysis, characterization, and phenotyping of EV populations and their cargo in a technique called nano-flow cytometry. While nano-flow cytometry has become more prevalent as an analysis tool and undergone standardization formats (Welsh et al., 2020), nano-sorting is neither well defined nor a commonly used technique. Historically, this is due to the complexity of the technique and instrumentation necessary for sorting of EVs (Morales-Kastresana et al., 2019, Kormelink et al., 2015). In this study, the CytoFLEX SRT, a semiconductor based benchtop cell sorter equipped with avalanche photodiodes and optimized signal detection, (Brittain et al., 2019) is utilized for the detection and nano-sorting of miRs in EV populations. Platelet EVs, red blood cell EVs, and neutrophil EVs are mixed with molecular beacons (MBs) targeted to miR495, miR451, and miR148 respectively. MBs are nucleic acid sequences locked in a hairpin conformation with a quencher signal and fluorophore attached to the end (Tyagi, 1996). Upon binding with a specific target RNA sequence, the quencher is spatially moved from the fluorophore and a fluorescent signal is produced. Upon analysis of the EV populations with MBs via VSSC and fluorescent thresholding, fluorescent positive signals corresponding to the particular miR target were sorted. Sorted samples were verified via microscopy and PCR analysis and compared to fluorescent negative miR EVs The presence of EV populations and subsequent increased target miRs, verified via dark field and fluorescent microscopy, confirm sorting efficiency compared to the negative population. Additionally, CT values from the PCR analysis show 8x difference in miR detection between positive and negative sorted samples. These results show the CytoFLEX SRT to be a viable instrument for nano-sorting.