Simultaneous Targeted Detection of Proteins and RNAs in Single Cells
Simultaneous detection of both RNA and protein in individual single cells offers a powerful tool for genotype-to-phenotype investigations. Proximity extension assay (PEA) is a quantitative, sensitive, and multiplex protein detection system that has superb
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troduction The recent advent of high-throughput single-cell analysis platforms has greatly accelerated the pace of understanding cell types and characterizing cellular states and heterogeneity. Much of the progress has been elucidated from single-cell transcriptomic analysis using targeted RT-qPCR assays and RNA sequencing regimens [1, 2]. Ideally, RNA levels should indicate protein levels. However, previous studies indicate that levels of individual mRNAs and their respective translated proteins often display discordance [3]. Consequently, direct profiling of cellular protein levels offers a more accurate picture of protein-related cellular activities, while the ability to link RNA levels to protein expression would generate the most complete view of genotype-to-phenotype relationships in dynamic biological pathways. Quantification of proteins at the single-cell level relies on antibody-based detection approaches. While methods based on fluorescence-activated cell sorting (FACS) are widely used, this technology is often limited to cell surface markers. Another
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_22, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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Aik T. Ooi and David W. Ruff
single-cell protein detection method based on proximity ligation assay has sensitivity limitations and cannot be multiplexed [4]. To overcome these limitations, an improved detection format for protein analysis has been developed using proximity extension assay (PEA) [5]. For a protein target, PEA requires two independent antibodies, each conjugated with an oligonucleotide that carries a short sequence that is complementary to the other. When the pair of antibody probes bind to their antigen in close proximity, the complementary ends anneal, followed by strand extension to generate a full-length RT-qPCR template. The two-antibody system reduces background and thus enables simultaneous detection of multiple targets. With RT-qPCR as the end-point analysis, PEA overcomes the limitation of overlapping fluorescence spectra faced by the FACS technology and in turn allows for a higher number of possible targets in a single experiment. A single-cell version of the method has been developed for the Fluidigm C1 system with RT-qPCR readout on the Biomark HD system [6, 7], where the use of custom PEA antibody probes for multiplex targets has been reported. Constructing custom panels requires time-consuming antibody–oligonucleotide conjugation procedures and verification [8]. The above-mentioned single-cell C1 microfluidic protocols can be readily adapted to using commercially available and validated PEA antibody panels from the Proseek® Multiplex96x96 Kit (Olink® Proteomics). We incorporate at the front end a mild cell lysis condition that allows the binding of PEA antibody probes to their antigen targets while preserving cellular RNA. Furthermore, by utilizing the polymerase activity of a reverse trans
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