Abstract
In situ hybridization of oligonucleotide probes to intracellular RNA allows quantification of predefined gene transcripts within millions of single cells using cytometry platforms. Previous methods have been hindered by the number of RNA that can be analyzed simultaneously. Here we describe a method called proximity ligation assay for RNA (PLAYR) that permits highly multiplexed RNA analysis that can be combined with antibody staining. Potentially any number of RNA combined with antigen can be analyzed together, being limited only by the number of analytes that can be measured simultaneously.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Bauman JG, Bayer JA, van Dekken H (1990) Fluorescent in-situ hybridization to detect cellular RNA by flow cytometry and confocal microscopy. J Microsc 157(Pt 1):73–81. https://doi.org/10.1111/j.1365-2818.1990.tb02948.x
Porichis F, Hart MG, Griesbeck M, Everett HL, Hassan M, Baxter AE, Lindqvist M, Miller SM, Soghoian DZ, Kavanagh DG, Reynolds S, Norris B, Mordecai SK, Nguyen Q, Lai C, Kaufmann DE (2014) High-throughput detection of miRNAs and gene-specific mRNA at the single-cell level by flow cytometry. Nat Commun 5:5641. https://doi.org/10.1038/ncomms6641
Lalli E, Gibellini D, Santi S, Facchini A (1992) In situ hybridization in suspension and flow cytometry as a tool for the study of gene expression. Anal Biochem 207(2):298–303. https://doi.org/10.1016/0003-2697(92)90015-y
Patterson BK, Till M, Otto P, Goolsby C, Furtado MR, McBride LJ, Wolinsky SM (1993) Detection of HIV-1 DNA and messenger RNA in individual cells by PCR-driven in situ hybridization and flow cytometry. Science 260(5110):976–979. https://doi.org/10.1126/science.8493534
Belloc F, Durrieu F (1994) Detection of mRNA species by flow cytometry. Methods Cell Biol 42(Pt B):59–69. https://doi.org/10.1016/s0091-679x(08)61068-7
Borzi RM, Piacentini A, Monaco MC, Lisignoli G, Degrassi A, Cattini L, Santi S, Facchini A (1996) A fluorescent in situ hybridization method in flow cytometry to detect HIV-1 specific RNA. J Immunol Methods 193(2):167–176. https://doi.org/10.1016/0022-1759(96)00070-1
Larsson C, Grundberg I, Soderberg O, Nilsson M (2010) In situ detection and genotyping of individual mRNA molecules. Nat Methods 7(5):395–397. https://doi.org/10.1038/nmeth.1448
Player AN, Shen LP, Kenny D, Antao VP, Kolberg JA (2001) Single-copy gene detection using branched DNA (bDNA) in situ hybridization. J Histochem Cytochem 49(5):603–612. https://doi.org/10.1177/002215540104900507
Frei AP, Bava FA, Zunder ER, Hsieh EW, Chen SY, Nolan GP, Gherardini PF (2016) Highly multiplexed simultaneous detection of RNAs and proteins in single cells. Nat Methods 13(3):269–275. https://doi.org/10.1038/nmeth.3742
Duckworth AD, Gherardini PF, Sykorova M, Yasin F, Nolan GP, Slupsky JR, Kalakonda N (2019) Multiplexed profiling of RNA and protein expression signatures in individual cells using flow or mass cytometry. Nat Protoc 14(3):901–920. https://doi.org/10.1038/s41596-018-0120-8
Fredriksson S, Gullberg M, Jarvius J, Olsson C, Pietras K, Gustafsdottir SM, Ostman A, Landegren U (2002) Protein detection using proximity-dependent DNA ligation assays. Nat Biotechnol 20(5):473–477. https://doi.org/10.1038/nbt0502-473
Soderberg O, Gullberg M, Jarvius M, Ridderstrale K, Leuchowius KJ, Jarvius J, Wester K, Hydbring P, Bahram F, Larsson LG, Landegren U (2006) Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat Methods 3(12):995–1000. https://doi.org/10.1038/nmeth947
Lizardi PM, Huang X, Zhu Z, Bray-Ward P, Thomas DC, Ward DC (1998) Mutation detection and single-molecule counting using isothermal rolling-circle amplification. Nat Genet 19(3):225–232. https://doi.org/10.1038/898
Brodin P (2019) The biology of the cell – insights from mass cytometry. FEBS J 286(8):1514–1522. https://doi.org/10.1111/febs.14693
Bendall SC, Simonds EF, Qiu P, Amir el AD, Krutzik PO, Finck R, Bruggner RV, Melamed R, Trejo A, Ornatsky OI, Balderas RS, Plevritis SK, Sachs K, Pe’er D, Tanner SD, Nolan GP (2011) Single-cell mass cytometry of differential immune and drug responses across a human hematopoietic continuum. Science 332(6030):687–696. https://doi.org/10.1126/science.1198704
Muftuoglu M, Li L, Liang S, Mak D, Lin AJ, Fang J, Burks JK, Chen K, Andreeff M (2021) Extended live-cell barcoding approach for multiplexed mass cytometry. Sci Rep 11(1):12388. https://doi.org/10.1038/s41598-021-91816-w
Hartmann FJ, Simonds EF, Bendall SC (2018) A universal live cell barcoding-platform for multiplexed human single cell analysis. Sci Rep 8(1):10770. https://doi.org/10.1038/s41598-018-28791-2
Mei HE, Leipold MD, Schulz AR, Chester C, Maecker HT (2015) Barcoding of live human peripheral blood mononuclear cells for multiplexed mass cytometry. J Immunol 194(4):2022–2031. https://doi.org/10.4049/jimmunol.1402661
Han G, Spitzer MH, Bendall SC, Fantl WJ, Nolan GP (2018) Metal-isotope-tagged monoclonal antibodies for high-dimensional mass cytometry. Nat Protoc 13(10):2121–2148. https://doi.org/10.1038/s41596-018-0016-7
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Duckworth, A.D., Slupsky, J.R., Kalakonda, N. (2024). Highly Multiplexed and Simultaneous Characterization of Protein and RNA in Single Cells by Flow or Mass Cytometry Platforms Using Proximity Ligation Assay for RNA. In: Gužvić, M. (eds) Single Cell Analysis. Methods in Molecular Biology, vol 2752. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3621-3_10
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3621-3_10
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3620-6
Online ISBN: 978-1-0716-3621-3
eBook Packages: Springer Protocols