Abstract
The systematic modulation of mRNA and proteins governs the complicated and intermingled biological functions of our cells. Traditionally, transcriptomic technologies such as DNA microarray and RNA-Seq have been used to identify, characterize, and profile gene expression data. These are, however, considered bulk methods as they are unable to measure gene expression at the single-cell level, unless the cells are pre-sorted. Branched DNA is a flow cytometry-based detection platform that has been developed recently to measure mRNA at the single-cell level. Originally adapted from microscopy, the current system has been modified to achieve compatibility with the detection of surface and intracellular antigens using monoclonal antibodies conjugated to fluorochromes, thus permitting simultaneous detection of mRNAs and proteins. The Branched DNA method offers a variety of advantages when compared to traditional or standard methods used for the quantification of mRNA, such as (a) the detection of specific mRNA on a per cell basis, (b) an alternate detection tool when the measurement of a protein is technically infeasible (i.e., no quality antibody exists) or the epitope is not assessable, and (c) correlate the analysis of mRNA with protein. Compared to earlier attempts at measuring nucleic acid by flow cytometry, the hybridization temperature applied in the Branched DNA assay is much lower, thus preserving the integrity of cellular structures for further characterization. It also has greatly increased specificity and sensitivity. Here, we provide detailed instruction for performing the Branched DNA method using it in a model system to correlate the expression of CD8 mRNA and CD8 protein by flow cytometry.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Bentley DL (2014) Coupling mRNA processing with transcription in time and space. Nat Rev Genet 15(3):163–175. doi:10.1038/nrg3662
Hocine S, Singer RH, Grunwald D (2010) RNA processing and export. Cold Spring Harb Perspect Biol 2(12):a000752. doi:10.1101/cshperspect.a000752
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
Patterson BK, Mosiman VL, Cantarero L, Furtado M, Bhattacharya M, Goolsby C (1998) Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype. Cytometry 31(4):265–274
Van Hoof D, Lomas W, Hanley MB, Park E (2014) Simultaneous flow cytometric analysis of IFN-gamma and CD4 mRNA and protein expression kinetics in human peripheral blood mononuclear cells during activation. Cytometry A 85(10):894–900. doi:10.1002/cyto.a.22521
Trask B, van den Engh G, Landegent J, in de Wal NJ, van der Ploeg M (1985) Detection of DNA sequences in nuclei in suspension by in situ hybridization and dual beam flow cytometry. Science 230(4732):1401–1403
Mutty CE, Timm EA Jr, Stewart CC (1999) Effects of thermal exposure on immunophenotyping combined with in situ PCR, measured by flow cytometry. Cytometry 36(4):303–311
Patterson BK, Goolsby C, Hodara V, Lohman KL, Wolinsky SM (1995) Detection of CD4(+) T-cells harboring human-immunodeficiency-virus type-1 DNA by flow cytometry using simultaneous immunophenotyping and PCR-driven in-situ hybridization - evidence of epitope masking of the CD4 cell surface molecule in-vivo. J Virol 69(7):4316–4322
Yu H, Ernst L, Wagner M, Waggoner A (1992) Sensitive detection of RNAs in single cells by flow cytometry. Nucleic Acids Res 20(1):83–88
Boyum A (1968) Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyteaggregating agents. Scand J Clin Lab Invest Suppl 97:31–50
Fuss IJ, Kanof ME, Smith PD, Zola H (2009) Isolation of whole mononuclear cells from peripheral blood and cord blood. Curr Protoc Immunol Chapter 7:Unit7 1. doi:10.1002/0471142735.im0701s85
Roederer M (2001) Spectral compensation for flow cytometry: visualization artifacts, limitations, and caveats. Cytometry 45(3):194–205
Soh KT, Tario JD Jr, Colligan S, Maguire O, Pan D, Minderman H, Wallace PK (2016) Simultaneous, single-cell measurement of messenger RNA, cell surface proteins, and intracellular proteins. Curr Protoc Cytom 75:7.45.1–7.45.33. doi:10.1002/0471142956.cy0745s75
Acknowledgment
The authors acknowledge Dylan Malayter and Castle Funatake (both from Affymetrix/eBioscience) for their contributions to this chapter. Paul K. Wallace is partially supported by the Roswell Park Cancer Institute Ovarian SPORE NIH Grant 1P50CA159981-01A1. Flow cytometry was performed at Roswell Park Cancer Institute’s Department of Flow and Image Cytometry, which was established in part by equipment grants from the NIH Shared Instrument Program, and receives support from the Core Grant (5 P30 CA016056-29) from the National Cancer Institute to the Roswell Park Cancer Institute.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media LLC
About this protocol
Cite this protocol
Soh, K.T., Wallace, P.K. (2018). RNA Flow Cytometry Using the Branched DNA Technique. In: Hawley, T., Hawley, R. (eds) Flow Cytometry Protocols. Methods in Molecular Biology, vol 1678. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7346-0_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7346-0_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7344-6
Online ISBN: 978-1-4939-7346-0
eBook Packages: Springer Protocols