Abstract
RNA in situ hybridization (ISH) is a widely used technique for the localization of mRNA in tissues. Limitations to traditional ISH include the number of targets that can be analyzed concurrently and the ability for many of these assays to be used in formalin-fixed, paraffin-embedded tissues (FFPE). Here, we describe the GeoMx™ RNA assay that is capable of the highly multiplexed detection of mRNA targets in FFPE tissues. This assay utilizes ISH probes linked to indexing oligo barcodes via a photocleavable linker and the GeoMx Digital Spatial Profiler (DSP) Instrument to enable profiling of RNA targets in a region-of-interest-based method. In brief, 5 μm FFPE sections are dewaxed, target retrieved, digested with proteinase K, post-fixed, and then incubated overnight with GeoMx RNA detection probes. Stringent washes are performed followed by the addition of fluorescently labeled antibodies for use as morphology markers. User-defined regions of interest are then profiled on the GeoMx DSP through region-specific cleaving and collecting the photocleaved indexing oligos. Cleaved indices are then quantified using NanoString nCounter® Technology generating digital quantification of RNA expression with spatial context.
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References
Chung JY, Braunschweig T, Williams R et al (2008) Factors in tissue handling and processing that impact RNA obtained from formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem 56(11):1033–1042. https://doi.org/10.1369/jhc.2008.951863
Merritt CR, Ong GT, Church S et al (2020) Multiplex digital spatial profiling of proteins and RNA in fixed tissue. Nature Biotechnology (in press) https://doi.org/10.1101/559021
Toki MI, Merritt CR, Wong PF et al (2019) High-plex predictive marker discovery for melanoma immunotherapy treated patients using Digital Spatial Profiling. Clin Can Res. https://doi.org/10.1158/1078-0432.CCR-19-0104
Nanostring Technologies, Inc. MAN-10087-03, GeoMx Digital Spatial Profiler Online User Manual
nCounter® Expression CodeSet Design Manual MAN-C0002-03. https://www.nanostring.com/download_file/view/247/3779. Accessed 25 June 2019
Raj A, van den Bogaard P, Rifkin SA et al (2008) Imaging individual mRNA molecules using multiple singly labeled probes. Nat Methods 5(10):877–879. https://doi.org/10.1038/nmeth.1253
Geiss GK, Bumgarner RE, Birditt B et al (2008) Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 26(3):317–325
Nanostring User manual: PlexSet™ Reagents for Gene Expression MAN-10040-05. https://www.nanostring.com/download_file/view/972/3778. Accessed 25 June 2019
Nuovo GJ, Elton TS, Nana-Sinkam P et al (2009) A methodology for the combined in situ analyses of the precursor and mature forms of microRNAs and correlation with their putative targets. Nat Protoc 4(1):107–115. https://doi.org/10.1038/nprot.2008.215
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Zollinger, D.R., Lingle, S.E., Sorg, K., Beechem, J.M., Merritt, C.R. (2020). GeoMx™ RNA Assay: High Multiplex, Digital, Spatial Analysis of RNA in FFPE Tissue. In: Nielsen, B.S., Jones, J. (eds) In Situ Hybridization Protocols . Methods in Molecular Biology, vol 2148. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0623-0_21
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DOI: https://doi.org/10.1007/978-1-0716-0623-0_21
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-0623-0
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