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ViewRNA® Cell Plus Assay

Simultaneously visualize RNA with single-molecule sensitivity and protein in single cells

The ability to simultaneously visualize protein and RNA species in individual cells is necessary to address important biological questions. Additionally, studying both protein and RNA can be useful in cases where expression levels vary between mRNA and protein, as well as in studies that require the detection of post-translational modifications. However, development of a combined technique has proved challenging due to the incompatibility of immunocytochemistry (ICC) and fluorescent in situ hybridization (FISH) protocols.

The ViewRNA® Cell Plus Assay is a novel method that combines ICC with the ViewRNA® technology, a proprietary FISH and sequential branched-DNA (bDNA) amplification technique, to visualize RNA with single-molecule sensitivity along with protein in individual cells. This assay enables simultaneous detection of up to three RNA targets in combination with immunophenotyping for cell surface and intracellular proteins using both indirect and direct ICC, which allows for in-depth characterization of specific cell subpopulations. The combined visualization of RNA and protein expression at the single-cell level is a valuable tool that has broad implications for advancing research across multiple fields of biology.

  • Analyze sample heterogeneity:phenotyping rat hippocampal primary cell culture data set
  • Golgin 97 eFluor® 660 (green) Ki-67 eFluor® 660 (white)
    Dissociated rat hippocampal primary cells (13 days in vitro) were processed using the ViewRNA® Cell Plus Assay to stain for parvalbumin mRNA (Pvalb type6, blue, VC6-15122) with antibodies for neurons (Anti-MAP2 eFluor® 570, red, cat. no. 41-9763) and astrocytes (Anti-GFAP Alexa Fluor® 488, green, cat. no. 53-9892). Pvalb is detected in a subset of neurons. Nuclei are stained with DAPI (also shown in blue). Data courtesy of the tom Dieck laboratory, Max Planck Institute for Brain Research. Dissociated rat hippocampal primary cells (13 days in vitro) were processed using the ViewRNA® Cell Plus Assay to stain two different mRNAs, one coding for CamK2a (CamK2a Type1, red, VC1-14239) and one coding for Map2 (Mtap2 Type6, blue, VC6-16200) together with an antibody marker for astrocytes (Anti-GFAP Alexa Fluor® 488, green, cat. no. 53-9892). While none of the mRNAs is expressed in astrocytes, Mtap2 is detected in all neurons and CamK2a in a subset. Nuclei are stained with DAPI (also shown in blue). Data courtesy of the tom Dieck laboratory, Max Planck Institute for Brain Research.
  • Track, visualize and detect genomic viral RNA: Zika virus RNA and tubulin protein imaging data set
  • Golgin 97 eFluor® 660 (green)
    Zika virus (MR766m)-infected Vero cells are visualized with the ViewRNA® Cell Plus Assay using ZIKV RNA-specific probe set Type 1 (red, cat. no. VF1-19981) and Anti-alpha Tubulin Alexa Fluor® 488 antibody (green, cat. no. 53-4502). Nuclei are stained with DAPI (blue). Data courtesy of A. Brass laboratory, University of Massachusetts Medical School.
  • Unmask rare cells: circulating tumor cell analysis data set
  • Merge
    RNA + DAPI CK/EpCAM DAPI HER2 mRNA GAPDH mRNA
    Golgin 97 eFluor® 660 (green)
    Ki-67 eFluor® 660 (white) Ki-67 eFluor® 660 (white) Ki-67 eFluor® 660 (white) Ki-67 eFluor® 660 (white) Ki-67 eFluor® 660 (white)
    Detection of SKBR3 cells within mixed blood cell population with the ViewRNA® Cell Plus Assay using ErbB2 mRNA-specific probe set Type 6 (red, cat. no. VA6-12652), GAPDH mRNA-probe set Type 4 (green, cat. no. VA4-10641) and Anti-Human Cytokeratin 7 and Anti-Human CD326 Epithelial cell adhesion molecule purified antibodies followed by Anti-Mouse IgG Alexa Fluor® 546 (yellow, cat. no. 14-9005, 14-9326, A10036). Nuclei are stained with DAPI (blue). Data courtesy of RARECYTE.
  • Single-cell analysis of non-coding RNAs: microRNA expression profiles in breast cancer cell line data set
  • Golgin 97 eFluor® 660 (green)

    Detection of microRNA (miR) expression along with tubulin, Ki-67, and survivin protein with the ViewRNA® Cell Plus Assay in ductal and adenocarcinoma breast cancer cell lines. (Top three rows) miR-145, miR-34a, and miR-143 (white, cat. no. VM1-10252, VM1-10243, VM1-10251) are expressed at similar levels between the different cell lines examined. miR is co-stained with Anti-alpha Tubulin Alexa Fluor® 488 (green, cat. no. 53-4502). Nuclei are stained with DAPI (blue). (Middle three rows) Let-7a and miR-19a (white, cat. no. VM1-10409, VM1-10350) are highly expressed across the different breast cancer cell lines. miR-221 (white, cat. no. VM1-10264) is expressed at very low levels, with MDA-MB-231 cells expressing slightly higher levels than the other cell lines. miR is co-stained with Anti-Ki-67 eFluor® 660 (red, cat. no. 50-5699). Nuclei are stained with DAPI (blue). (Bottom three rows) miR-17 (white, cat. no. VM1-10233) is highly expressed across the different breast cancer cell lines, with SKBR3 cells exhibiting slightly lower expression than the others. miR-30d and miR-107 (white, cat. no. VM1-10241, VM1-10246) is expressed at low levels. miR is co-stained with Anti-survivin Alexa Fluor® 488 (green, cat. no. 53-9176). Nuclei are stained with DAPI (blue).