ViewRNA Cell Plus Assay

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

The complex interactions between transcription, translation, and post-translational modifications are hidden from view in typical endpoint assays that measure either RNA or protein levels. While in situ hybridization (ISH) provides a method for examining the levels of specific RNA transcripts in individual cells, and immunocytochemistry (ICC) utilizes antibodies to visualize the localization of specific proteins (and protein modifications), the ability to simultaneously observe RNA and protein in a single cell has been thwarted by the incompatibility of ICC and ISH protocols.

Further complicating this analysis, traditional ISH methods lack sensitivity for detecting low-abundance RNA species and have limited multiplexing capability that further constrain its compatibility with other cell assays.

Invitrogen ViewRNA Cell Plus Assay is a novel method that combines ICC with the Invitrogen ViewRNA technology, a proprietary fluorescence ISH (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.

Technology overview

FISH is a powerful technique that enables the specific detection of target RNA localization in fixed cells. Traditional FISH techniques using large oligonucleotide sequences labeled with one to five fluorophores are generally limited by high background and low sensitivity due to non-specific binding and insufficient signal amplification.

ViewRNA Cell Assays incorporate a proprietary probe set design and bDNA signal amplification technology. A target-specific Probe Set of approximately 20 oligonucleotide pairs hybridizes to the target RNA of interest. Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures, which are formed by Pre-amplifiers, Amplifiers, and fluorochrome-conjugated Label Probes. This methodology results in greater specificity, lower background, and higher signal-to-noise ratios. ViewRNA Cell Assays are the most sensitive and specific RNA ISH methods on the market.

In comparison, to create images with similar discernible spots, traditional FISH techniques can require a 600-times longer exposure and a 100-times greater camera gain than ViewRNA ISH Cell Assays. Furthermore, under equivalent imaging conditions, ViewRNA ISH Cell Assay is 100-times brighter, creating a minimum two- to three-times higher signal-to-noise ratio (Nat Methods 10:1127 (2013) ).

Search for available (in stock) probe sets

Assay kits and supporting products

The ViewRNA Cell Plus Assay contains all the reagents needed to conduct the assay. Positive control probes and target probes for genes of interest are sold separately.

Antibody compatibility

To date, we have evaluated the performance of more than 100 ICC-compatible antibodies (comprised of a mix of unconjugated/purified, biotinylated, and fluorophore-conjugated format; see table below) for use in ViewRNA Cell Plus Assay. To accomplish this, antibody performance was tested either in the context of the full assay (antibody staining and RNA detection) or by using Invitrogen ViewRNA Cell Plus Fixation/Permeabilization Buffer Set (Cat. No. 00-19001) according to the antibody staining portion of the protocol. We found that 95% of the antibodies tested that perform optimally with formaldehyde-fixed cells also perform well with the ViewRNA Cell Plus Assay. Antibodies that require methanol fixation were generally found to be unsuitable for this assay.

In this evaluation, we have assessed a broad panel of antibodies with specificities against structural proteins, transcription factors, organelles, and surface markers. Our results show that signal intensity and resolution of detail is maintained with ViewRNA Cell Plus Assay compared to our standard immunocytochemistry protocol. Based on our studies, we conclude that antibodies which are suitable for ICC or immunohistochemistry (IHC) (i.e., optimized for formaldehyde fixation) are likely to be compatible with ViewRNA Cell Plus Assay. Nevertheless, we recommend using ViewRNA Cell Plus Fixation/Permeabilization Buffer Set (Cat. No. 00-19001) to establish acceptable performance criteria prior to running the complete ViewRNA Cell Plus Assay.

Species Specificity Clone Cat. No. Conjugate Concentration tested
Recommended
Human, Mouse, Rat, Canine, Bovine, Guinea Pig, Pig, Horse Actin HHF35 14-6496 Purified 10 µg/mL
Human, Mouse, Rat, Canine, Bovine, Guinea Pig, Pig, Horse Actin HHF35 53-6496 Invitrogen Alexa Fluor 488 30 µg/mL
Human AFP (alpha fetoprotein) 1E8 14-9499 Purified 5 µg/mL
Human AHR FF3399 14-9854 Purified 20 µg/mL
Human ALDH1L1 7G8 14-9595 Purified 1 µg/mL
Human alpha-Catenin 1G5 14-9120 Purified 5 µg/mL
Human Arginase-1 sl6arg 53-9779 Alexa Fluor 488 20 µg/mL
Human Arginase-1 sl6arg 50-9779 Invitrogen eFluor 660 5 µg/mL
Human Arginase-1 sl6arg 14-9779 Purified 5 µg/mL
Human, Mouse B220 RA3-6B2 53-0452 Alexa Fluor 488 20 µg/mL
Human, Mouse, Rat, Bovine Beta tubulin class III 2G10-TB3 14-4510 Purified 5 µg/mL
Human, Mouse, Rat, Bovine Beta tubulin class III 2G10-TB3 53-4510 Alexa Fluor 488 10 µg/mL
Human, Mouse, Rat, Bovine Beta tubulin class III 2G10-TB3 50-4510 eFluor 660 10 µg/mL
Human, Mouse, Rat, Bovine Beta tubulin class III 2G10-TB3 41-4510 Invitrogen eFluor 570 5 µg/mL
Human Brachyury X1AO2 14-9770 Purified 5 µg/mL
Mouse CD3 17A2 50-0032 eFluor 660 10 µg/mL
Human, Mouse and Canine CD324, E-Cadherin DECMA-1 53-3249 Alexa Fluor 488 20 µg/mL
Mouse CD4 RM4-5 13-0042 Biotin 10 µg/mL
Mouse CD4 RM4-5 41-0042 eFluor 570 5 µg/mL
Mouse CD8 53-6.7 50-0081 eFluor 660 10 µg/mL
Mouse CD8a 4SM15 13-0808 Biotin 10 µg/mL
Mouse CD11b M1/70 50-0112 eFluor 660 5 µg/mL
Human, Mouse, Rat, Chicken Connexin 32 2A 14-9759 Purified 5 µg/mL
Human, Mouse, Rat Connexin 43 1A 50-4759 eFluor 660 10 µg/mL
Human Cox IV IB52C31H10 14-9775 Purified 1 µg/mL
Human Cox IV IB52C31H10 41-9775 eFluor 570 1 µg/mL
Human CoxIV IB52C31H10 53-9775 Alexa Fluor 488 10 µg/mL
Human Cytokeratin 7 LP5K 14-9005 Purified 10 µg/mL
Human, Mouse, Rat Early endosome antigen 1 1G11 14-9114 Purified 1 µg/mL
Human CD326 EpCAM 1B7 14-9326 Purified 10 µg/mL
Human ER alpha tbykt 14-9740 Purified 1 µg/mL
Human Fibronectin FN-3 14-9869 Purified 10 µg/mL
Human, Mouse, Rat, Pig GFAP GA5 53-9892 Alexa Fluor 488 10 µg/mL
Human Golgin-97 CDF4 53-9767 Alexa Fluor 488 10 µg/mL
Human Golgin-97 CDF4 50-9767 eFluor 660 5 µg/mL
Human Golgin-97 CDF4 14-9767 Purified 1 µg/mL
Human, Mouse, Rat Grim-19 1A8 14-9937 Purified 5 µg/mL
Human Her2 MJD2 14-9757 Purified 5 µg/mL
Human Her2 MJD2 41-9757 eFluor 570 5 µg/mL
Human, Mouse Her4 HFR1 14-9687 Purified 10 µg/mL
Human Ki-67 20Raj1 42-5699 Invitrogen eFluor 615 1 µg/mL
Human Ki-67 20Raj1 50-5699 eFluor 660 20 µg/mL
Human Ki-67 20Raj1 11-5699 FITC 10 µg/mL
Human Ki-67 20Raj1 14-5699 Purified 1 µg/mL
Human Ki-67 20Raj1 13-5699 Biotin 5 µg/mL
Human Ki-67 20Raj1 41-5699 eFluor 570 5 µg/mL
Mouse, Rat Ki-67 SolA15 41-5698 eFluor 570 0.5 µg/mL
Mouse, Rat Ki-67 SolA15 50-5698 eFluor 660 10 µg/mL
Hu, m, Rt MAP2 AP20 41-9763 eFluor 570 5 µg/mL
Mouse, Rat MASH1 24B72D11 14-5794 Purified 20 µg/mL
Human, Mouse, Rat, Dog Myogenin F5D 53-5643 Alexa Fluor 488 5 µg/mL
Human, Mouse, Rat, Dog Myogenin F5D 14-5643 Purified 1 µg/mL
Human, Mouse, Rat, etc. Myosin MF20 53-6503 Alexa Fluor 488 5 µg/mL
Human, Mouse, Rat, etc. Myosin MF20 50-6503 eFluor 660 10 µg/mL
Mouse Nanog eBioMLC-51 50-5761 eFluor 660 10 µg/mL
Mouse Nanog eBioMLC-51 53-5761 Alexa Fluor 488 10 µg/mL
Human Nestin 10C2 53-9843 Alexa Fluor 488 10 µg/mL
Human Nestin 10C2 14-9843 Purified 5 µg/mL
Human, Mouse OCT3/4 EM92 41-5841 eFluor 570 5 µg/mL
Human, Mouse, Rat PCNA PC10 14-9910 Purified 1 µg/mL
Human, Mouse, Rat Phospho-ATM 10H11.E12 14-9046 Purified 10 µg/mL
Human, Mouse Phospho-ERK1/2 MILAN8R 14-9109 Purified 5 µg/mL
Human, Mouse Phospho-H2AX CR55T33 14-9865 Purified 5 µg/mL
Human Progesterone receptor KMC912 13-9764 Biotin 2.5 µg/mL
Human Progesterone receptor KMC912 14-9764 Purified 1 µg/mL
Human Progesterone receptor KMC912 50-9764 eFluor 660 20 µg/mL
Human Snail1 20C8 53-9859 Alexa Fluor 488 20 µg/mL
Human Snail1 20C8 14-9859 Purified 5 µg/mL
Human, Mouse Sox2 Btjce 41-9811 eFluor 570 5 µg/mL
Human, Mouse Sox2 Btjce 50-9811 eFluor 660 10 µg/mL
Human, Mouse Sox2 Btjce 14-9811 Purified 5 µg/mL
Human, Mouse Sox9 GMPR9 53-9765 Alexa Fluor 488 10 µg/mL
Human, Mouse Sox9 GMPR9 14-9765 Purified 1 µg/mL
Human Survivin STLALYV 53-9176 Alexa Fluor 488 10 µg/mL
Human, Mouse, Rat Thrombospondin-1 A6.1 14-9756 Purified 1 µg/mL
Human, Mouse, Rat, etc. Tubulin DM1A 42-4502 eFluor 615 1 µg/mL
Human, Mouse, Rat, etc. Tubulin DM1A 53-4502 Alexa Fluor 488 5 µg/mL
Human, Mouse, Rat, etc. Tubulin DM1A 14-4502 Purified 5 µg/mL
Human, Mouse, Rat, etc. Tubulin DM1A 13-4502 Biotin 10 µg/mL
Human, Rat, Chicken, Canine Vimentin V9 41-9897 eFluor 570 1 µg/mL
Human, Rat, Chicken, Canine Vimentin V9 50-9897 eFluor 660 5 µg/mL
Human, Mouse, Rat, Avian Vinculin 7F9 41-9777 eFluor 570 10 µg/mL
Human, Mouse, Rat, Avian Vinculin 7F9 53-9777 Alexa Fluor 488 20 µg/mL
Human, Mouse, Rat, Avian Vinculin 7F9 14-9777 Purified 5 µg/mL
Mouse, Rat, Canine ZO-1 R26.4C 41-9776 eFluor 570 1 µg/mL
Human CD3 OKT3 50-0037 eFluor 660 0.12 µg/ml
Human CD4 N1UG0 41-2444 eFluor 570 20 µg/mL
Mouse FoxP3 FJK-16s 53-5773 Alexa Fluor 488 10 µg/mL
Not recommended
Human, Mouse B220 RA3-6B2 50-0452 eFluor 660 10 µg/mL
Mouse CD4 GK1.5 53-0041 Alexa Fluor 488 10 µg/mL
Mouse CD4 GK1.5 50-0041 eFluor 660 5 µg/mL
Mouse, Rat Nestin Rat-401 14-5843 Purified 10 µg/mL
Mouse, Human, Rat, etc. Cytokeratin AE1/AE3 42-9003 eFluor 615 5 µg/mL
Mouse, Human, Rat, etc. Cytokeratin AE1/AE3 41-9003 eFluor 570 1 µg/mL
Mouse, Human, Rat, etc. Cytokeratin AE1/AE3 53-9003 Alexa Fluor 488 1 µg/mL
Human, Mouse, Rat, Chicken Connexin 32 2A 53-9759 Alexa Fluor 488 1 µg/mL
Human, Mouse, Rat Connexin 43 1A 14-4759 Purified 5 µg/mL
Human Heat shock protein 27 STRSN 14-9112 Purified 1 µg/mL
Mouse MHC Class II M5/114.15.2 11-5321 FITC 20 µg/mL
Human, Mouse, Rat PCNA PC10 42-9910 eFluor 615 0.5 µg/mL
Human CD8 AMC908 50-0008 eFluor 660 5 µg/mL

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