ViewRNA Cell Plus Assay

Ultra-Sensitive RNA and Protein Detection

ViewRNA ISH Cell and Cell Plus Assays utilize RNA FISH, a powerful technique that combines fluorescence ISH (FISH) and sequential branched-DNA (bDNA) amplification technique to visualize RNA with single-molecule sensitivity along with protein in individual cells. ViewRNA ISH Cell Assay (RNA detection) and Cell Plus Assays (RNA and protein detection) are increasingly important for tracking RNA and protein and understanding the relationship between transcription and translation at the single cell level–technologies that are fundamental to a viral research and a broad range of biological application areas.

Order your ViewRNA ISH Cell or Cell Plus Assay  Find SARS-CoV-2 probes

ViewRNA ISH Cell and Cell Plus Assays offer:

  • Visualization of RNA (ISH Cell Assay) or simultaneous visualization or RNA and protein (Cell Plus Assay) through fluorescence in situ hybridization
  • Single mRNA molecule detection without RNA isolation, reverse transcription, or PCR amplification
  • Visualization of targets not easily localized using other technologies (see also ViewRNA Tissue Assay for hard to work with tissue samples)

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ViewRNA ISH Cell Assays using bDNA technology

There are four parts to the ViewRNA ISH Cell and Cell Plus assay workflow (Figure 1). In the first step, the sample is fixed, permeabilized, and prepared to allow target accessibility (Note: For ViewRNA Cell Plus Assay antibody staining should occur prior to in situ hybridization. It is recommended to use antibodies that have been tested for assay compatibility using ViewRNA Cell Plus Fixation/Permeabilization Buffer Set–see table below for antibodies that have been tested for compatibility). In Step 2, a proprietary, target-specific Probe Set of approximately 20 oligonucleotide pairs hybridizes to each target mRNA (Table 1). Signal amplification is achieved through specific hybridization of adjacent oligonucleotide pairs to bDNA structures (Step 3), which provides much higher specificity of signal than traditional Stellaris FISH assays. ViewRNA Assays have proven successful for detection of RNA with a length as small as 20 nucleotides, which is particularly useful when target RNA length is very small, such as microRNA, circular RNA, splice variants, etc.

Figure 1. Overview of ViewRNA ISH Cell Assay workflow.

Overview of ViewRNA ISH cell assay workflow
 Click image to enlarge

Signal amplification (Step 3) using bDNA technology is achieved via a series of sequential hybridization steps that require Pre-Amplifier molecules, Amplifier molecules and Fluorescent Label Probes that all hybridize to each other form a fully assembled signal amplification 'tree', which has ~400 binding sites for each Label Probe, resulting in an ~8,000-fold amplification for each individual transcript. Target mRNAs are visualized and detected (Step 4) using a standard fluorescent microscope such as an EVOS Cell Imaging System or with the required filter sets (see Ordering Information, Step 1).  For accurate and quantitative detection of ViewRNA spots per cell, or intensity of spots, a high-content imager such as a CellInsight High-Content Screening Instrument can also be used for 96-well, 384-well, or 1536-well plate high-content format.

Note for ViewRNA Cell Plus Assay: As with ViewRNA ISH Cell assay, the ViewRNA Cell Plus assay uses ISH bDNA technology, but has been formatted and optimized for use with antibody-based protein detection to simultaneously visualize RNA and protein in individual cells. The ViewRNA Cell Plus assay enables detection of up to four RNA targets in combination with immunophenotyping for cell surface or intracellular proteins using both indirect and direct immunocytochemistry. Antibody compatibility with this assay must be determined using either the ViewRNA Cell Plus Fixation/Permeabilization Buffer Set–see table below for antibodies that have been tested for compatibility. As with any immunocytochemistry experiment, antibodies should be titrated for optimal performance.

Table 1. Probe sets available for ViewRNA ISH Cell and Cell Plus Assays.

Probe setFluorescent labelViewRNA ISH Cell Assay compatibility
Type 1Alexa Fluor 546 ISH Cell and Cell Plus Assays
Type 4Alexa Fluor 488ISH Cell and Cell Plus Assays
Type 6Alexa Fluor 647 ISH Cell and Cell Plus Assays
Type 10Alexa Fluor 750ISH Cell and Cell Plus Assays

 

ViewRNA ISH Cell and Cell Plus Assays at-a-glance

 ISH Cell AssayCell Plus Assay
Use
  • Detect single RNA molecules at single-cell resolution and quantify gene expression with a limit of detection of single RNA.
  • Visualization of RNA (Cell Assay) or RNA and protein (Cell Plus Assay) through fluorescence in situ hybridization
  • Detection of Micro-RNA, circular RNA, exon junctions/splice variants, gene fusion, gene knockout, pre-miRNA, gene editing/CRISPR, CAR-T cell validation and detection
DifferencesOptimized for cultured cells or circulating tumor cells (CTCs)Compatible with multiplex mRNA detection by immunocytochemistry (immunostaining)
Sample typeOptimized for detection of mRNA in cell culture and circulating tumor cells (CTCs)
  • Compatible with detection of mRNA in cell culture and circulating tumor cells (CTCs)
  • Compatible with tissue and embryo, with minor modifications to the protocol
Compatible
  • Coverslips mounted on slides 
  • 96-well compatible
  • High content kits available for 384-well plates
  • Coverslips mounted on slides 
  • 96-well compatible
  • ICC/IF compatible (1 to 2-plex protein)
Not compatibleNot ICC/IHC compatibleNot compatible with 384-well plates
Plex level and fluorophore options
  • 1 to 4-plex RNA
     
  • Alexa Fluor 488
  • Alexa Fluor 546
  • Alexa Fluor 647
  • Alexa Fluor 750
  • 1 to 4-plex RNA and one or more protein targets, or any combination of RNA and protein as long as the fluorescence readout is optically distinct
     
  • Alexa Fluor 488
  • Alexa Fluor 546
  • Alexa Fluor 647
  • Alexa Fluor 750
Instrumentation
  • Hybridization oven
  • Fluorescence or microscope/scanner
  • Hybridization oven
  • Fluorescence or microscope/scanner

Need to assay tissue samples? A separate kit has been developed for assaying tissue. Learn more about the ViewRNA ISH Tissue Assay which can be run as a chemiluminescent or fluorescent assay.
 

Featured ViewRNA ISH Cell Assay applications

Single RNA molecule visualization using ViewRNA ISH Cell Assay

Visualize single RNA molecules at the single cell level

Simultaneous visualization of protein and RNA targets with ViewRNA Cell Plus

Simultaneous visualization of protein and RNA targets with ViewRNA Cell Plus


Combining FISH and ICC with the ViewRNA Cell Plus Assay

Combining FISH and ICC with the ViewRNA Cell Plus Assay

Identification of breast cancer CTCs using the ViewRNA ISH Cell Assay

Figure 1. Identification of breast cancer CTCs from human bone marrow using the ViewRNA ISH Cell Assay.A putative breast cancer tumor cell from a bone marrow sample was fixed and analyzed using the ViewRNA ISH Cell Assay for ERBB2 (green), pan-keratin panel (cytokeratin 8, 18, and 19) (yellow), and BACT (purple). The technology was used to identify the only two such CTCs on the slide. Data courtesy of Radium Hospital, Oslo, Norway.

HeLa

Visualization and quantitation of transcript heterogeneity in cell lines using the ViewRNA ISH Cell Assay

SKBR3

Visualization and quantitation of transcript heterogeneity in cell lines using the ViewRNA ISH Cell Assay

Figure 2. Visualization and quantitation of transcript heterogeneity in cell lines using the ViewRNA ISH Cell Assay. Multiplex analysis of Her2 mRNA (green) and control 18S rRNA (red) in HeLa cells and SKBR3 cells. Nuclei were stained with DAPI (blue) and visualized by fluorescence microscopy.
 

Time course of gene expression at the single-cell level using the ViewRNA ISH Cell Assay

Figure 3. Time course of gene expression after cytokine induction, at the single-cell level using the ViewRNA ISH Cell Assay. HeLa cells were treated with PMA for 0–8 hours. Then the cells were fixed and analyzed using the ViewRNA ISH Cell Assay for induction of IL-6 (red) and IL-8 expression (green). Nuclei were stained with DAPI (blue). The results show post-induction peaks at 1–2 hours for IL-6 and 4 hours for IL-8. Furthermore, the assay reveals the heterogeneity of individual cell responses.

Detection of H1N1 influenza A RNA in cells using the ViewRNA ISH Cell Assay

Figure 4. Detection of H1N1 Influenza A RNA migration in cells using the ViewRNA ISH Cell Assay. Nuclear translocation of viral RNA is necessary for replication of the influenza virus. Murine embryonic fibroblasts (MEFs) were incubated on ice with influenza A virus (H1N1, PR8 strain). At time 0, virus was removed and medium (warmed to 37°C) was added. At the indicated time points after warming, the cells were fixed and processed using the ViewRNA ISH Cell Assay Kit with a probe set against the nucleoprotein (NP) viral genomic segment (green). Nuclei are stained blue with DAPI. Time dependent nuclear translocation of the NP genome is seen at 90 min post-infection, and by 180 min the export of newly synthesized viral genomes from the nuclei to the cytosol is also observed. Data courtesy of Dr. Abraham L Brass, Ragon Institute of MGH, MIT, and Harvard.

Multiplexing of viral RNA and mRNA in Huh7 cells using View RNA Assays

Figure 5. Multiplexing of viral RNA and mRNA in Huh7 cells. Multiplex fluorescence in situ detection of HCV genomic viral RNA (green) and 18S rRNA (red) in Huh7 cells lacking (–HCV) or containing (+HCV) a hepatitis C virus (HCV) replicon. Nuclei are stained with DAPI (blue).

Multiplex analysis of miRNA and mRNA expression levels using the ViewRNA Cell Assay.

Figure 6. Multiplex analysis of miRNA and mRNA of various expression levels using the ViewRNA Cell Assay. Simultaneous detection of miRNA (Fast Red substrate, Cy3/550 channel) and mRNA (in FITC channel or in Cy7 channel) expression in any given cell. Shown are duplex assays showing expression of different miRNA and mRNA targets in HeLa cells. HPRT, ACTB, and PPIB were detected in the FITC channel, and GAPD was detected in the Cy7 channel. Based on known expression levels, the data confirm accurate and precise study and visualization of miRNA and mRNA of various expression levels, where one distinct spot corresponds to one molecule of an miRNA or mRNA target.

Compound dose-response profiling

Visualization and quantitation of cultured cells using the ViewRNA Cell Assay for high-content screening

Figure 7. Visualization and quantitation of induction in cultured cells using the ViewRNA Cell Assay for high-content screening. HeLa cells were cultured in a 384-well clear bottom plate and treated with various concentrations of PMA. Following a 4-hour PMA treatment, cells were fixed and analyzed for IL-8 expression (green). Nuclei were stained with DAPI (blue).

Compound screening with multiple markers

Multiplex analysis of compound screens using the ViewRNA Cell Assay for high-content screening

Figure 8. Multiplex analysis of compound screens using the ViewRNA Cell Assay for high-content screening. Individual images show nuclei and RNA targets: gene 1, gene 2, and gene 3. The merged image shows simultaneous detection of the three RNA targets. Nuclei were stained with DAPI (blue). Data courtesy of Dr. Dan Garza, Proteostasis, Inc.

Ordering information

Follow the five steps below to begin your ViewRNA ISH Cell or Cell Plus Assay

InstrumentSpecification
Fluorescent microscope or high-content screening instrument

Dependent upon fluorophores used in your experiment. Recommendations per fluorophore are listed

Alexa Fluor 488
Ex/Em: 490/525
Suggested filter: FITC

Alexa Fluor 546
Ex/Em: 556/573
Suggested filter: TRITC 
Alexa Fluor 647
Ex/Em: 650/665
Suggested filter: Cy5
Alexa Fluor 750
Ex/Em: 740/775
Suggested filter: Cy7
IncubatorCapable of maintaining temperature at 40 ± 1°C
Metal heat blockFor 1.5 mL microcentrifuge tube, placed inside the validated incubator
Temperature validation kitUse a calibrated thermometer to verify the incubator temperature

Instructions: Choose one assay from the two available kits in step 1. Following selection in step 1 choose any accessory kits and reagents that might be needed for the chosen assay. Make sure to select from the appropriate table of accessory kits and reagents for the chosen assay.

  • A maximum of four types of probe sets are currently available for RNA detection.
  • Select different Probe Sets depending on the RNA expression level and kit selected.
  • For multiplex analysis with immunolabeling of both cell-surface and intracellular proteins, find fluorophore-conjugated antibodies.
Probe Set typeExpression level of target RNAFluorochrome labelLaser excitation wavelength (nm)Suggested bandpass filter (nm)
Type 1LowAlexa Fluor 546561610/20
Type 4Medium-to-highAlexa Fluor 488488530/30
Type 6LowAlexa Fluor 647633, 635660/20
Type 10Medium-to-highAlexa Fluor 750633, 635780/60

Search for available (in stock) Probe Sets

Custom Branched DNA Probe Set Tool

Not finding what you’re looking for? Order Custom Branched DNA Probe Sets for PrimeFlow, QuantiGene Singleplex, and ViewRNA Cell and Tissue assays using the custom ordering tool if your target of interest is not found in our web catalog.

Customer bDNA Probe Set Ordering Tool

  • Positive control—use positive control probe sets in every experiment to ensure proper assay performance: Housekeeping genes such as ACTB, GAPD, PPIB or UBC probe sets are commonly used as positive controls.  For micro-RNA experiments, probe set for Let7a works for many sample types. 
  • Negative control—such as samples with the target specific probe omitted, or samples labeled with a probe against a target not expressed in the cells of interest (e.g., dapB, a bacterial gene); highly recommended.
  • Search probes used for positive and negative controls in probe search tool in Step 4, for the species.  Please note that ViewRNA probes are very specific, and probe set for one species, commonly does not detect targets in another species.

Antibody compatibility for ViewRNA Cell Plus Assay

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.

SpeciesSpecificityCloneCat. No.ConjugateConcentration tested
Recommended
Human, Mouse, Rat, Canine, Bovine, Guinea Pig, Pig, HorseActinHHF3514-6496Purified10 µg/mL
Human, Mouse, Rat, Canine, Bovine, Guinea Pig, Pig, HorseActinHHF3553-6496Alexa Fluor 48830 µg/mL
HumanAFP (alpha fetoprotein)1E814-9499Purified5 µg/mL
HumanAHRFF339914-9854Purified20 µg/mL
HumanALDH1L17G814-9595Purified1 µg/mL
Humanalpha-Catenin1G5MA1-2000Purified5 µg/mL
HumanArginase-1sl6arg53-9779Alexa Fluor 48820 µg/mL
HumanArginase-1sl6arg50-9779eFluor 6605 µg/mL
HumanArginase-1sl6arg14-9779Purified5 µg/mL
Human, MouseB220RA3-6B253-0452Alexa Fluor 48820 µg/mL
Human, Mouse, Rat, BovineBeta tubulin class III2G10-TB314-4510Purified5 µg/mL
Human, Mouse, Rat, BovineBeta tubulin class III2G10-TB353-4510Alexa Fluor 48810 µg/mL
Human, Mouse, Rat, BovineBeta tubulin class III2G10-TB350-4510eFluor 66010 µg/mL
Human, Mouse, Rat, BovineBeta tubulin class III2G10-TB341-4510eFluor 5705 µg/mL
HumanBrachyuryX1AO214-9770Purified5 µg/mL
MouseCD317A250-0032eFluor 66010 µg/mL
Human, Mouse and CanineCD324, E-CadherinDECMA-153-3249Alexa Fluor 48820 µg/mL
MouseCD4RM4-513-0042Biotin10 µg/mL
MouseCD4RM4-541-0042eFluor 5705 µg/mL
MouseCD853-6.750-0081eFluor 66010 µg/mL
MouseCD8a4SM1513-0808Biotin10 µg/mL
MouseCD11bM1/7050-0112eFluor 6605 µg/mL
Human, Mouse, Rat, ChickenConnexin 322A14-9759Purified5 µg/mL
HumanCox IVIB52C31H1014-9775Purified1 µg/mL
HumanCoxIVIB52C31H1053-9775Alexa Fluor 48810 µg/mL
HumanCytokeratin 7LP5K14-9005Purified10 µg/mL
Human, Mouse, RatEarly endosome antigen 11G1114-9114Purified1 µg/mL
HumanCD326 EpCAM1B714-9326Purified10 µg/mL
HumanER alphatbykt14-9740Purified1 µg/mL
HumanFibronectinFN-314-9869Purified10 µg/mL
Human, Mouse, Rat, PigGFAPGA553-9892Alexa Fluor 48810 µg/mL
HumanGolgin-97CDF414-9767Purified1 µg/mL
Human, Mouse, RatGrim-191A814-9937Purified5 µg/mL
HumanHer2MJD214-9757Purified5 µg/mL
Human, MouseHer4HFR114-9687Purified10 µg/mL
HumanKi-6720Raj142-5699eFluor 6151 µg/mL
HumanKi-6720Raj150-5699eFluor 66020 µg/mL
HumanKi-6720Raj111-5699FITC10 µg/mL
HumanKi-6720Raj114-5699Purified1 µg/mL
HumanKi-6720Raj113-5699Biotin5 µg/mL
HumanKi-6720Raj141-5699eFluor 5705 µg/mL
Mouse, RatKi-67SolA1541-5698eFluor 5700.5 µg/mL
Mouse, RatKi-67SolA1550-5698eFluor 66010 µg/mL
Mouse, RatMASH124B72D1114-5794Purified20 µg/mL
Human, Mouse, Rat, DogMyogeninF5D53-5643Alexa Fluor 4885 µg/mL
Human, Mouse, Rat, DogMyogeninF5D14-5643Purified1 µg/mL
Human, Mouse, Rat, etc.MyosinMF2053-6503Alexa Fluor 4885 µg/mL
Human, Mouse, Rat, etc.MyosinMF2050-6503eFluor 66010 µg/mL
MouseNanogeBioMLC-5150-5761eFluor 66010 µg/mL
MouseNanogeBioMLC-5153-5761Alexa Fluor 48810 µg/mL
HumanNestin10C253-9843Alexa Fluor 48810 µg/mL
HumanNestin10C214-9843Purified5 µg/mL
Human, MouseOCT3/4EM9241-5841eFluor 5705 µg/mL
Human, Mouse, RatPCNAPC1014-9910Purified1 µg/mL
Human, Mouse, RatPhospho-ATM10H11.E1214-9046Purified10 µg/mL
Human, MousePhospho-ERK1/2MILAN8R14-9109Purified5 µg/mL
Human, MousePhospho-H2AXCR55T3314-9865Purified5 µg/mL
HumanProgesterone receptorKMC91213-9764Biotin2.5 µg/mL
HumanProgesterone receptorKMC91214-9764Purified1 µg/mL
HumanProgesterone receptorKMC91250-9764eFluor 66020 µg/mL
HumanSnail120C853-9859Alexa Fluor 48820 µg/mL
HumanSnail120C814-9859Purified5 µg/mL
Human, MouseSox2Btjce41-9811eFluor 5705 µg/mL
Human, MouseSox2Btjce50-9811eFluor 66010 µg/mL
Human, MouseSox2Btjce14-9811Purified5 µg/mL
Human, MouseSox9GMPR953-9765Alexa Fluor 48810 µg/mL
Human, MouseSox9GMPR914-9765Purified1 µg/mL
HumanSurvivinSTLALYV53-9176Alexa Fluor 48810 µg/mL
Human, Mouse, RatThrombospondin-1A6.114-9756Purified1 µg/mL
Human, Mouse, Rat, etc.TubulinDM1A42-4502eFluor 6151 µg/mL
Human, Mouse, Rat, etc.TubulinDM1A53-4502Alexa Fluor 4885 µg/mL
Human, Mouse, Rat, etc.TubulinDM1A14-4502Purified5 µg/mL
Human, Mouse, Rat, etc.TubulinDM1A13-4502Biotin10 µg/mL
Human, Rat, Chicken, CanineVimentinV941-9897eFluor 5701 µg/mL
Human, Rat, Chicken, CanineVimentinV950-9897eFluor 6605 µg/mL
Human, Mouse, Rat, AvianVinculin7F941-9777eFluor 57010 µg/mL
Human, Mouse, Rat, AvianVinculin7F953-9777Alexa Fluor 48820 µg/mL
Human, Mouse, Rat, AvianVinculin7F914-9777Purified5 µg/mL
Mouse, Rat, CanineZO-1R26.4C41-9776eFluor 5701 µg/mL
HumanCD3OKT350-0037eFluor 6600.12 µg/ml
HumanCD4N1UG041-2444eFluor 57020 µg/mL
MouseFoxP3FJK-16s53-5773Alexa Fluor 48810 µg/mL
Not recommended
Human, MouseB220RA3-6B250-0452eFluor 66010 µg/mL
MouseCD4GK1.553-0041Alexa Fluor 48810 µg/mL
MouseCD4GK1.550-0041eFluor 6605 µg/mL
Mouse, RatNestinRat-40114-5843Purified10 µg/mL
Mouse, Human, Rat, etc.CytokeratinAE1/AE342-9003eFluor 6155 µg/mL
Mouse, Human, Rat, etc.CytokeratinAE1/AE341-9003eFluor 5701 µg/mL
Mouse, Human, Rat, etc.CytokeratinAE1/AE353-9003Alexa Fluor 4881 µg/mL
Human, Mouse, Rat, ChickenConnexin 322A53-9759Alexa Fluor 4881 µg/mL
Human, Mouse, RatConnexin 431A14-4759Purified5 µg/mL
HumanHeat shock protein 27STRSN14-9112Purified1 µg/mL
MouseMHC Class IIM5/114.15.211-5321FITC20 µg/mL
Human, Mouse, RatPCNAPC1042-9910eFluor 6150.5 µg/mL
HumanCD8AMC90850-0008eFluor 6605 µg/mL

Do you need SARS-CoV-2 probes?

Use the step-by-step ordering information above to order your kit and probes, including controls. In step 4, if you need SARS-CoV-2 probes, see the table below, and follow the ‘How to order SARS-CoV-2 probes’ instructions provided.

DyeTypeScaleCatalog
number
Assay ID
AF5461120 μLCVX-06VPNKRHH
AF5461440 μLCVX-01VPNKRHH
AF54611440 μLCVX-02VPNKRHH
AF4884120 μLCVX-06VP9HHYZ
AF4884440 μLCVX-01VP9HHYZ
AF48841440 μLCVX-02VP9HHYZ
AF6476120 μLCVX-06VPAAADR
AF6476440 μLCVX-01VPAAADR
AF64761440 μLCVX-02VPAAADR
AF75010120 μLCVX-06VPCE3XN
AF75010440 μLCVX-01VPCE3XN
AF750101440 μLCVX-02VPCE3XN

ViewRNA ISH Cell and Cell Plus Probe Sets were designed using the NCBI strain and sequence from Accession Number LC521925, and cover multiple regions of the viral transcript. Find in the table below the binding sites and transcript region for the probe sets included in the assay. If you need a custom probe designed for a specific RNA region use the Custom Branched DNA Probe Set Tool.

 Position (5' to 3')Region
1705-1676orf1ab
24356-5607orf1ab
312808-13942orf1ab
420571-21759orf1ab
526646-27763M
628212-29152orf8 and N
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