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.
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)
On this page:
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.
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 set||Fluorescent label||ViewRNA ISH Cell Assay compatibility|
|Type 1||Alexa Fluor 546||ISH Cell and Cell Plus Assays|
|Type 4||Alexa Fluor 488||ISH Cell and Cell Plus Assays|
|Type 6||Alexa Fluor 647||ISH Cell and Cell Plus Assays|
|Type 10||Alexa Fluor 750||ISH Cell and Cell Plus Assays|
ViewRNA ISH Cell and Cell Plus Assays at-a-glance
|ISH Cell Assay||Cell Plus Assay|
|Differences||Optimized for cultured cells or circulating tumor cells (CTCs)||Compatible with multiplex mRNA detection by immunocytochemistry (immunostaining)|
|Sample type||Optimized for detection of mRNA in cell culture and circulating tumor cells (CTCs)||
|Not compatible||Not ICC/IHC compatible||Not compatible with 384-well plates|
|Plex level and fluorophore options||
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
Simultaneous visualization of protein and RNA targets with ViewRNA Cell Plus
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.
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.
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.
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.
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).
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
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
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.
Follow the five steps below to begin your ViewRNA ISH Cell or Cell Plus Assay
|Fluorescent microscope or high-content screening instrument||
Dependent upon fluorophores used in your experiment. Recommendations per fluorophore are listed
Alexa Fluor 488
|Alexa Fluor 546
Suggested filter: TRITC
|Alexa Fluor 647
Suggested filter: Cy5
|Alexa Fluor 750
Suggested filter: Cy7
|Incubator||Capable of maintaining temperature at 40 ± 1°C|
|Metal heat block||For 1.5 mL microcentrifuge tube, placed inside the validated incubator|
|Temperature validation kit||Use 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 type||Expression level of target RNA||Fluorochrome label||Laser excitation wavelength (nm)||Suggested bandpass filter (nm)|
|Type 1||Low||Alexa Fluor 546||561||610/20|
|Type 4||Medium-to-high||Alexa Fluor 488||488||530/30|
|Type 6||Low||Alexa Fluor 647||633, 635||660/20|
|Type 10||Medium-to-high||Alexa Fluor 750||633, 635||780/60|
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.
- 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.
|Species||Specificity||Clone||Cat. No.||Conjugate||Concentration tested|
|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||Alexa Fluor 488||30 µg/mL|
|Human||AFP (alpha fetoprotein)||1E8||14-9499||Purified||5 µg/mL|
|Human||Arginase-1||sl6arg||53-9779||Alexa Fluor 488||20 µg/mL|
|Human||Arginase-1||sl6arg||50-9779||eFluor 660||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||eFluor 570||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||41-0042||eFluor 570||5 µg/mL|
|Mouse||CD8||53-6.7||50-0081||eFluor 660||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||Cox IV||IB52C31H10||14-9775||Purified||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, Mouse, Rat, Pig||GFAP||GA5||53-9892||Alexa Fluor 488||10 µg/mL|
|Human, Mouse, Rat||Grim-19||1A8||14-9937||Purified||5 µg/mL|
|Human, Mouse||Her4||HFR1||14-9687||Purified||10 µg/mL|
|Human||Ki-67||20Raj1||42-5699||eFluor 615||1 µg/mL|
|Human||Ki-67||20Raj1||50-5699||eFluor 660||20 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|
|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, 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, 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|
|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|
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.
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|
|6||28212-29152||orf8 and N|
For Research Use Only. Not for use in diagnostic procedures.