What are ViewRNA ISH Assays?
Invitrogen ViewRNA ISH Cell and Tissue Assays incorporate branched-DNA (bDNA) amplification technology to visualize RNA with single-molecule sensitivity. bDNA uses a proprietary probe set design and signal amplification technology, offering a unique, robust, and sensitive in situ hybridization assay for RNA localization.
ViewRNA reagents now available
Fast Red and DAB-based ViewRNA tissue staining kits with enhanced sensitivity and sharpness are now available.
Overview of ViewRNA ISH Assays
ViewRNA ISH assay selection guide
Use the table below to find the ViewRNA ISH assay that’s right for your research needs.
| ISH Cell Assay | ISH Cell Plus Assay | ISH Colorimetric Tissue Assay | ISH Fluorescence Tissue Assay | |
|---|---|---|---|---|
| Use |
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| ||
| Differences | Optimized for cultured cells or circulating tumor cells (CTCs) | Compatible with multiplex mRNA detection by immunocytochemistry (immunostaining) | Optimized for fixed tissue sections. Multiplex mRNA detection by immunohistochemistry using colorimetric visualization. Multiplexing up to three RNA targets. | Optimized for fixed tissue sections. Multiplex mRNA detection by immunohistochemistry using fluorescence visualization. Multiplexing up to four RNA targets. |
| Sample type | Optimized for detection of mRNA in cell culture | Optimized to use with:
| ||
| Compatible sample formats |
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| |
| Not compatible | Not ICC/IHC compatible | Not compatible with 384-well plates | Not validated in ICC/IHC | |
| Plex level and readout options | 1 to 4-plex RNA
| 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
| Colorimetric: up to 3-plex Fast Red and Fast Blue substrates are also compatible with traditional TRITC/RFP and CY5.5/CY7 filter sets, respectively, for fluorescent readouts. | Up to 4-plex
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| Instrumentation required |
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| |
ViewRNA ISH probe set types
Probe sets are required for ViewRNA Assays and must be purchased separately. Each probe set type is specific to the detection label and brightfield/fluorescence readout. For multiplexing experiments, it is critical to avoid using more than one of the same probe set type per sample. Use the following table to determine which probe set type is needed for your experiment.
| Probe Set | Expression level of target RNA | Detection Label | Fluorescence Readout |
|---|---|---|---|
| 1 | Low | Alexa Fluor 546 | TRITC/RFP |
| 4 | Medium | Alexa Fluor 488 | FITC/GFP |
| 6 | Low | Alexa Fluor 647 | Deep Red/Cy5 |
| 10 | Abundant | Alexa Fluor 750 | Near infrared/Cy7 |
| Probe Set | Expression level of target RNA | Detection Label | Colorimetric Readout | Fluorescence readout |
|---|---|---|---|---|
| 1 | Low | Fast Red | Red color | TRITC/RFP |
| 4 | Low | DAB | Brown color | Not applicable |
| 6 | Low | Fast Blue | Blue color | Cy5.5 or Cy7 |
| Probe Set | Expression level of target RNA | Detection Label | Fluorescence readout |
|---|---|---|---|
| 1 | Low | Alexa Fluor 594 | Texas Red |
| 1 | Low | Alexa Fluor 546 | TRITC/RFP |
| 4 | Medium | Alexa Fluor 488 | FITC/GFP |
| 6 | Low | Alexa Fluor 647 | Deep Red/Cy5 |
| 10 | Abundant | Alexa Fluor 750 | Near Infrared/Cy7 |
| Probe Set | Expression level of target RNA | Detection Label | Fluorescence Readout |
|---|---|---|---|
| 1 | Low | Alexa Fluor 546 | TRITC/RFP |
| 4 | Medium | Alexa Fluor 488 | FITC/GFP |
| 6 | Low | Alexa Fluor 647 | Deep Red/Cy5 |
| 10 | Abundant | Alexa Fluor 750 | Near infrared/Cy7 |
| Probe Set | Expression level of target RNA | Detection Label | Colorimetric Readout | Fluorescence readout |
|---|---|---|---|---|
| 1 | Low | Fast Red | Red color | TRITC/RFP |
| 4 | Low | DAB | Brown color | Not applicable |
| 6 | Low | Fast Blue | Blue color | Cy5.5 or Cy7 |
| Probe Set | Expression level of target RNA | Detection Label | Fluorescence readout |
|---|---|---|---|
| 1 | Low | Alexa Fluor 594 | Texas Red |
| 1 | Low | Alexa Fluor 546 | TRITC/RFP |
| 4 | Medium | Alexa Fluor 488 | FITC/GFP |
| 6 | Low | Alexa Fluor 647 | Deep Red/Cy5 |
| 10 | Abundant | Alexa Fluor 750 | Near Infrared/Cy7 |
ISH Colorimetric Tissue Assay Workflow
Figure 1A. ViewRNA ISH assay. The ViewRNA ISH Tissue Assay, based on branched-DNA signal amplification technology, can be used to measure three targets simultaneously and has the sensitivity and robustness to measure single mRNA molecules in single cells.
ISH Fluorescence Cell and Tissue Assay Workflow
Figure 1B. ViewRNA FISH assay. The ViewRNA Fluorescence Assay, based on branched-DNA signal amplification technology, can be used to measure four targets simultaneously and has the sensitivity and robustness to measure single mRNA molecules in single cells.
ViewRNA ISH cell assays
ViewRNA ISH Cell and Cell Plus Assays offer:
- Visualization of RNA (ISH Cell Assay) or simultaneous visualization of 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
Ordering ViewRNA ISH cell assays
| Kit | Probe set types | Cat. No. | Size |
|---|---|---|---|
| ViewRNA ISH Cell Assay Kit | 1, 4, 6 | QVC0001 | 24 assays |
| ViewRNA Cell Plus Assay Kit | 1, 4, 6 | 88-19000-99 | 24 assays |
| ViewRNA ISH Cell 750 Module | 10 | QVC0200 | 24 assays |
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 controls: For ViewRNA Cell Assays, 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 samples.
Negative controls: Negative controls should be used in every experiment to help ensure proper evaluation of specific versus non-specific binding. Samples with the target specific probe omitted or samples labeled with a probe against a target not expressed in cells of interest are highly recommended.
Search probes used for positive and negative controls in probe search tool for the species. Please note that ViewRNA probes are very specific, and a probe set for one species commonly does not detect targets in another species.
ViewRNA ISH tissue assays
ViewRNA ISH tissue assays offer:
- Robust, single mRNA molecule detection in tissue sections
- Accurate, specific, and simultaneous detection of up to four mRNA, micro-RNA, or other non-coding RNA
- Direct, spatial visualization of RNA in complex tissue architectures while retaining tissue morphology
- Eliminates unnecessary steps and biases associated with miRNA, RNA, or DNA purification, cDNA synthesis, and PCR amplification
Ordering ViewRNA ISH tissue assays
| Kit | Probe set types | Cat. No. | Size |
|---|---|---|---|
| View RNA Tissue Core Kit | 1, 4, 6 | QVT0400 | 24 slides |
| ViewRNA Tissue Assay Fast Red Module | 1 | QVT0410 | 24 slides |
| ViewRNA Tissue Assay Fast Blue Module | 6 | QVT0420 | 24 slides |
ViewRNA Tissue Assay DAB Module | 4 | QVT0430 | 24 slides |
| For additional RNA multiplex ability, ViewRNA Tissue Assay Core Kit can be combined with ViewRNA Tissue Assay Modules | |||
| ViewRNA Fast Red 1‑Plex Kit | 1, 6 | QVT0410C | 24 slides |
| QVT4410C | 96 assays | ||
| ViewRNA Fast Blue 1‑Plex Kit | 1, 6 | QVT0420C | 24 slides |
| QVT4420C | 96 assays | ||
| ViewRNA DAB 1‑Plex Kit | 4 | QVT0430C | 24 slides |
| QVT4430C | 96 assays | ||
| ViewRNA Red‑Blue 2‑Plex Kit | 1, 6 | QVT0412C | 24 slides |
| QVT4412C | 96 assays | ||
| ViewRNA Red‑Brown 2‑Plex Kit | 1, 4, 6 | QVT0422C | 24 slides |
| QVT4422C | 96 assays | ||
| ViewRNA Blue‑Brown 2‑Plex Kit | 1, 4, 6 | QVT0433C | 24 slides |
| QVT4433C | 96 assays | ||
| Kit | Probe set types | Cat. No. | Size |
|---|---|---|---|
| ViewRNA Tissue Fluorescence 4-Plex Assay Core Kit, with (Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 750) | 1,4,6,10 | QVT0700 | 24 slides |
| QVT4700 | 96 slides | ||
| ViewRNA Tissue Fluorescence 4-Plex Assay Core Kit, with (Alexa Fluor 488, Alexa Fluor 594, Alexa Fluor 647, Alexa Fluor 750) | 1,4,6,10 | QVT0800 | 24 slides |
| QVT4800 | 96 slides | ||
| ViewRNA Tissue Fluorescence 1-Plex Assay Core Kit, with Alexa Fluor 647 (Type 6) label probe | 6 | QVT0600C | 24 slides |
| QVT4600C | 96 slides | ||
| For additional RNA multiplex ability, ViewRNA Tissue Florescence modules can be combined with ViewRNA Tissue Fluorescence 1-Plex Assay Core Kit and with Alexa Fluor 647 (Type 6) label probe kit | |||
| ViewRNA Tissue Alexa Fluor 488 (Type 4) Module | 4 | QVT0688B | 24 slides |
| QVT4688B | 96 slides | ||
| ViewRNA Tissue Alexa Fluor 546 (Type 1) Module | 1 | QVT0646B | 24 slides |
| QVT4646B | 96 slides | ||
| ViewRNA Tissue Alexa Fluor 594 (Type 1) Module | 1 | QVT0694B | 24 slides |
| QVT4694B | 96 slides | ||
| ViewRNA Tissue Alexa Fluor 750 (Type 10) Module | 10 | QVT0640B | 24 slides |
| QVT4640B | 96 slides | ||
| Choose either ViewRNA Tissue Alexa Fluor 546 (Type 1) Module or ViewRNA Tissue Alexa Fluor 594 (type 1) Module. Both uses Type 1 probes and are not compatible with each other. | |||
For colorimetric kits, please find probe sets using the “ViewRNA Cell” filter in the search tool below.
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 probe sets should be used in every experiment to ensure proper performance. For ViewRNA Tissue Assays, one of the following kits can be used for a positive control:
ViewRNA Tissue Positive Control Kit (2-plex) (4 rat kidney slides + probes)
ViewRNA Tissue Positive Control Kit (1-plex) (4 rat kidney slides + probe)
Negative controls should be used in every experiment to help ensure proper evaluation of specific versus non-specific binding. Samples with the target specific probe omitted or samples labeled with a probe against a target not expressed in cells of interest are highly recommended.
Search probes used for positive and negative controls in probe search tool for the species. Please note that ViewRNA probes are very specific, and a probe set for one species commonly does not detect targets in another species.
ViewRNA ISH assay image gallery
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
SKBR3
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.
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