ViewRNA ISH Cell Assays

• 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
• ViewRNA Cell and Cell Plus Assays at-a-glance
• Featured applications and image gallery
• Ordering information
   

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.

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.
 

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.

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.

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