Introducing New RNA-Protein Co-Detection Assays

For those of you who are not familiar with RNAscope technology, it's an in-situ hybridization platform designed for detecting specific RNA biomarkers. On this slide, you can see I have described the three major assays falling under the RNAscope technology umbrella for detecting RNA biomarkers.

The first is the RNAscope assay, which is specifically for targets that are over 300 nucleotides in length. This assay is available as a chromogenic or fluorescent assay and in different configurations of automated or manual assays. The second is our BaseScope assay, which is a specialized assay designed for targets that are between 50 to 300 nucleotides in length. These include your small molecules like splice variants, exon-exon junctions, highly homologous sequences, and point mutations. This is again either a single plex or duplex assay available in automated or manual platforMS

Our newest assay is the microRNAscope assay, which, as the name suggests, is for targets even smaller than 50 nucleotides. And these are for targets like microRNAs, ASOs, or siRNAs. And currently, this is available as a single plex assay in both automated and manual platform. In addition to our RNA detection assays, we've also launched a DNAScope assay this summer, which allows you to visualize DNA biomarkers.

Why is it important for us to look at RNA biomarkers with a highly precise tool like RNAScope? In the last few years, we have understood the importance of spatial resolution since it allows us to study highly heterogeneous tissues like the embryo or the brain or tumor, wherein you have multiple cell types that are constantly interacting with each other, communicating and inter-determining the function of the target tissue. Tools like RNAscope are highly specific and allow you to visualize your target RNA biomarker or target of interest with high sensitivity and specificity at single cell and single molecule resolution and thereby allowing you to elucidate the heterogeneity of these tissues and study the cellular interactions.

While it's important to study spatial expression of RNA biomarkers, it's even more important to get a multiomic analysis of your complex tissues, thereby understanding the dynamic interactions happening between different cell types. And realizing this, we have enabled combining our RNAscope-ISH assays with immunofluorescence or immunohistochemistry thereby allowing detection of your RNA biomarkers in combination with target protein biomarkers. This allows us to visualize target gene expressions in target cells. It allows us to identify cellular sources of secreted factors like chemokines, cytokines, and growth factors. It also allows us to get better spatial visualization of target cells, by looking at by marking the cell border using a specific antibody and visualizing gene expression with single cell resolution using RNAscope probes. And therefore, by combining RNAscope with either immunohistochemistry or immunofluorescence, we can leverage the best of proteomic and transcriptomic approaches with high resolution and specificity.

In the next few slides, let's figure out a few examples of how RNAscope can be combined with immunohistochemistry or immunofluorescence for a range of different applications, starting with examining cell type specific gene expression. In this example, they're studying the expression and rule of PRDM12, which is a transcriptional regulator, and its role in development of pain-sensing neurons. For this example, they used a specific PRDM12 RNAscope probe and combined it with three different antibodies to study which of the subtype of these neurons were expressing PRDM12 and therefore responsible for the development of these pain sensing neurons. We observed that when they combined it with TRKA, they saw a very strong co-expression of PRDM12 with TRKA. While on the other hand, the correlation was not as strong when they combined it with either TRKC or TRKB antibodies. And this told them that there was a strong correlation of PRDM12 with TRKA shows that PRDM12 is expressed in almost all the nociceptive neurons, but the expression was not as strong in proprioceptive or mechanoreceptive neurons. This revealed that the presence of the PRDM12 transcription regulator in these TRKA positive neurons suggested that it was essential for the development of these pain sensing neurons.

In this next example, they have used the BaseScope assay in combination with IHC to validate single nuclear RNA sequencing data. Here they are studying multiple sclerosis and trying to understand the differential cell populations in MS (multiple sclerosis) brain versus a normal brain. They were able to obtain postmortem MS brain and compare it to a normal brain and study expression of different RNA markers. Here you can see that they have identified different sets of neuronal and non-neuronal populations in the brain, and they see some differential expression between wild type and MS brain. They used a few markers specific to oligodendrocyte populations and studied their expression in this control brain versus brain with MS, and here you can see with the BaseScope assay, the dual BaseScope assay, they were able to identify these different populations of oligodendrocytes by using subpopulation specific markers.

Next, they wanted to also identify what are the different populations of cells that are potentially present in a control brain versus MS brain. They compared a MS lesion and visualized what they are already seen as a part of the single nuclear seq RNA sequencing. That is that they identified a hybrid cell called oligodendroglia cell having characteristics of both the oligodendrocyte and the microglia, and therefore expressed markers belonging to both cell types. And they confirmed this by doing dual BaseScope IHC by using CD74 microglial marker and olig-1 to oligodendrocyte marker and showed that these two markers, which are specific to different types of cells, were co-localizing in this hybrid oligodendroglia cell. And this cell was present in abundance in the MS lesion. suggesting that this hybrid cell population could have something to do with the MS pathology and might contribute to certain pathological features associated with MS.

In the next example, let's look at how RNAscope can be used in combination with immunofluorescence to identify soluble factors and the cell of origin secreting these soluble factors. This is an example where the researchers were studying pancreatic cancer and trying to identify whether LIF, which is leukocyte inhibitory factor, had a role to play in progression of pancreatic cancers. They identified that in patients with pancreatic cancer; there are high levels of circulating LIF in their brain compared to normal individuals. Similarly, they also identified that presence of LIF mRNA correlates to decreased disease-free survival in these patients. What they wanted to identify is how this LIF was contributing to growth and progression of pancreatic cancer. They used RNAscope in combination with immunofluorescence to study the expression of LIF and identify how it might impact growth of pancreatic cancer cells.

In this image, you can see that they combined immunofluorescence for keratin-19 with that of LIF RNAscope probe and PTPRC or CD45 RNAscope probe. And you can see that LIF and PTPRC were present in the stromal region of this tumor, while keratin-19 expression was, as you can imagine, specifically located or localized to the tumor cells, suggesting that LIF is expressed by potentially by the pancreatic stellate cells in the stroma and was exerting its impact in a more paracrine fashion to promote growth of pancreatic cancer cells.

Let's look at another example of how RNAscope is combined with IHC to identify a specific population of immune cells. In this paper that was published in Nature Medicine, these researchers were treating a melanoma patient with anti-PD-1 therapy, and this patient unfortunately reacted adversely and led to encephalitis in this patient, which was ultimately fatal. But these researchers were interested in understanding which immune cell populations were responsible for inducing encephalitis in this patient. They performed TCR sequencing on this inflamed brain tissue and identified sequence that was most frequently occurring and perform and designed a specific BaseScope probe against this TCR sequence. They then localized the sequence with CD4 and CD8 antibodies to identify which type of T cells were present in this encephalized brain. And surprisingly, they were able to show that this TCR sequence co-localized with CD4 positive T cells and not with CD8 positive T cells, suggesting that it was a CD4 memory T cell population that was responsible for inducing this encephalitis triggered by PD-1 treatment. This was a very important finding because it's important to understand the type of adverse events that can happen due to immunotherapies and the type of cell populations responsible for induction of these adverse events.

In this next example, they want to identify regulatory mechanisms of PD-L1 mRNA. Although checkpoint therapies have been extremely successful for a number of malignancies, we don't yet completely understand how these immune checkpoint receptors and markers are regulated at the mRNA level. Franchini and colleagues identified a post-transcriptional regulatory mechanism by which PD-1 mRNA was regulated in T cells. They identified that PD-1 mRNA is transported via microtubules and stress granules before they are translated in the cytoplasm. And to demonstrate that the utilized RNAscope in combination with immunofluorescence and showed us in this image, as you can see, when the cells were untreated, you can see nice tubulin staining and PD-1 staining, as well as presence of PD-1 protein in yellow, and a nice co-localization of the mRNA and protein, as you would imagine, and presence of intact tubulin. But when the cells were treated with a microtubule inhibitor, you can see that the expression of mRNA is present as expected, the tubulin marker is not present anymore, and also the PD-1 protein is very low or almost negligible in expression, suggesting that the mRNA was not successfully transported to the cytoplasm and therefore did not get translated efficiently. Furthermore, they also wanted to show that this was happening via the stress granules and used stress granule specific marker G3BP1 antibody and combined it with the PD-1 transcript probe, showing nice co-localization of the stress granule biomarker, sorry stress granule marker with that of PD-1 mRNA, suggesting that the transport was indeed via stress granules and microtubules and is important regulatory mechanism of the PD-1 mRNA. In this next example, we'll see how this workflow can be successfully used to identify certain biomarkers and drug targets.

In this example, these researchers are studying the Alzheimer's disease model and although we have made significant advances in understanding Alzheimer's disease, we still don't understand the association of senescent cells with that of certain physiological characteristics associated with Alzheimer's disease, including cognitive impairment. These researchers wanted to demonstrate the presence of these senescent cells in the amyloid beta plaques found in the Alzheimer's brain. They used RNAscope P16 probe in combination with the AB antibody to demonstrate that P16 positive cells were indeed present in these amyloid beta plaques in this mouse model. They went a step further to show that with 3D microscopy and imaging, they were able to study the 3D structure of the plaque, showcasing that the cells were dispersed throughout the plaques, the senescence cells at P16 were present throughout the plaques.

Next, they also wanted to show that by reducing senescence in these animal models, they could potentially suppress neuropathology associated with Alzheimer's disease and other degenerative conditions. For that, they treated these Alzheimer's disease mice with two senolytic compounds that the FDA approved, that is two compounds that would prevent senescence. And when they've treated these mice with these senolytic compounds, you can see compared to the vehicle, the treated sample had very low to no expression of P16, suggesting that the senescence was significantly reduced in these treated models.

Next, we're going to look at an example of how this technique is specific enough to look at the spatial organization of a tissue and identify niche-specific expression of RNA biomarkers. Here, in this example, they are studying the role of CD19-positive fibroblasts in differentiation and homeostasis of the intestinal stem cells. As you all know, intestinal stem cells are important as they renew the intestinal epithelium, which is important for a healthy gut function. They combined CD19 specific RNAscope probe with that of various antibodies for cell type specific markers.

In this first image they've combined CD90 with GP38, which is the fibroblast specific marker. And as expected, there is nice co-localization of this GP38 with the CD9 positive cells and these intestinal grips. They also combined CD90 with BRDU, which is an indicator of proliferating cells. And by using BRDU antibody, they were showing that CD19 was in proximity to these epithelial cells that were proliferating. And finally, they were able to show that CD19 was also proximal to LGR5 positive cells in the crypt, which are potentially your intestinal stem cells.

How does CD19 positive fibroblasts exert their effect on these stem cells and regulate differentiation of these epithelial cells? They were able to show that by expressing or by secreting soluble factors CMA3, they were the fibroblasts, the CD90 positive fibroblasts are able to exert this impact on the epithelial cells and promote differentiation and homeostasis of these stem cells. And they were also able to show that NPR2, which is a receptor for CMA3 was also found on the epithelial cells of these intestines. With this workflow of combining RNAscope with immunofluorescence, they were able to establish the role of the CD90 positive fibroblasts and demonstrate how they were able to regulate differentiation of these stem cells in the intestinal crypts.

Finally, the last example, I will show you how. In this last example, let's study how these researchers used RNAscope in combination with immunofluorescence to study infection by the Rift Valley virus. The Rift Valley fever virus is a zoonotic infection, is a livestock disease that can potentially have zoonotic transmission to humans. We're not clear on how this might impact a pathology in the brain if it impacts humans. These researchers studied the infection of this virus by using a rat model. They utilized specific RNAscope probe to the viral RNA and combined that with IBA1 positive antibody. to study infiltration of immune cells post-infection in this rat brain. As you can see, compared to the uninfected cell, uninfected image, there is increased presence of the viral mRNA and immune infiltration post-infection day from one to day seven, suggesting that there can be potentially significant neuroinflammation related to this viral infection. With that, I would like to transfer the platform back to Madhura and thank you for your attention today.
Thank you, Anushka, for that excellent overview of the need to detect RNA in protein and for showing us some great examples of applicable research questions addressed with this. And with that, I would like to introduce you to our reagents that enable these applications. The new reagents in the form of the RNA protein co-detection anciliary kit, when used with the new workflow, will allow researchers to simultaneously examine cell type specific gene expression and identify cellular sources of secreted proteins. Some of you may have already tested certain antibodies and found them incompatible with our RNAscope and BaseScope assays. Our aim with this new assay is to enable more antibodies to be compatible with our assays, and we will see later some examples of key antibodies that were previously incompatible and are now compatible with this new workflow. The new assays essentially contain three parts: the new RNA protein co-detection ancillary kit, which includes the co-detection blocker, co-detection target retrieval solution, and the co-detection antibody diluent, which is also available as a standalone item. This kit has been validated with our chromogenic red and multiplex fluorescent assays on both the manual and the Leica platforms and has been validated with our BaseScope assays.

Along with this kit, we would also like to introduce you to the new protocol where the key primary antibody step is now performed right after the target retrieval step, following which we go on to perform the rest of the RNA-ISH steps. We then follow this up with a secondary antibody to detect the protein of interest on the same section, and this workflow has been designed to be completed within two days, with the primary antibody incubation lasting overnight as the breakpoint. With this new integrated workflow approach, we have now enabled more antibodies to be compatible with RNA-protein co-detection and reduced the need for extensive screening for compatible antibody clones, resulting in faster time-to-results.

For those of you who may have tested the sequential workflow before, these new reagents and workflows support equivalent or better performance of antibodie,s and also supports equivalent sensitivity to ISH-only detection and more faithfully retains specificity of the antibody in the IHC alone. In this slide, I'm showing what a signal from an IHC-only run looks like in the left panel. And in the final panel on the right, with our integrated workflow and the new pre-treatment reagents, we can now rescue the IHC signal, and this, in a sense, makes it easier to combine your antibody of choice with RNA-ISH.

To enable this compatibility, our team has developed some key reagents, the first of which is the target retrieval solution. The target retrieval with the current RNAscope and BaseScope assays has been thoroughly validated and is intended for use with the RNA-ISH assays alone. However, in some instances, these may have impacted the protein detection. This new co-detection target retrieval reagent has been formulated for improved protein detection while maintaining equivalent ISH conditions or ISH detection. The second reagent is the co-detection antibody diluent. In our experience, certain commercially available antibody diluents have negatively impacted RNA-ISH staining, as can be seen from the image in the middle panel on this slide. This drove us to develop a new diluent which maintains both RNA-ISH and IHC staining. Lastly, to ensure no cross-detection between the two chemistries, a blocker is needed, which is only required for the manual chromogenic assays.

The main difference between the previous workflow and the new reagents in the workflow is that with the sequential workflow, extensive prior optimization was needed to ensure antibody compatibility. Whereas now a pre-validated list is no longer needed, and most IHC-compatible antibodies can now be combined with the RNA-ISH assay. An example is the CD8 antibody shown here in the left panel with standard IHC, in the middle panel with ISH and IHC without the new reagents in the protocol where we lose the signal, and in the right panel we are now able to visualize both signals with the new co-detection reagents.

And with that, I would like to do a quick recap. If you're running our manual assay, you would need the ancillary kit. And if you're running this on the Leica platform or the Leica BOND RX instrument, you only need the antibody diluent. The assays compatible on the Leica BOND RX are the 2.5 assay, the red assay, the LS-multiplex fluorescent assay, as well as the BaseScope LS-red assay. In this slide, we create a reference for the reagents you would need for each protocol that we have validated with our assays. As an example, if you're running our manual assay, red assay, with the new reagents and workflow, you would need all three reagents provided in the ancillary kit. Please reach out to our excellent support team to get help with getting started with these new assays.

Now, I would like to take you through some examples of staining generated with the new reagents in each of our validated assays with IHC antibodies. In the first example here, we show some data with CD4, CD5, and FOXP3. CD markers are cell surface markers, which are very useful for the identification and characterization of leucocytes and the different subpopulations of leucocytes. CD marker-specific antibodies have been widely used for cell sorting, identification, and cancer diagnosis. FOXP3 is a protein involved in immune system responses and known to be a master regulator of the regulatory pathway in the development and function of regulatory T-cells. These represent fairly standard proteins utilized in oncology research, and shown here, combined with RNA-ISH can be used to get more data out of precious samples. The samples shown here are tonsil, head and neck cancer. And as you can see from the panel on the right, both RNA-ISH and IHC signal is maintained with the new workflow and reagent with our Leica red assay.

Another example of staining here, the protein detection is performed using a green chromogen, giving a great contrast on tissues where an alternate to the brown DAB is preferred, perhaps due to tissue artifacts. Shown here are examples of CD3, CD8, and KI67 antibodies in the left panel as control IHC alone. In the middle panel, combined with RNA-ISH without the new reagents and workflow in the right panel, we observed optimal signal detection with both the RNA and protein. This run was performed with our manual assay.

In this slide, we show compatibility of these antibodies and workflow with our BaseScope assays on the Leica platform. Moving on to fluorescent ISH and IF, these new reagents and workflow have also been validated with our multiplex fluorescent assays, where we use the TSA detection system to visualize the signals. Shown as an example here are the CD3 and CD8 antibodies on lung cancer tissue with control probe PPIB for RNA ISH. The antibody signal is in white and the RNAscope signal is in red. Here is an example of 3Plex-ish with Ki67 antibody with this new reagents and workflow, a key example of how you can get much more data with more targets and detect both RNA and protein on the same slide. Here, we're highlighting some antibodies, specifically CD markers that may have previously been incompatible in combining with RNA-ISH and required extensive screening and optimization.

And lastly, here are some more examples of CD markers detected with the brown or the green chromogen along with the red-ISH signal. And with all that data, I would like to conclude and summarize today's presentation. In today's session, we have shown some key examples of how multi-omics profiling with single-cell resolution can be achieved with the new reagents and workflow. With this new ancillary kit, you can now incorporate RNA-ISH into your existing IHC workflow to gain deeper insights and preserve precious samples. This new reagent and workflow have been extensively validated with the red chromogenic and multiplex fluorescent assays on manual and the Leica platforms. And lastly, we hope that with this product and key research examples we have shown in this deck, you're now able to utilize the power of two technologies, IHC and RNA-ISH, to answer your research questions. With that, we will now address some questions that we have coming through. Thank you.