In Situ Hybridization: Ingredients for Success

What is in situ hybridization?

In situ hybridization (ISH) is a method which allows the localization of specific DNA or RNA sequences within a section of tissue using a labeled complementary strand. The target gene can be visualized in brightfield or fluorescence. Compared to an immunohistochemistry assay, which is used more broadly to localize proteins within tissue, an ISH assay allows scientists to understand the organization and function of genes in a section of tissue with greater sensitivity. An ISH assay not only allows researchers to determine whether cells are positive for a specific DNA/RNA sequence, but it can allow them to understand the magnitude of positivity within an individual cell for the target gene.

It is evident that in situ hybridization is a useful tool which can provide key information across preclinical research and within clinical trials. Additionally, an in situ hybridization assay can be used in combination with an immunohistochemistry assay to provide researchers with unique insights into cellular and molecular mechanisms within tissue.

Dual ISH + IHC assays have a wide variety of useful applications:

Brightfield images of ISH  (left) and IHC (right) assays on human colon tissue.
  • Using ISH + IHC on unique but related targets can provide information about the relationship between the targets.
  • Using a dual assay can help identify the location of a target protein within tissue and identify the cells that originally secreted the protein of interest.
  • Using both ISH + IHC assays on the same target can provide insight into the gene regulation mechanism within tissue.
  • Using an IHC cytokeratin marker to delineate tumor regions in tandem with an ISH assay can highlight the regulation mechanism of a target gene as it relates to tumor regions within tissue.
  • ISH + IHC dual assays can provide useful insights about protein degradation.

In situ hybridization is made an even more powerful tool when AI-powered algorithms are applied to digital whole slide ISH images to generate data and insights that cannot otherwise be discerned by the human eye. However, there are some key ingredients to a successful ISH assay that must first be addressed to ensure robust and reliable ISH data and insights.

Maximize the Value of In situ Hybridization Assays

When preparing to perform an ISH assay, it is important to know that RNA and DNA degrade quickly in collected tissue, limiting the researcher’s ability to detect and visualize their target. There are some important steps during sample preparation that can help minimize RNA degradation in tissue:

  • Tissue fixation – Tissues should be fixed shortly after collection in 10% Neutral Buffered Formalin (NBF). Volume of NBF for fixation should be 20x the size of the tissue sample. Once fixed, tissue should be transferred to 70% ethanol. Make sure your fixed tissue is kept in the 70% EtOH for no more than 48 hours.
    • Flash frozen tissue may also be used instead of fixed tissue.
  • RNA preservation – Sections should be cut fresh in RNase-free conditions from FFPE or OCT tissue onto charged slides for greater tissue adhesion. Sections designated for ISH should be used within 3 months of sectioning. While in storage, tissue sections should be kept at a constant 4°C.
    • Alternatively, tissue sections may be kept at room temperature if stored with a desiccant.

Another key aspect of a successful ISH assay is optimization. Each unique combination of species + tissue type + gene target must first be optimized to test if the target is being correctly detected, and to hone in on the ideal conditions for detecting and visualizing the target. It can help to have pre-existing IHC data about a species + related protein combination as a clue to whether the analogous RNA target can be detected via in situ hybridization in a similar sample.

Additionally, when performing digital whole slide imaging of an ISH assay, imaging settings must be optimized for each set of samples to account for differences in tissue types, stain intensity, tissue autofluorescence, and coloration. Every assay is different, and for each one it is important to narrow detection to the true ISH signal and to reduce the presence of misleading imaging artifacts as much as possible. At Reveal Biosciences, our expert imaging specialists dedicate time and attention to optimizing image settings so that the ISH signal is neither undercounted nor overestimated.

Leica BondRX in the Reveal Biosciences lab.

Finally, consistent application of the ISH protocol itself across multiple samples is key to reliable results. Many ISH protocols are performed manually, where slight differences in treatment due to human error may lead to inconsistent results. Some technologies, such as the Leica BondRX used by the Reveal Biosciences lab, offer an automated solution. Once programmed, the BondRX consistently applies the ISH protocol to each sample with calculated precision to maximize confidence in any insights and data generated from the ISH assay.

Applying In Situ Hybridization Technology

At Reveal Biosciences, our lab has developed strong expertise in applying in situ hybridization methods across many species and tissue types. As Advanced Cell Diagnostics (ACD) Certified Providers, we have access to a wealth of resources and expert knowledge to help you apply the latest ISH technologies to your preclinical research project. ACD ISH products that are ready for use now at Reveal Bio include:

  • RNAscope – Targets mRNA and long non-coding RNA over 300 base pairs; chromogenic or fluorescent detection, capable of single and multiplex .
  • BaseScope – Targets exon junctions/splice variants, RNA sequences between 50 – 300 base pairs, and validated point mutations; chromogenic detection, single or duplex.
  • miRNAscope – Targets antisense oligonucleotides, microRNA, small interfering RNA, and other small RNAs between 17 – 50 base pairs; chromogenic detection, single plex.

Our close relationship with ACD allows us rapid access to pre-made ISH probes for common targets, while custom probes for less common targets can be developed upon request.

Capturing Advanced, AI-Powered Data from an In Situ Hybridization Assay

Fluorescent in situ hybridization whole slide image (left) and cell segmentation mask over the same image (right).

The steps described above are all critical to producing reliable, consistent data within and across in situ hybridization assays. Our team of experts will guide you through planning an ISH assay based on your unique project goals. Reveal Bio’s full-service precision pathology lab is equipped for every step of an ISH assay from sample preparation and sectioning, to optimization and application of the optimized ISH protocol, to digital whole slide imaging with cloud-based image management. Finally, our image data specialists apply advanced, AI-powered models to your ISH images to generate quantitative RNA expression data from whole sections, regions of interest, and tissue microarray cores. These data hold the key to deep insights that otherwise could not be discerned from a qualitative assessment of an in situ hybridization assay.

Data outputs include binned positive cell counts and density values, where positive cells are grouped into bins according to the number of positive ISH dots detected per cell. For multiplex assays, we also generate co-localization data to provide quantitative information on the relationship between multiple gene targets. In a dual ISH + IHC protocol, we can simultaneously generate protein expression data along with RNA localization data on the same slide, providing powerful insights into the target’s regulation of expression.

Heatmap visualization of binned ISH density values across a sample set.

Our automated workflow makes in situ hybridization easy for you. With our expertise in sample preparation, our access to advanced tools such as the Leica BondRX for consistent application of ISH protocols, to robust optimization steps and image quality checkpoints, Reveal Biosciences provides the most reliable preclinical ISH data solution for your project. To learn more about applying the power of AI to in situ hybridization assays, please visit our AI-powered in situ hybridization webpage.

Written by:

Jeremy Warner, Scientific Marketing Associate, Reveal Biosciences

Pathology Revealed

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