Multiplexed in situ Hybridization in Human Tissues

Asterand Bio’s PhaseZERO® Research Services has established expertise in both ViewRNA® and RNAscope® branched chain in situ hybridization techniques (ISH). Thorough assay development and optimization approaches allow us to effectively use these techniques to deliver high quality, biologically relevant data supporting human tissue based target and biomarker validation projects and diagnostic assay development.

ISH-ISH

We have recently expanded our capabilities in delivery of automated multiplexed data sets through the introduction of RNAscope 2.5 (Figure 1). This approach is particularly suited to the evaluation of multiple targets in human tissues where robust antibodies are not available and increases both throughput and reproducibility through full automation.

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Figure 1.  mRNAs for glucagon (red) and insulin (brown) are shown demonstrating the separate localization of cells expressing these pancreatic islet targets. Data were generated using automated, multiplexed ISH. We also have experience detecting low abundance mRNAs using this approach.

 

ISH-IHC

In addition, we have developed an approach to allow sequential ISH and IHC in the same human tissue section. The advantage of multiplexing ISH and IHC is to allow use of validated antibodies together with mRNA detection for target localization (e.g. for validation of protein and mRNA expression) and where an appropriate antibody for one of the targets is not available or not trusted. This again is performed in fully-automated environment, providing an increased throughput and reproducibility versus a manual approach (Figure 2 and 3).

 

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Figure 2. Glucagon mRNA (red) and pro-insulin protein (brown), demonstrating the separate localization of cells expressing these pancreatic islet targets. Panels A–C: consecutive FFPE sections of human pancreas. Panel A: glucagon mRNA with IgG negative control. Panel B: multiplexed detection of both targets. Panel C:  pro-insulin p rotein with DapB mRNA negative control. Panel D: multiplexed IgG and DapB mRNA negative controls.

 

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Figure 3. Insulin mRNA (red) and pro-insulin protein (brown), demonstrating co-localization of mRNA and insulin protein immuno-reactivity confirming antibody specificity. Panels A–C: consecutive FFPE sections of human pancreas. Panel A: insulin mRNA with IgG negative control. Panel B: co-localized insulin mRNA signal and pro-insulin immunoreactivity. Panel C: pro-insulin protein with DapB mRNA negative control. Panel D: multiplexed IgG and DapB mRNA negative controls

 

Using multiplexed approaches such as these has clear application across many areas of disease biology, in particular for novel immuno-oncology approaches where understanding of specific immune cell interactions with solid tumors is paramount.

For further information, contact us at advantage@asterandbio.com.