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Transcriptomic biomarker discoveries lead to a more thorough investigation requiring scientists to validate the location of RNA expression using RNAscope™ technology.

Biomarker Research MoleculesWhen is it best to explore RNA as a Biomarker?


+ Many biomarkers discovered from transcriptomic studies.
– In situ measurement techniques previously unsuitable for routine use.

Useful when:

• Differential RNA expression identified as biomarker
• Protein measurement not possible.
• Limited correlation with protein levels: direct RNA measurement is desirable.


• Transcriptomic discovery phase identifies biomarker:
ncRNA must be measured directly, as no protein counterparts

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Protein limitations as a biomarker

• Limited antibody availability
• Antibodies can lack specificity or have unknown specificity
• Low sensitivity of antibody based methods makes it challenging to interrogate secreted and scarce proteins
• Antibody development & validation can be time consuming, challenging and cost-prohibitive
• Antibody batches can exhibit high batch-to-batch variation
• RNA and protein levels may not correlate linearly
• Limited to protein-coding genes
• Lack of standardization in both assay and data interpretation (IHC)

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The Value of RNA Biomarkers

Our understanding of RNA is constantly evolving, with discoveries over the last few decades including alternative splicing and the catalytic activity of some RNAs, along with the identification of many small non-coding and long noncoding RNAs (lncRNAs). It has become increasingly clear that RNA is a key player in a myriad of both normal and disease-related cellular activities, and genetic regulatory control. For this reason, RNA has become a molecule of great interest and value across the research, diagnostic and pharmaceutical arenas, and since RNA expression reflects the state of a biological system, it presents an ideal choice of biomarker. RNA expression levels are highly dynamic and integrate both genetic and epigenetic mechanisms of gene regulation, serving as an excellent molecular phenotypic readout of the functional state of the cell. In order to utilize the full potential of these biomarkers, there is an unprecedented

demand for the next generation of more effective tools for routine RNA biomarker analysis. This demand is particularly acute when it comes to in situ techniques, in order to map important pathways and networks within the morphological context (Figure 2).

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Figure 2: Revealing the RNA expression signature of individual cells within the tissue architecture. Human breast cancer FFPE tissue is probed for MMP9 mRNA expression using RNAscope® 2.0 HD Reagent Kit-BROWN. Overexpression of this matrix metalloprotease facilitates tumor growth and invasion; and although signal (brown) is abundant in scattered stromal cells of the tumor microenvironment, it is barely detectable within tumor cells.

RNA Biomarkers LP

Sensitivity: Detection of each single RNA molecule requires only three double Z probe pairs to bind to target RNA. The 20 double Z probe pairs provide robustness against partial target RNA accessibility or degradation.

Specificity: The double Z probe design prevents background noise. Single Z probes binding to nonspecific sites will not produce a full binding site for the pre-amplifier, thus preventing amplification of non-specific signals and enhancing specificity.

Single molecule visualization and single-cell quantitation: Hybridization of three or more double Z probe pairs is visualized as a punctate signal dot under a standard microscope. Analysis software quantifies RNA expression levels for each single-cell.

Compatible with degraded RNA: The double Z probe design, with its relatively short target region (36-50 bases of the lower region of the double Z), allows for successful hybridization of partially degraded RNA.

Widespread application: As long as at least a 300-base unique sequence is available, RNAscope can be applied to virtually any gene, species or tissue.

RNAscope® ISH

RNAscope is a new multiplex nucleic acid in situ hybridization technology, based on ACD’s (Advanced Cell Diagnostics Inc., Hayward, CA) unique probe design and signal amplification methodology. The RNAscope approach is an alternative to conventional ISH/FISH in situ RNA detection, and the method provides the opportunity to profile single-cell gene expression in situ with single-molecule detection sensitivity, unlocking the full potential of RNA biomarkers (Figure 4). To date, this method is the only platform that has the sensitivity to detect most genes in the human transcriptome in situ, as well as simultaneously quantifying multiple RNA transcripts at a single-cell resolution. In order to substantially improve the signal-to-noise ratio of RNA ISH, RNAscope employs a probe design strategy very similar to fluorescence resonance energy transfer (FRET), in which two independent probes (double Z probes) must hybridize to the target sequence in tandem in order for signal amplification to occur (Figure 5). Since it is highly unlikely that two independent probes will hybridize to a nonspecific target right next to each other, this design concept ensures selective amplification of target specific signals.

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Figure 4: Quantitative RNA ISH profiles in situ at single-cell resolution. A. HER2 mRNA was visualized with RNAscope Multiplex Fluorescent Kit and quantified by counting signal dots in individual HeLa cells. Nuclei were counterstained with DAPI (blue) and a probe set to 18S rRNA was used as internal control for RNA detection. B. HER2 mRNA in the same HeLa cell culture was quantified using a “grind-and-bind” method (QuantiGene2®) to estimate the absolute copy number of HER2 mRNA in single cells. These two estimates were in close agreement, demonstrating the quantitative capacity of RNAscope. [21]

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Figure 5: The RNAscope assay procedure is completed within a single day. Once the sample is prepared, oligo probes complementary to the RNA target of interest are added and hybridization takes place. Specific signal is subsequently amplified with the double Z probe system, and immediately detected and quantified via standard brightfield microscope or multi-spectral fluorescent imaging system.


The current RNAscope probe design method of 20 double Z probe pairs requires 1KB of unique sequence. It can be applied to targets as few as 300 bases long but but this sequence length requirement does exclude the ability to interrogate small coding or non-coding RNAs such as snoRNAs, microRNAs, siRNAs, snRNAs, exRNAs, piRNAs - which can be as small as 18 bases. Classified as 200 bases or more, long non-coding RNAs (lncRNAs) are suitable targets for RNAscope technology, which is by far the most sensitive in situ method available for this gene class. This breakthrough method confers numerous advantages including increased sensitivity, specificity, single molecule visualization and quantification – in addition to compatibility with partially degraded RNA samples (common in FFPE tissue sections), The method also enables multiplex staining of RNA transcripts (Figure 7).

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Whether searching for gene expression information or studying the function of RNA species themselves, it is evident that there is no better marker of RNA than RNA itself. DNA and protein surrogates do not always correlate with their RNA expression patterns due to pre- and post-translational modifications, but until now these biomarkers have been predominantly analysed following the discovery phase, since routine RNA analysis just wasn’t good enough. A method was required that was both high-throughput and would fit simply into the current pathology workflow for validation and translation into diagnosis. ACD’s RNAscope assay technology fills that gap by overcoming the pitfalls of other methods, while providing a direct path from discovery to clinical assays by maintaining biomarkers at the RNA level. Next generation sequencing approaches will continue to fuel RNA biomarker discoveries and the need for RNA biomarker validation within tissue morphology will continue to increase in order to fully understand disease relevance and the complex biology of the marker. RNAscope is an ideal platform that can be used downstream of NGS and microarrays for translating new scientific advances into clinical research. 

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