The human tumor samples used in this study were deidentified and acquired from commercial sources in accordance with the local ethical guidelines for human research.
1. Sample, Equipment, and Reagent Preparation
<ol>
	<li>FFPE Sample Preparation
	<ol>
		<li>Tissues
		<ol>
			<li>Immediately following dissection, fix the tissue (cut into blocks of 3&#8211;4 mm in thickness) in 10% neutral-buffered formalin (NBF) for 16&#8211;32 h at room temperature (RT). 
			NOTE: Fixation time will vary depending on tissue type and size.</li>
			<li>Wash the sample with 1x phosphate-buffered saline (PBS) and dehydrate using a standard ethanol (EtOH) series (70% EtOH for 30&#8211;60 min, 80% EtOH for 30&#8211;60 min, 90% EtOH for 30&#8211;60 min, 95% EtOH for 30&#8211;60 min, 3x 100% for EtOH 30&#8211;60 min) followed by xylene.</li>
			<li>Embed the sample in paraffin using standard procedures6 and trim the paraffin blocks as needed to remove excess paraffin. 
			NOTE: Block size will vary depending on the tissue sample size, but a typical size is 0.75&#160;x 0.75 inches2&#160;or smaller.</li>
		</ol>
		</li>
		<li>Cell lines
		<ol>
			<li>Collect and pellet cells according to the recommended methods for the specific cell line.</li>
			<li>Fix cells in 10% formaldehyde at RT for 24 h on a rotator.</li>
			<li>Prepare cells as a pellet in prewarmed processing gel (e.g., Histogel). Allow the pellet to solidify by placing the gel-cell pellet onto a piece of parafilm on ice, and let it sit for 2&#8211;3 min. Submerge the gel-cell pellet in 1x PBS.</li>
			<li>Dehydrate and embed the cell pellets as described in step 1.1.1.</li>
		</ol>
		</li>
		<li>Section preparation
		<ol>
			<li>Cut the embedded tissue/cells into 5 &#177; 1 &#956;m sections using a microtome, mount the sections on electrostatically adhesive glass slides, and air-dry them overnight at RT. 
			NOTE: The slides can be stored at RT under desiccation for up to 3 months.</li>
			<li>Place the slides in a slide rack and bake the mounted tissue slides in a circulating air oven at 60 &#176;C for 1 h before performing the assay. 
			NOTE: Use the slides immediately or store them at RT with desiccants for up to 1 week. Prolonged storage may result in RNA degradation.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Equipment preparation
	<ol>
		<li>Set the hybridization oven to 40 &#176;C. Thoroughly wet the humidifying paper and remove any residual dH2O. Insert the paper into the humidity control tray, and insert the tray into the hybridization oven to prewarm for at least 30 min before use.</li>
	</ol>
	</li>
	<li>Reagent preparation
	<ol>
		<li>Fill two clearing agent dishes with 200 mL of xylene and fill two staining dishes with 200 mL of 100% EtOH, which will be used for deparaffinization of sections.</li>
		<li>Prepare 200 mL of commercially available 1x target retrieval reagent by adding 180 mL of dH2O to 20 mL of 10x target retrieval reagent. Place two slide holders in a steamer. Fill one slide holder with 200 mL of 1x target retrieval reagent and fill the other slide holder with 200 mL of dH2O. Heat both solutions to a boil using the steamer.</li>
		<li>Warm 50x wash buffer to 40 &#176;C for 10&#8211;20 min. Prepare 3 L of 1x wash buffer by diluting 60 mL of prewarmed 50x wash buffer with 2.94 L of dH2O.</li>
		<li>In a fume hood, prepare 50% Gill&#8217;s Hematoxylin counterstaining solution by adding 100 mL of Gill&#8217;s Hematoxylin to 100 mL of dH2O. In the fume hood, prepare bluing reagent (0.02% (w/v) ammonia water) by adding 1.43 mL of 28&#8211;30% ammonium hydroxide to 250 mL of dH2O.</li>
		<li>Prewarm target probes at 40 &#176;C for 10 min prior to probe hybridization and bring the amplifying reagents (AMP 0&#8211;6) to RT.</li>
	</ol>
	</li>
</ol>
2. RNA In Situ Hybridization Assay
<ol>
	<li>Deparaffinization and dehydration
	<ol>
		<li>After baking as described in step 1.1.3.2 (optional stopping point 1), deparaffinize the sections in xylene for 5 min with agitation. Deparaffinize again in fresh xylene for 5 min. Dehydrate in 100% EtOH for 2 min with agitation, and repeat again in fresh 100% EtOH for 2 min.</li>
		<li>Air dry the slides for 5 min at 60 &#176;C in a circulating air oven or at RT until they are completely dry (optional stopping point 2).</li>
	</ol>
	</li>
	<li>Sample pretreatments
	<ol>
		<li>Incubate the sections with ~4 drops of ready-to-use hydrogen peroxide for 10 min at RT to quench the endogenous peroxidase activity. Decant the solution from the slides and rinse them twice with dH2O.</li>
		<li>Incubate the sections with 200 mL of the target retrieval reagent for 15&#8211;30 min at 100 &#176;C in a steamer. 
		NOTE: Incubation time may vary depending on tissue type. In this protocol, target retrieval was performed for 15 min for both tumor samples and cell pellets.</li>
		<li>Decant the solution from the slides, rinse twice with dH2O, dip in 100% EtOH for 3 min, and dry the slides at 60 &#176;C in a circulating air oven or at RT until completely dry.</li>
		<li>Draw a hydrophobic barrier around the section using a hydrophobic pen, approximately 0.75&#160;x 0.75 inches2. 
		NOTE: It is not recommended to draw a smaller barrier. For larger sections, a larger barrier will need to be drawn.</li>
		<li>Let the barrier dry completely for 1 min, or leave overnight at RT (optional stopping point 3).</li>
		<li>Place the slides in a slide holder and place the slide holder in the humidity control tray. Add ~4 drops of Protease III to each slide and incubate them for 15&#8211;30 min at 40 &#176;C in the hybridization oven for protein digestion. 
		Note: Incubation time may vary depending on the tissue type. In this protocol, protease digestion was performed for 30 min for tumor samples and 15 min for cell pellets.</li>
		<li>Decant the solution from the slides and rinse them twice with dH2O.</li>
	</ol>
	</li>
	<li>Probe hybridization
	<ol>
		<li>Add ~4 drops of the appropriate ready-to-use probe solution to cover the entire section. If using larger sections, add ~5&#8211;6 drops.</li>
		<li>Hybridize the probes for 2 h at 40 &#176;C in the hybridization oven. Decant the solution from the slides and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure in this step.</li>
	</ol>
	</li>
	<li>Signal amplification
	<ol>
		<li>Incubate the sections with ~4 drops of AMP 0 per slide at 40 &#176;C in the hybridization oven for 30 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure in this step.</li>
		<li>Incubate the sections with ~4 drops of AMP 1 per slide at 40 &#176;C in the hybridization oven for 15 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure.</li>
		<li>Incubate the sections with ~4 drops of AMP 2 per slide at 40 &#176;C in the hybridization oven for 30 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure.</li>
		<li>Incubate the sections with ~4 drops of AMP 3 per slide at 40 &#176;C in the hybridization oven for 30 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure.</li>
		<li>Incubate the sections with ~4 drops of AMP 4 per slide at 40 &#176;C in the hybridization oven for 15 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure.</li>
		<li>Incubate the sections with ~4 drops of AMP 5 per slide at RT for 30 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure.</li>
		<li>Incubate the sections with ~4 drops of AMP 6 per slide at RT for 15 min. Decant the solution and wash the slides in 200 mL of 1x wash buffer for 2 min at RT with occasional agitation. Repeat the washing procedure.</li>
	</ol>
	</li>
	<li>Signal detection
	<ol>
		<li>Prepare the Fast Red dye working solution. For one slide with a 0.75 x 0.75 inches2 barrier, add 2 &#956;L of the Fast Red-B dye to 120 &#956;L of Fast Red-A into a tube and mix well. 
		NOTE: Depending on the size of the hydrophobic barrier and number of slides, volumes of the Fast Red working solution will vary.</li>
		<li>Decant the excess liquid from the slides and add the Fast Red dye working solution to the slides. Incubate the slides for 10 min at RT in the tray with a cover to avoid light exposure. 
		NOTE: Use the Fast Red working solution within 5 min of preparation, and do not expose it to direct sunlight or UV light.</li>
		<li>Decant the Fast Red solution and rinse the slides twice with tap water. Place the slides back in a slide rack.</li>
	</ol>
	</li>
	<li>Counterstaining
	<ol>
		<li>Counterstain the tissue sections with 50% Gill&#8217;s Hematoxylin solution for 2 min at RT. Wash the slides with tap water and repeat this several times until the slides are clear, while the sections remain purple. Dip the slides into 0.02% ammonia water for bluing (dip 2&#8211;3 times). Replace the ammonia water with tap water and wash the slides 3&#8211;5 times.</li>
	</ol>
	</li>
	<li>Slide mounting
	<ol>
		<li>Dry the slides in a circulating air oven at 60 &#176;C for 15 min or at RT until completely dry. Place 1&#8211;2 drops of mounting reagent on each slide and place coverslips over each section. Avoid any trapping of air bubbles. Air dry the slides for at least 5 min. 
		NOTE: The Fast Red substrate is alcohol-sensitive. Do not dehydrate the slides in alcohol.</li>
	</ol>
	</li>
	<li>Visualization
	<ol>
		<li>Observe the slides under a standard brightfield microscope.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Bolha, L., Ravnik-Glava&#269;, M., Glava&#269;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs:+Biogenesis,+Function,+and+a+Role+as+Possible+Cancer+Biomarkers.">Circular RNAs: Biogenesis, Function, and a Role as Possible Cancer Biomarkers.</a> International Journal of Genomics. 2017, 6218353 (2017).</li><li>Yamada, A., Yu, P., Lin, W., Okugawa, Y., Boland, C. R., Goel, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+RNA-Sequencing+approach+for+the+identification+of+novel+long+non-coding+RNA+biomarkers+in+colorectal+cancer.">A RNA-Sequencing approach for the identification of novel long non-coding RNA biomarkers in colorectal cancer.</a> Scientific Reports. 8 (1), 575 (2018).</li><li>Erben, L., He, M. X., Laeremans, A., Park, E., Buonanno, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Ultrasensitive+In+Situ+Hybridization+Approach+to+Detect+Short+Sequences+and+Splice+Variants+with+Cellular+Resolution.">A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution.</a> Molecular Neurobiology. , (2017).</li><li>Mahmood, R., Mason, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In-situ+hybridization+of+radioactive+riboprobes+to+RNA+in+tissue+sections.">In-situ hybridization of radioactive riboprobes to RNA in tissue sections.</a> Methods in Molecular Biology. 461, 675-686 (2008).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> Journal of Molecular Diagnostics. 14 (1), 22-29 (2012).</li><li>Paraffin processing of tissue. Protocols Online Available from: <a target="_blank" href="https://www.protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/">https://www.protocolsonline.com/histology/sample-preparation/paraffin-processing-of-tissue/</a> (2016)</li><li>An, Z., Aksoy, O., Zheng, T., Fan, Q. W., Weiss, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+growth+factor+receptor+and+EGFRvIII+in+glioblastoma:+signaling+pathways+and+targeted+therapies.">Epidermal growth factor receptor and EGFRvIII in glioblastoma: signaling pathways and targeted therapies.</a> Oncogene. , (2018).</li><li>Frampton, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activation+of+MET+via+diverse+exon+14+splicing+alterations+occurs+in+multiple+tumor+types+and+confers+clinical+sensitivity+to+MET+inhibitors.">Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors.</a> Cancer Discovery. 5 (8), 850-859 (2015).</li><li>Awad, M. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MET+Exon+14+Mutations+in+Non-Small-Cell+Lung+Cancer+Are+Associated+With+Advanced+Age+and+Stage-Dependent+MET+Genomic+Amplification+and+c-Met+Overexpression.">MET Exon 14 Mutations in Non-Small-Cell Lung Cancer Are Associated With Advanced Age and Stage-Dependent MET Genomic Amplification and c-Met Overexpression.</a> Journal of Clinical Oncology. 34 (7), 721-730 (2016).</li><li>Hammond, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=American+Society+of+Clinical+Oncology/College+of+American+Pathologists+guideline+recommendations+for+immunohistochemical+testing+of+estrogen+and+progesterone+receptors+in+breast+cancer.">American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer.</a> Archives of Pathology and Laboratory Medicine. 134 (6), 907-922 (2010).</li><li>Gan, H. K., Cvrljevic, A. N., Johns, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidermal+growth+factor+receptor+variant+III+(EGFRvIII):+where+wild+things+are+altered.">The epidermal growth factor receptor variant III (EGFRvIII): where wild things are altered.</a> Federation of European Biochemical Societies Journal. 280 (21), 5350-5370 (2013).</li><li>Wheeler, S. E., Egloff, A. M., Wang, L., James, C. D., Hammerman, P. S., Grandis, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+EGFRvIII+detection+in+head+and+neck+squamous+cell+carcinoma.">Challenges in EGFRvIII detection in head and neck squamous cell carcinoma.</a> Public Library of Science One. 10 (2), e0117781 (2015).</li><li>Zhu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Junction-specific+and+Quantifiable+In+Situ+Detection+of+AR-V7+and+its+Clinical+Correlates+in+Metastatic+Castration-resistant+Prostate+Cancer.">Novel Junction-specific and Quantifiable In Situ Detection of AR-V7 and its Clinical Correlates in Metastatic Castration-resistant Prostate Cancer.</a> European Urology. , (2017).</li><li>Piwecka, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+a+mammalian+circular+RNA+locus+causes+miRNA+deregulation+and+affects+brain+function.">Loss of a mammalian circular RNA locus causes miRNA deregulation and affects brain function.</a> Science. , (2017).</li><li>Rev&#234;chon, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+progerin-expressing+preadipocytes+and+adipocytes+contribute+to+tissue+depletion+over+time.">Rare progerin-expressing preadipocytes and adipocytes contribute to tissue depletion over time.</a> Scientific Reports. 7 (1), 4405 (2017).</li><li>Fu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+ectopic+neuritogenesis+by+retinal+rod+bipolar+cells+is+regulated+by+miR-125b-5p+during+retinal+remodeling+in+RCS+rats.">Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats.</a> Scientific Reports. 7 (1), 1011 (2017).</li><li>Yates, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subclonal+diversification+of+primary+breast+cancer+revealed+by+multiregion+sequencing.">Subclonal diversification of primary breast cancer revealed by multiregion sequencing.</a> Nature Medicine. 21 (7), 751-759 (2015).</li><li>Baker, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Robust+RNA-based+in+situ+mutation+detection+delineates+colorectal+cancer+subclonal+evolution.">Robust RNA-based in situ mutation detection delineates colorectal cancer subclonal evolution.</a> Nature Communications. 8 (1), 1998 (2017).</li></ol>

All authors are employed by Advanced Cell Diagnostics, Inc.

In this report, the novel ISH assay protocol and its applications were discussed in detail. The assay allows for the direct visualization of exon junctions, short-target and highly homologous sequences, and point mutations in the tissue context. The assay is based on RNAscope technology5 and is therefore capable of single molecule detection. However, due to an advanced amplification system, the signal can be detected with probes containing as little as one double-Z pair or with a target template length of just 50 nucleotides. Because the probes may be as short as a single double-Z in length, this allows for the detection of exon junctions, short-target and highly homologous sequences, and point mutations (Table 1).
For successful performance of the assay, there are several technical recommendations. First, tissues should be fixed in fresh 10% neutral-buffered formalin (NBF) at room temperature for 16&#8211;32 h10. Underfixation (&#60;16 h) or overfixation (&#62;32 h) will impair performance of the assay and may require additional optimization. Second, to ensure optimal control of temperature and humidity, which are required for robust probe hybridization and signal amplification, the slide processing system and hybridization oven should be used for protocol steps 2.3 to 5 (protease pretreatment, probe hybridization, signal amplification, and signal detection). Third, excess residual buffers should be properly decanted before each step throughout the protocol (but not so much so that the tissue sections dry out). If the slides dry out, significant non-specific signal will develop. Fourth, depending on the tissue type, pretreatment optimization may be necessary. Using the wrong protease or performing for a suboptimal length of time may result in under- or over-digestion and will negatively affect the signal. Lastly, it is important to always run positive and negative controls with the test probes. Negative control probes ensure that there is no background signal, and positive control probes ensure that the assay has been done correctly and that the RNA quality in the sample is optimal for interpreting test probe results. If there is no signal with the positive control probe, then the RNA quality in the sample is likely suboptimal and a signal may not be seen with the test probe.
In addition to the manual assay, the ability to perform the assay on automated stainers was also demonstrated (Figure 6). This automated ISH assay yields a high signal-to-noise ratio and is applicable for the same applications as shown in Table 1; however, benefits of an automated assay include standardization of assay conditions, minimization of inter-user variability and hands-on time, and allowance for high-throughput screening of tissue samples in a reliable manner.
While immunohistochemistry (IHC) and qRT-PCR allow for detection of splice variants (particularly EGFRvIII), in FFPE clinical specimens, these techniques can lack the necessary specificity and do not offer insight into the spatial resolution of the splice variant expression, respectively11,12. A key advantage of the assay in this protocol is its highly sensitive and specific visualization of splice junctions while preserving the morphological tissue context. Here, the assay&#39;s ability to precisely target unique exon junctions for several splice variants, including EGFRvIII and MET&#916;14, was demonstrated (Figures 3 and 6). In addition, the assay has been shown to detect the splice variant AR-V7 in prostate cancer, multiple isoforms of ErbB4 in the brain, and confirmation of knockout of circular RNA Cdr1as in the mouse brain3,13,14.
Short target sequence detection by this ISH assay allows for visualization of RNA sequences as short as 50 nucleotides in length, as demonstrated by the detection of CDR3 sequences derived from Jurkat cells (Figure 3). The assay can also detect gene sequences that are highly homologous to other family members or species, as shown by Rev&#234;chon et al., who used the short target assay to detect human progerin expressed in mouse subcutaneous white adipose tissue15. In addition, small nucleolar RNA (snoRNA), CRISPR-mediated gene editing, and precursor-microRNA can be detected in situ with the short target assay. More recently, Fu et al. combined this ISH assay with IHC to identify precise cells in the retina expressing the pre-miRNA mir125b16.
Mutation profiling in tumors is critical for studying the progression of tumors and for developing targeted therapies. While mutation profiling can be achieved via high-throughput sequencing, this technology cannot fully address intratumoral heterogeneity or link genetic alterations with cellular morphology17,18. The detection of point mutations using this ISH assay allows for distinction of RNA target sequences at a single-base resolution, as validated by the detection of EGFR L858R and KRAS G12A single nucleotide variations in cell lines (Figure 5). Furthermore, Baker et al. used the point mutation assay to target multiple mutations in the BRAF, KRAS, and PIK3CA oncogenes in colorectal cancer18. They were able to identify and spatially map rare mutant subclones of tumor cells, ultimately showing how they contribute to intra-tumor heterogeneity.
In summary, a specialized RNA ISH assay has been developed. This methodology allows for the detection of splice variants, short sequences, and mutations in situ. It is sensitive, specific, quantifiable, and adaptable to performance both by manual methods and on automated stainers.

High-throughput transcriptomic technologies such as microarrays and next-gen RNA sequencing (RNAseq) have exponentially improved the discovery of RNA biomarkers with diagnostic, prognostic, and predictive clinical value for various diseases including cancer1,2. To advance the use of these biomarkers in precision medicine, there is a high need for standardized and robust technologies that can measure RNA biomarkers within the tissue context of clinical samples. While widely established grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, the required tissue homogenization and RNA isolation imply the loss of in vivo cell-type specificity and morphological information3. Conventional in situ RNA detection methodologies lack the sensitivity and specificity required to reliably measure rare or low-expressing RNA biomarkers within the tissue context4.
A commercial in situ hybridization (ISH) assay (e.g., the&#160;RNAscope assay) is one technology that has tackled these challenges and also enables the highly sensitive and specific visualization of single RNA molecules greater than 300 nucleotides within the tissue morphological context. This type of assay uses a unique oligonucleotide probe design of approximately 6&#8211;20 double-Z probe pairs combined with an advanced hybridization-based signal amplification5.
This study describes a specialized RNA ISH assay, BaseScope, complementary to the previously designed commercial technology that can detect RNA target sequences as short as 50 nucleotides at&#160;single nucleotide resolution. This assay addresses the intricate complexities of the transcriptome and is applicable for the accurate detection of exon junctions, short target sequences, and point mutations in the tissue context (Table 1) using as little as one double-Z probe pair. This report demonstrates the complete assay protocol and its use in the detection of splice variants, CDR3 sequences for T-cell receptor clones, and single-nucleotide mutations in FFPE cell lines and tumor tissues.

<table border="0" cellpadding="0" cellspacing="0" width="1692">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">HybEZ Oven (110 or 220 VAC) or HybEZ II Oven (110 or 220V)</td>
			<td style="width:255px;">ACD</td>
			<td style="width:172px;">310010 or 310013 (HybEZ&#8482;), 321710 or 321720 (HybEZ&#8482; II)</td>
			<td style="width:571px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">HybEZ Humidity Control Tray (with lid)</td>
			<td style="width:255px;">ACD</td>
			<td style="width:172px;">310012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">ACD EZ-Batch Slide Rack (20 slide capacity) 1 rack 310017</td>
			<td style="width:255px;">ACD</td>
			<td style="width:172px;">310017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">HybEZ Humidifying Paper</td>
			<td style="width:255px;">ACD</td>
			<td style="width:172px;">310015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">ImmEdge Hydrophobic Barrier Pen (required)&#160;</td>
			<td style="width:255px;">Vector Laboratory&#160;</td>
			<td style="width:172px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">SuperFrost Plus Slides (required)&#160;</td>
			<td style="width:255px;">Fisher Scientific&#160;</td>
			<td style="width:172px;">12-550-15</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">10% neutral-buffered formalin (NBF)&#160;</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Paraffin wax</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Microtome</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:695px;">Gill&#8217;s Hematoxylin I&#160;</td>
			<td style="width:255px;">American Master Tech Scientific/MLS</td>
			<td style="width:172px;">&#160;HXGHE1LT</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Xylene&#160;</td>
			<td style="width:255px;">Fisher Scientific/MLS</td>
			<td style="width:172px;">X3P-1GAL</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Tissue-Tek Vertical 24 Slide Rack&#160;</td>
			<td style="width:255px;">American Master Tech Scientific/MLS</td>
			<td style="width:172px;">LWSRA24</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:695px;">Tissue-Tek Staining Dishes&#160;</td>
			<td style="width:255px;">American Master Tech Scientific/MLS</td>
			<td style="width:172px;">LWT4457EA</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:695px;">Tissue-Tek Clearing Agent Dishes, xylene resistant&#160;</td>
			<td style="width:255px;">American Master Tech Scientific/MLS</td>
			<td style="width:172px;">LWT4456EA</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:695px;">100% alcohol (EtOH)&#160;</td>
			<td style="width:255px;">American Master Tech Scientific/MLS</td>
			<td style="width:172px;">ALREACS</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">VectaMount Permanent Mounting Medium (required)</td>
			<td style="width:255px;">Vector Labs&#160;</td>
			<td style="width:172px;">H-5000</td>
			<td></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:695px;">Cover Glass, 24 x 50 mm</td>
			<td style="width:255px;">Fisher Scientific/MLS&#160;</td>
			<td style="width:172px;">12-545-F</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:695px;">Ammonium hydroxide, 28&#8211;30%</td>
			<td style="width:255px;">Sigma-Aldrich/MLS</td>
			<td style="width:172px;">320145-500mL</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Carboy (&#62;3L)</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:695px;">Oster Steamer Model 5712, Black and Decker Steamer HS3000, or the Braun Multiquick FS 20 Steamer</td>
			<td style="width:255px;">/</td>
			<td style="width:172px;">/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Digital thermometer</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Water bath or incubator, capable of holding temperature at 40 +/&#8211; 1&#176;C</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Pipettors and tips, 1&#8211;1000 &#956;L</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Distilled water</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Tubes (various sizes)</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Fume hood</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Graduated cylinder</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Parafilm</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Paper towel or absorbent paper</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Microcentrifuge</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Microscope and accessories</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Drying oven, capable of holding temperature at 60 +/&#8211; 1&#176;C</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Formaldehyde</td>
			<td style="width:255px;">MLS</td>
			<td style="width:172px;">/</td>
			<td>MLS Major Laboratory Supplier in North America. For other regions, please check Catalog Numbers with your local lab supplier.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">Histogel</td>
			<td style="width:255px;">Fisher Scientific/MLS</td>
			<td style="width:172px;">22-110-678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Reagent Kit - RED</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">322900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Hs-EGFR-E1E2</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">701701</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Hs-EGFR-E1E8</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">701711</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Hs-EGFR-E7E8</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">701721</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Hs-EGFR-E8E9</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">701731</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Hs-MET-E14E15</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">701811</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Hs-MET-E13E15</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">701801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BaseScope Hs-KRAS-G12A</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td>705491</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BaseScope Hs-KRAS-G12-nt35WT</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td>705531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BaseScope Hs-EGFR-L858R</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td>705451</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BaseScope Hs-EGFR-L858WT</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td>705461</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:695px;">BaseScope Control Probe Pack Human</td>
			<td style="width:255px;">Advanced Cell Diagnostics</td>
			<td style="width:172px;">322975</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualizing genetic variants, short targets, and point mutations in the morphological tissue context with an rna in situ hybridization assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

The in situ hybridization assay workflow: 
The workflow is depicted in Figure 1 and consists of four parts: permeabilization of cells or tissues with target retrieval and protease solutions, hybridization of the probes to the target RNA, signal amplification, and visualization of the signal. The signal can also be quantified using digital imaging software systems or in a semi-quantitative manner based on the number of dots per cell. The manual procedure described in Figure 2 has also been fully automated in commercial auto-staining systems.
Representative staining for exon junction detection (EGFRvIII splice variant): EGFRvIII is a variant of the epidermal growth factor receptor that arises from an in-frame genomic deletion of exons 2 to 7, leading to constitutively active oncogenic signaling7. The assay was used to identify EGFRvIII status in FFPE glioblastoma (GBM) tumor samples. Single double-Z probes were designed to span the exon junctions in order to detect either WT, mutant, or both transcripts (Figure 3A). The WT EGFR probes span the junctions of either exons 1 and 2 (E1/E2) or exons 7 and 8 (E7/E8), while the EGFRvIII-specific probes span the junction of exons 1 and 8 (E1/E8). A common probe that spans the junction of exons 8 and 9 (E8/E9) was also used to detect total EGFR (both WT and EGFRvIII transcripts). All the probes were then used to determine EGFR status in FFPE GBM samples. The two representative examples shown in Figure 3B were taken from a larger study. EGFR status was confirmed by an independent method, RT-PCR. Both WT probes detected signal in both samples, indicating that the two samples express WT EGFR (Figure 3B). However, the mutant probe only showed signal detection in the EGFRvIII+ sample, confirming that this sample is indeed positive for the EGFRvIII variant (Figure 3B, left panels). Conversely, the mutant probe did not detect signal in the EGFRvIII- sample (Figure 3B, right panels). Taken together, these results demonstrate that the exon junction assay can identify EGFRvIII status in GBM FFPE tumor samples.
Representative staining for short targets: 
The CDR3, or complementary determining region 3, is a highly variable domain in T-cell receptors. Typically, the CDR3 sequence are quite short; for example, the CDR3 &#945; and &#946; sequences from the Jurkat T-cell line are 51 and 48 nucleotides in length, respectively (Figure 4A). To identify the specific CDR sequences expressed in Jurkat cells, antisense probes for CDR3 &#945; and &#946; that are expressed in the Jurkat T-cell line were generated, in addition to sense probes for CDR3 &#945; and &#946; to serve as negative control probes. All the probes were then tested in FFPE-prepared Jurkat cells with the assay. Robust staining was observed with anti-sense probes for both CDR3 &#945; and &#946; in the Jurkat cells, whereas sense probes detected little to no signal (Figure 4B). These results demonstrate the ability of the short target assay to discern between highly variable but short CDR sequences for T-cell receptor clones.
Representative staining for point mutation EGFR L858R: 
Point mutation probes were developed to detect single nucleotide variations and small insertions or deletions (INDELs) in the tumor context. Figure 5A demonstrates the ability for in situ detection of the point mutation EGFR L858R (2573T&#62;G). Two probes were designed: one to detect the L858R mutated EGFR sequence, and another to detect the EGFR L858 WT sequence. Both probes were tested in two FFPE-prepared cell lines: H2229, which only expresses EGFR L858 WT; and H1975, which is heterozygous for the EGFR L858R mutation. The L858R mutant probe detected signal only in the H1975 cell line but not in the H2229 cell line. However, the WT probe detected signal in both cell lines. Similarly, Figure 5B visualizes in situ detection of the point mutation KRAS G12A (35 G&#62;C). Two probes were designed to detect the KRAS G12A MT and KRAS G12 WT sequences and then tested on the HuT78 cell line (which only expresses KRAS G12 WT) and the SW116 cell line (which is heterozygous for the KRAS G12A mutation). While the KRAS G12 WT probe detected signal in both cell lines, the KRAS G12A probe only detected signal in the SW116 cell line. Taken together, these data demonstrate the technical capability of the point mutation assay in detecting single nucleotide polymorphisms in the cell and tissue context.
Representative staining for the automated in situ assay: 
Automated assays allow for a greater number of samples to be run more reliably, minimizing inter-user variability and hands-on time and generating consistently reproducible results. Therefore, an automated version of the assay was developed. To demonstrate automated staining with this assay, detection of the splice variant MET&#916;14 was examined. This variant is the result of exon 14 in the MET gene being skipped during pre-mRNA splicing, which leads to constitutive activation and oncogenic transformation of the MET receptor8,9. To specifically detect the MET&#916;14 variant, two exon junction probes were designed: one that spans the junction of exons 13 and 15 (E13/E15) to detect the MET&#916;14 variant transcript, and another that spans the junction of exons 14 and 15 (E14/E15) to detect the WT MET transcript (Figure 6A). Both probes were then tested in 2 FFPE-prepared cell lines: H596, which expresses the MET&#916;14 variant, and A549, which expresses the WT MET gene. Both probes showed mutually exclusive expression patterns, with the E13/E15 probe only detecting signal in the H596 cells and the E14/E15 probe only detecting signal in the A549 cells (Figures 6B and 6C). Lastly, the probe for dapB showed no signal, indicating no background signal (Figures 6B and 6C). Overall, this data demonstrates specific detection of the MET variant MET&#916;14 in situ utilizing the automated BaseScope assay.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58097/58097fig1.jpg" /> 
Figure 1: The assay workflow. The workflow consists of 4 major steps: pretreatment to permeabilize cells or tissue, probe hybridization to target RNA, signal amplification, and signal detection by visualization under brightfield or fluorescent microscope. Individual dots can be quantified using a digital image analysis platform. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58097/58097fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58097/58097fig2.jpg" /> 
Figure 2: Illustration of the manual assay protocol. The entire assay can be completed in 9 h. Pretreatment times may vary depending on tissue type, so it is advised to consult Appendix A in the user manual for tissue pretreatment recommendations regarding incubation time. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58097/58097fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58097/58097fig3.jpg" /> 
Figure 3: Representative images of exon junction detection. (A) Shown here are the exon organization for EGFR WT and EGFRvIII transcripts and a schematic depicting double-Z exon junction probes that straddle the junction to detect EGFR WT or EGFRvIII. (B) The exon junction assay was performed on two FFPE glioblastoma samples using the probes listed in (A), as well as a positive control probe, POLR2A, and negative control probe, dapB. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58097/58097fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58097/58097fig4.jpg" /> 
Figure 4: Representative images of short target sequence detection. (A) Shown here are CDR3 sequences in Jurkat cells. The sequence in black is a flanking common sequence and the sequence in red is unique to either CDR3&#945; or CDR3&#946;. Single double-Z short target sequence probes were designed against these sequences. (B) The short target assay was performed on Jurkat cells prepared as an FFPE cell pellet using anti-sense or sense probes targeting the sequences in (A), and dapB was used as a negative control. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58097/58097fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58097/58097fig5.jpg" /> 
Figure 5: Representative images of point mutation detection. (A) The assay was performed on H2229 cells (homozygous for EGFR L858) and H1975 cells (heterozygous for the EGFR L858R mutation) prepared as an FFPE cell pellet using single double-Z point mutation probes targeting the EGFR L858 WT sequence or EGFR L858R mutated sequence. (B) The point mutation assay was performed on HuT78 cells (homozygous for KRAS G12A) and SW116 cells (heterozygous for the KRAS G12A mutation) prepared as an FFPE cell pellet using single double-Z point mutation probes targeting the KRAS G12 WT sequence or KRAS G12A mutated sequence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58097/58097fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58097/58097fig6.jpg" /> 
Figure 6: Representative images of automated staining with the exon junction assay. (A) Shown here is the exon organization for MET WT and MET&#916;14 transcripts and a schematic depicting single double-Z exon junction probes that straddle the junction to detect MET WT or MET&#916;14. (B) and (C) The automated exon junction assay was performed on H596 cells (expressing MET&#916;14) and A549 cells (expressing MET WT) prepared as an FFPE cell pellet using the probes listed in (A), as well as negative control probe dapB. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58097/58097fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Exon Junction</td>
			<td>Short Sequence</td>
			<td>Point Mutation</td>
		</tr>
		<tr>
			<td>Splice variant/isoform</td>
			<td>Sequences between 50 and 300 nt</td>
			<td>Point mutation</td>
		</tr>
		<tr>
			<td>Circular RNA (circRNA)</td>
			<td>Highly homologous sequences</td>
			<td>Short indel</td>
		</tr>
		<tr>
			<td>Gene fusion</td>
			<td>CDR3 sequence for TCR clones&#160;</td>
			<td>Gene editing</td>
		</tr>
		<tr>
			<td>Gene knockout (KO)</td>
			<td>Pre-miRNA</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Small nucleolar RNA (snoRNA)</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td>Gene editing</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Applications of the in situ assay. There are 3 main categories for applications of this assay: exon junction, short target sequence, and point mutation. Listed in each column are some specific application examples for each category.

Watch this Scientific Journal Video about Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay at JoVE.com

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

visualizing-genetic-variants-short-targets-point-mutations

Nanyang Technological University

Research

JoVE Journal

Biology

10.5K Views.  Advanced Cell Diagnostics, Inc. Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

Video: Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Double Whole Mount in situ Hybridization of Early Chick Embryos

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

Assay for Neural Induction in the Chick Embryo

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying the formation and initial patterning of the nervous system. This video demonstrates how to graft organizer tissue into a host, a method by which Hensen s node (the organizer in the chick embryo) is grafted to a host competent ectoderm. The organizer graft instructs overlying na ve tissue to adopt a neural fate via neural inducing signals. This mechanism is referred to as neural induction, and constitutes the initial step in the formation of brain and spinal cord in amniotes. This method is essentially used for the characterization of putative neural inducing molecules in chick. This video demonstrates the different steps in the assay for neural induction; First, the donnor embryo is explanted and pinned on a dish. Then, the host embryo is prepared for New culture. The graft is excised and transplanted to the host area pellucida margin. The host is cultured for 18-22 hrs. The assembly is fixed and processed for further applications (e.g. in situ hybridization). This method was originally devised by Waddington 1,2 and Gallera 3,4.

Neural induction is the first step in the formation of the brain. It is a mechanism by which Hensen's node (organizer), instructs adjacent tissue to adopt a neural fate, i.e. to give rise to the nervous system. This video demonstrates an assay for neural induction in chick embryo.

Visualizing RNA Localization in Xenopus Oocytes

RNA localization is a conserved mechanism of establishing cell polarity. Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to spatially restrict gene expression of Vg1 protein. Tight control of Vg1 distribution in this manner is required for proper germ layer specification in the developing embryo. RNA sequence elements in the 3' UTR of the mRNA, the Vg1 localization element (VLE) are required and sufficient to direct transport. To study the recognition and transport of Vg1 mRNA in vivo, we have developed an imaging technique that allows extensive analysis of trans-factor directed transport mechanisms via a simple visual readout. 
To visualize RNA localization, we synthesize fluorescently labeled VLE RNA and microinject this transcript into individual oocytes. After oocyte culture to allow transport of the injected RNA, oocytes are fixed and dehydrated prior to imaging by confocal microscopy. Visualization of mRNA localization patterns provides a readout for monitoring the complete pathway of RNA transport and for identifying roles in directing RNA transport for cis-acting elements within the transcript and trans-acting factors that bind to the VLE (Lewis et al., 2008, Messitt et al., 2008). We have extended this technique through co-localization with additional RNAs and proteins (Gagnon and Mowry, 2009, Messitt et al., 2008), and in combination with disruption of motor proteins and the cytoskeleton (Messitt et al., 2008) to probe mechanisms underlying mRNA localization.

Visualizing RNA Localization in Xenopus Oocytes

Visualization of in vivo RNA transport is accomplished by microinjection of fluorescently labeled RNA transcripts into Xenopus oocytes, followed by confocal microscopy.

RNA localization is a conserved mechanism of establishing cell polarity.  Vg1 mRNA localizes to the vegetal pole of Xenopus laevis oocytes and acts to ...

Mutagenesis and Analysis of Genetic Mutations in the GC-rich KISS1 Receptor Sequence Identified in Humans with Reproductive Disorders

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in adulthood 1,2,3. Inactivating mutations in KISS1R identified in patients have been associated with iodiopathic hypogonadotropic hypogonadism (IHH) 1,2 and precocious puberty 4. Functional studies of these mutants are crucial for our understanding of the mechanisms underlying the regulation of reproduction by this receptor as well as those shaping the disease outcomes, which result from abnormal KISS1R signaling and function. However, the highly GC-rich sequence of the KISS1R gene makes it rather difficult to introduce mutations or amplify the gene encoding this receptor by PCR.
Here we describe a method to introduce mutations of interest into this highly GC-rich sequence that has been used successfully to generate over a dozen KISS1R mutants in our laboratory. We have optimized the PCR conditions to facilitate the amplification of a range of KISS1R mutants that include substitutions, deletions or insertions in the KISS1R sequence. The addition of a PCR enhancer solution, as well as of a small percentage of DMSO were especially helpful to improve amplification. This optimized procedure may be useful for other GC-rich templates as well.
The expression vector encoding the KISS1R is been used to characterize signaling and function of this receptor in order to understand how mutations may change KISS1R function and lead to the associated reproductive phenotypes. Accordingly, potential applications of KISS1R mutants generated by site-directed mutagenesis can be illustrated by many studies 1,4,5,6,7,8. As an example, the gain-of-function mutation in the KISS1R (Arg386Pro), which is associated with precocious puberty, has been shown to prolong responsiveness of the receptor to ligand stimulation 4 as well as to alter the rate of degradation of KISS1R 9. Interestingly, our studies indicate that KISS1R is degraded by the proteasome, as opposed to the classic lysosomal degradation described for most G protein-coupled receptors 9. In the example presented here, degradation of the KISS1R is investigated in Human Embryonic Kidney Cells (HEK-293) transiently expressing Myc-tagged KISS1R (MycKISS1R) and treated with proteasome or lysosome inhibitors. Cell lysates are immunoprecipitated using an agarose-conjugated anti-myc antibody followed by western blot analysis. Detection and quantification of MycKISS1R on blots is performed using the LI-COR Odyssey Infrared System. This approach may be useful in the study of the degradation of other proteins of interest as well.

Mutations in the kisspeptin receptor (KISS1R) are associated with reproductive disorders in patients. Here we describe how to introduce mutations of interest in the GC-rich sequence of KISS1R as well as the use of KISS1R constructs to characterize the degradation pathway of the receptor by immunoprecipitation and western blot.

The kisspeptin receptor (KISS1R) is a G protein-coupled receptor recognized as the trigger of puberty and a regulator of reproductive competence in ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...