This work was supported by the American Heart Association [Grant-in-Aid 0365274B to H.L.], National Cancer Institute [GI SPORE P50CA150964 to Z.W.], National Institutes of Health [Office of Research Infrastructure Shared Instrumentation Grant S10RR031845 to the Light Microscopy Imaging Facility at Case Western Reserve University], and China Scholarship Council [to X.G.]. 
The authors thank Richard Lee in the Light Microscopy Imaging Core for his help with slide scanning.

All of the experiments described here that involve mice have been approved by the Case Western Reserve University Institutional Animal Care and Use Committee. The title of the protocol 2016-0068 (PI: Hua Lou) is &#8220;Role of alternative pre-mRNA splicing in vertebrate development&#8221;.
NOTE: Information of all of the equipment, reagents and supplies used in this protocol is included in Table of Materials.
1. Prepare Formalin Fixed Paraffin Embedded (FFPE) Sections
<ol>
	<li>Euthanize 1 CD1 mouse and 1 C57BL/6J mouse by cervical dislocation. Carefully cut open the skull with surgical scissors and forceps. Remove the brain with a scoop. 
	NOTE: 5-month-old CD1 male mice and 3-month-old C57BL/6J male mice were used and showed similar results.
	<ol>
		<li>Wash the brain with PBS. Fix the whole brain with a solution containing 10% formalin at room temperature (RT) for 30 h by putting the whole brain in 50 mL of the formalin solution.</li>
		<li>Change the fixing solution to PBS and incubate the brain in PBS overnight at 4 &#176;C. Dehydrate and embed the brain with xylene and paraffin using standard procedures18.</li>
	</ol>
	</li>
	<li>Use a microtome to cut embedded tissue into 5 &#181;m sections. Put the paraffin ribbon in a RT water bath first, and then in a 45 &#176;C water bath to spread the ribbon.
	<ol>
		<li>When the wrinkles and folds in the ribbon disappear, immediately mount sections on glass slides. Air dry slides overnight at RT. Bake slides for 1 h at 60 &#176;C before use.</li>
	</ol>
	</li>
</ol>
2. Sample Pretreatment
<ol>
	<li>Deparaffinize tissue sections. Carry out these steps in a fume hood.
	<ol>
		<li>Fill two washing dishes (Figure 1A) with 250 mL of fresh xylene and two with 250 mL of 100% ethanol. Insert tissue slides into a washing rack (Figure 1A) and place the washing rack in washing dishes following the order and incubation time indicated in Table 1.</li>
		<li>During each incubation, lift the washing rack up and down more than 3 times in the washing dish. After each incubation, quickly move the washing rack into the next washing dish to prevent the tissue sections from drying out.</li>
		<li>After completing the incubation steps, remove the washing rack with slides from the washing dish. Place the rack in a 60 &#176;C incubator for 5 min or until the slides are completely dry.</li>
	</ol>
	</li>
	<li>Draw a 0.75&#8217;&#8217; x 0.75&#8217;&#8217; barrier surrounding the tissue section with a hydrophobic barrier pen. Air dry the barrier for 3 min at RT.</li>
	<li>Pretreat slices
	<ol>
		<li>Prepare reagents and equipment
		<ol>
			<li>Turn on the hybridization oven (Figure 1B) and set temperature to 40 &#176;C 30 min before use.</li>
			<li>Wet the humidifying paper (Figure 1E) with distilled water completely and put it in humidity control tray (Figure 1D, left). Keep the humidity control tray in the hybridization oven.</li>
			<li>Prepare 700 mL of fresh 1x Target Retrieval Reagents by adding 630 mL of dH2O to 70 mL of 10x Target Retrieval Reagents.</li>
		</ol>
		</li>
		<li>Apply hydrogen peroxide.
		<ol>
			<li>Put the deparaffinized slides on the bench. Add 5&#8211;8 drops of hydrogen peroxide to a section to completely cover the tissue. Incubate for 10 min at RT.</li>
			<li>Tap the slides on a piece of absorbent paper to remove the hydrogen peroxide solution, one slide at a time. Immediately insert the slide into a washing rack and move the rack to a washing dish filled with dH2O.</li>
			<li>Wash the slides by moving the rack up and down in distilled water for 3&#8211;5 times. Move the washing rack to a washing dish filled with fresh distilled water and wash slides one more time.</li>
		</ol>
		</li>
		<li>Perform target retrieval
		<ol>
			<li>Place a 1,000 mL glass beaker on a hot plate. Add 700 mL of fresh 1x Target Retrieval Reagents into the beaker and cover it with foil. Insert a thermometer in the beaker through the foil cover (Figure 1C). Turn on the hot plate and set on high for 10&#8211;15 min.</li>
			<li>When the temperature of retrieval reagents reaches 98 &#176;C, switch the temperature control of the hot plate from high to low to maintain a mild boil. Do not boil more than 15 min.</li>
			<li>Meanwhile, carefully open the foil and put the washing rack with slices inside the retrieval regent. Cover the beaker again and incubate for 15 min.</li>
			<li>Use forceps to remove the rack from the beaker to a washing dish filled with 200 mL of dH2O and rinse for 15 s.</li>
			<li>Move the rack to a washing dish containing 100% fresh ethanol and let sit for 3 min. Remove slides and dry them in a 60 &#176;C incubator for 10 min.</li>
		</ol>
		</li>
		<li>Apply Protease III
		<ol>
			<li>Place the slides on the stain rack (Figure 1D, right). Add 5 drops of Protease III to cover the tissue. Carefully put the rack in the humidity control tray (Figure 1D, left) and insert the tray into the 40 &#176;C hybridization oven. Incubate for 30 min.</li>
			<li>Remove the slide rack and put the tray back into the oven. Tap the slides to remove the excess liquid, one slide at a time. Insert the slide in a washing rack and place the rack in a washing dish filled with dH2O. Move the rack up and down to wash the slides for 3-5 times.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. ISH Assay
<ol>
	<li>Prepare materials
	<ol>
		<li>Pre-warm 50x wash buffer in a 40 &#176;C incubator for 20 min. Mix 2.94 L of dH2O and 60 mL of 50x wash buffer to make 1x wash buffer.</li>
		<li>Prepare 50% Hematoxylin staining solution by adding 100 mL of Gill&#8217;s hematoxylin I to 100 mL of dH2O. Prepare 0.02% (w/v) ammonia water by adding 1.43 mL of 1 N ammonium hydroxide to 250 mL of dH2O. Mix well in a container.</li>
		<li>Put the probes and AMP 0&#8211;6 reagents at RT 30 min before use. Keep the humidity control tray in the 40 &#176;C hybridization oven.</li>
	</ol>
	</li>
	<li>ISH procedure 
	NOTE: To examine the expression of the Nf1 splicing isoforms and calculate the percent spliced in (PSI) value for exon 23a, three different ISH probes that target exon 23a-included isoform, exon 23a-excluded isoform or both isoforms are used (Figure 2). The probes are used on adjacently cut tissue sections. To ensure the accuracy of the calculation, it is critical that the three tissue sections are treated exactly the same in the hybridization, amplification and signal detection steps.
	<ol>
		<li>Probe hybridization.
		<ol>
			<li>Remove the slides from the washing rack and tap the excess liquid from the slides. Place the slides in the stain rack.</li>
			<li>Use a pencil to label the slides with the probe name. Put the three adjacently cut brain tissue slides to be hybridized with three different Nf1 probes (Figure 2) together. Carry out the subsequent steps in the same order of slides.</li>
			<li>Add 4 drops of probe to cover the tissue. Do this one slide at a time to prevent the sections from drying out. Place the stain rack in the humidity control tray and insert it into the 40 &#176;C oven for 2 h.</li>
			<li>Wash the slides twice with washing buffer. For each wash, fill the staining dish with 200 mL of 1x wash buffer and place a washing rack in it. Tap excess liquid on a paper towel one slide at a time and quickly insert it into the washing rack. Move the slide rack up and down in the dish for 3&#8211;5 times for 2 min at RT.</li>
			<li>For the second wash, use fresh wash buffer.</li>
		</ol>
		</li>
		<li>Signal amplification.
		<ol>
			<li>Tap the excess wash buffer from the slides and place them in the stain rack. Add 4 drops of AMP 0 to cover the tissue. Place the stain rack in the humidity control tray and insert it in the oven for 30 min at 40 &#176;C.</li>
			<li>Wash the slides twice with washing buffer as described above.</li>
			<li>Repeat this step with AMP 1&#8211;6 following the order and condition of Table 2. Wash the slides twice after each hybridization step.</li>
		</ol>
		</li>
		<li>Signal Detection
		<ol>
			<li>Tap the excess wash buffer from the slides and place them in stain rack. Add 2 &#181;L of Fast Red B to 120 &#181;L of Fast Red A (1:60 ratio) in a microcentrifuge tube. Mix well and add to fully cover the tissue section. 
			NOTE: The total volume of Red A/B mix is based on the number and size of tissue sections. For a 0.75&#8217;&#8217; x 0.75&#8217;&#8217; area, 120 &#181;L is enough for one section. Use the mix within 5 min and avoid exposure of the mix to direct sunlight or UV light.</li>
			<li>Close the tray and incubate for 10 min at RT. Remove the solution to a waste container and immediately insert the slides into a washing rack and place the rack in a washing dish filled with tap water. Rinse again with fresh tap water.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Counterstaining of the slides
	<ol>
		<li>Remove the washing rack from tap water, and quickly tap on absorbent paper to remove extra water. Put the rack into a 50% hematoxylin staining solution and immediately move the rack up and down several times. Incubate for 2 min at RT.</li>
		<li>Transfer the slide rack back into the tap water dish. Wash 3&#8211;5 times by moving the rack up and down and changing fresh tap water until the slides become clear, while the brain tissues remain purple.</li>
		<li>Move the rack to a dish containing 0.02% ammonia water. Move the rack up and down 2&#8211;3 times until the tissues turn blue. Wash the slides 3&#8211;5 times with fresh tap water in a staining dish.</li>
	</ol>
	</li>
	<li>Mounting of the samples
	<ol>
		<li>Move the slide rack from the staining dish to a 60 &#176;C incubator and incubate for at least 15 min until the slides are completely dry.</li>
		<li>Place 40 &#181;L of mounting medium on the slide and carefully place a 24 mm x 50 mm coverslip over the tissue section. Air dry slides for 30 min at RT.</li>
	</ol>
	</li>
</ol>
4. Data Collection and Analysis
NOTE: Use a slide scanner to scan the images at 40X magnification.
<ol>
	<li>Positive and negative controls
	<ol>
		<li>For each experiment, include positive and negative controls (Figure 3). As a good positive control, signal should be visible as punctate dots at 20-40X magnification.</li>
		<li>Use Mouse (Mm)-PPIB-1ZZ as a positive control. This probe targets a common housekeeping gene PPIB. For negative control, one dot to every 20 cells displaying background staining per 20X microscope field is acceptable.</li>
		<li>Use the DapB-1ZZ probe as a negative control. This probe targets the bacterial gene dapB. These control probes have been used in many ISH assays19.</li>
	</ol>
	</li>
	<li>Signal detection and analysis of AS expression patterns of Nf1 exon 23a
	<ol>
		<li>As the hybridization signals are indicated as punctate dots, count the dots manually. The number of dots is correlated with the level of the transcripts that is detected by a specific probe. Note that the number of dots can also be analyzed by ImageJ or SpotStudio software.</li>
		<li>Use three probes to hybridize to exon 23a-containing, exon 23a-lacking isoforms, or total Nf1 transcripts (Figure 2). As the three probes cannot be used simultaneously, use three adjacent brain tissue sections to hybridize to each probe.
		<ol>
			<li>To calculate percent spliced in (PSI) for exon 23a, count the number of cells and dots in several brain regions. For each region of interest, count more than 400 cells from several sub-regions as indicated in Figure 4.</li>
			<li>Collect data from the same sub-regions for each of the three probes. Calculate PSI as number of dots per cell with the exon 23a inclusion probe/(number of dots per cell with the exon 23a inclusion probe + number of dot per cell with the exon23a skipping probe) x 100%.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Wang, E. T., 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=Alternative+isoform+regulation+in+human+tissue+transcriptomes.">Alternative isoform regulation in human tissue transcriptomes.</a> Nature. 456, 470-476 (2008).</li><li>Pan, Q., Shai, O., Lee, L. J., Frey, B. J., Blencowe, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deep+surveying+of+alternative+splicing+complexity+in+the+human+transcriptome+by+high-throughput+sequencing.">Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.</a> Nature Genetics. 40, 1413-1415 (2008).</li><li>Cieply, B., Carstens, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+roles+of+alternative+splicing+factors+in+human+disease.">Functional roles of alternative splicing factors in human disease.</a> Wiley Interdisciplinary Reviews: RNA. 6, 311-326 (2015).</li><li>Xiong, H. 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=RNA+splicing.+The+human+splicing+code+reveals+new+insights+into+the+genetic+determinants+of+disease.">RNA splicing. The human splicing code reveals new insights into the genetic determinants of disease.</a> Science. 347, 1254806 (2015).</li><li>Shirai, Y., Watanabe, M., Sakagami, H., Suzuki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+splice+variants+in+the+5'UTR+of+Gtf2i+expressed+in+the+rat+brain:+alternative+5'UTRs+and+differential+expression+in+the+neuronal+dendrites.">Novel splice variants in the 5'UTR of Gtf2i expressed in the rat brain: alternative 5'UTRs and differential expression in the neuronal dendrites.</a> Journal of Neurochemistry. 134, 578-589 (2015).</li><li>Cui, Y., Liu, J., Irudayaraj, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+quantification:+in+situ+analysis+of+transcriptome+and+pre-mRNA+alternative+splicing+at+the+nanoscale.">Beyond quantification: in situ analysis of transcriptome and pre-mRNA alternative splicing at the nanoscale.</a> Wiley Interdisciplinary Reviews: Nanomedicine and Nanobiotechnology. 9, (2017).</li><li>Trifonov, S., Yamashita, Y., Kase, M., Maruyama, M., Sugimoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glutamic+acid+decarboxylase+1+alternative+splicing+isoforms:+characterization,+expression+and+quantification+in+the+mouse+brain.">Glutamic acid decarboxylase 1 alternative splicing isoforms: characterization, expression and quantification in the mouse brain.</a> BMC Neuroscience. 15, 114 (2014).</li><li>Hollander, D., Naftelberg, S., Lev-Maor, G., Kornblihtt, A. R., Ast, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Are+Short+Exons+Flanked+by+Long+Introns+Defined+and+Committed+to+Splicing?.">How Are Short Exons Flanked by Long Introns Defined and Committed to Splicing?.</a> Trends in Genetics. 32, 596-606 (2016).</li><li>Cassidy, A., Jones, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+in+situ+hybridisation.">Developments in in situ hybridisation.</a> Methods. 70, 39-45 (2014).</li><li>Meshorer, 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=Alternative+splicing+and+neuritic+mRNA+translocation+under+long-term+neuronal+hypersensitivity.">Alternative splicing and neuritic mRNA translocation under long-term neuronal hypersensitivity.</a> Science. 295, 508-512 (2002).</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, 22-29 (2012).</li><li>Wang, H., 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+for+in+situ+detection+of+transcriptionally+active+human+papillomavirus+in+head+and+neck+squamous+cell+carcinoma.">RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.</a> Journal of Visualized Experiments. , (2014).</li><li>Zhu, H., Hinman, M. N., Hasman, R. A., Mehta, P., Lou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+neuron-specific+alternative+splicing+of+neurofibromatosis+type+1+pre-mRNA.">Regulation of neuron-specific alternative splicing of neurofibromatosis type 1 pre-mRNA.</a> Molecular and Cellular Biology. 28, 1240-1251 (2008).</li><li>Hinman, M. N., Sharma, A., Luo, G., Lou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+type+1+alternative+splicing+is+a+key+regulator+of+Ras+signaling+in+neurons.">Neurofibromatosis type 1 alternative splicing is a key regulator of Ras signaling in neurons.</a> Molecular and Cellular Biology. 34, 2188-2197 (2014).</li><li>Nguyen, H. T., 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=Neurofibromatosis+type+1+alternative+splicing+is+a+key+regulator+of+Ras/ERK+signaling+and+learning+behaviors+in+mice.">Neurofibromatosis type 1 alternative splicing is a key regulator of Ras/ERK signaling and learning behaviors in mice.</a> Human Molecular Genetics. 26, 3797-3807 (2017).</li><li>Zhou, H. L., 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=Hu+proteins+regulate+alternative+splicing+by+inducing+localized+histone+hyperacetylation+in+an+RNA-dependent+manner.">Hu proteins regulate alternative splicing by inducing localized histone hyperacetylation in an RNA-dependent manner.</a> Proceedings of the National Academy of Sciences of the United States of America. 108, E627-E635 (2011).</li><li>Sharma, A., 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=Calcium-mediated+histone+modifications+regulate+alternative+splicing+in+cardiomyocytes.">Calcium-mediated histone modifications regulate alternative splicing in cardiomyocytes.</a> Proceedings of the National Academy of Sciences of the United States of America. 111, E4920-E4928 (2014).</li><li>Adzemovic, M. Z., 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=Immunohistochemical+Analysis+in+the+Rat+Central+Nervous+System+and+Peripheral+Lymph+Node+Tissue+Sections.">Immunohistochemical Analysis in the Rat Central Nervous System and Peripheral Lymph Node Tissue Sections.</a> Journal of Visualized Experiments. , (2016).</li><li>Shokry, I. M., Callanan, J. J., Sousa, J., Tao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+In+Situ+Hybridization+using+Oligonucleotide+Probes+on+Paraformaldehyde-prefixed+Brain+of+Rats+with+Serotonin+Syndrome.">Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome.</a> Journal of Visualized Experiments. , (2015).</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></ol>

The authors have nothing to disclose.

This communication reports the use of BaseScope RNA ISH to examine AS expression patterns in mouse brain sections. It is demonstrated that anti-sense exon-exon junction probes shorter than 50 nucleotides can target exon inclusion and skipping isoforms robustly and specifically. Furthermore, the resulting signals can be used to calculate PSI of an alternative exon.
A few variations were tested in the procedure. For example, frozen tissue sections generated by cryostat sectioning were tested and...

Alternative splicing (AS) is a common process that occurs during pre-mRNA maturation. In this process, an exon can be differentially included in mature mRNA. Thus, through AS, one gene can generate many mRNAs that code for different protein products. It is estimated that 92&#8211;94% of human genes undergo alternative splicing1,2. Abnormal alternative splicing patterns resulted from genetic mutations have been linked to a large number of diseases, including amyotrophic lateral sclerosis, myotonic dystrophy, and cancer3,4. It is thus crucial to investigate and better understand alternative splicing regulatory mechanisms in an attempt to find new treatments of human diseases.
AS is often regulated in a cell type-specific fashion. It is important to determine the AS expression pattern of a specific gene in a given biological system. However, this becomes complicated when a complex organ that contains many different types of cells, such as the brain or heart, is studied. In this case, an ideal choice of assay system is RNA in situ hybridization (ISH) using tissue sections so the AS expression pattern of a specific gene can be detected in many cell types simultaneously. Indeed, exon-specific probes have been used to assess expression levels of an alternative exon5,6,7. However, this approach is not well suited for AS pattern analysis for the following reasons. First, conventional ISH methods usually use probes longer than 300 bp, while the average size of vertebrate internal exons (not first or last exon) is 170 nucleotides8,9. Second, when an exon-specific probe is used to examine the splicing pattern of an internal alternative exon, the only mRNA isoform detected by the probe is the one that contains the exon, while the mRNA isoform without the exon cannot be detected. Thus, calculation of the percent spliced in (PSI) value for the alternative exon is complicated. Furthermore, conventional fluorescent ISH often combines ISH with immunostaining, which reduces the detection efficiency and robustness. For example, in a study that investigated the stress-induced splicing isoform switching of the acetylcholinesterase (AChE) mRNA, digoxigenin was incorporated into the ISH probe and detected using anti-digoxigenin antibody. Alternatively, biotin-labeled probe was detected by an alkaline phosphatase/streptavidin conjugate and a substrate for alkaline phosphatase10. Neither method uses any amplification strategy to increase the sensitivity of detection. As a result, it is challenging to detect mRNA transcripts that are expressed at low levels. Thus, a simpler and more robust ISH assay system is needed to analyze AS expression patterns in situ.
BaseScope was recently developed based on the platform of RNAscope, a well-established and widely used ISH assay system. Both assay systems employ a target-specific amplification technology that increases the sensitivity of detection11,12. What distinguishes one from the other is the length of the target sequence, which is as short as 50 nucleotides for BaseScope, and 300&#8211;1,000 nucleotides for RNAscope. Thus, it is possible to design probes that target exon-exon junctions to detect specific alternative mRNA isoforms. In the current study, a procedure was established to examine AS expression patterns of neurofibromatosis type 1 (Nf1) exon 23a, an alternative exon extensively studied in the same laboratory13,14,15,16,17, in mouse brain sections. The results demonstrate that BaseScope is an ideal system to study expression patterns of Nf1 exon 23a in situ. As this assay system can be adapted to analyze AS expression patterns of many alternative exons, it represents a powerful new tool in the studies of AS.

<table>
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization Oven</td>
			<td>Advanced Cell Diagnostics</td>
			<td>241000ACD</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidity Control Tray (with lid)</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310012</td>
			<td></td>
		</tr>
		<tr>
			<td>Stain Rack</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310017</td>
			<td></td>
		</tr>
		<tr>
			<td>Hot plate</td>
			<td>Fisher Scientific</td>
			<td>1160049SH</td>
			<td></td>
		</tr>
		<tr>
			<td>Imperial III General Purpose Incubator</td>
			<td>Lab-Line</td>
			<td>302</td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Leica</td>
			<td>SCN400</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pretreatment kit</td>
			<td>Advanced Cell Diagnostics</td>
			<td>322381</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2000899</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease III*</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2000901</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Target Retrieval</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2002555</td>
			<td></td>
		</tr>
		<tr>
			<td>BaseScope Detection Reagent Kit</td>
			<td>Advanced Cell Diagnostics</td>
			<td>332910</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 0</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001814</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 1</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001815</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 2</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001816</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 3</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001817</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 4</td>
			<td>Advanced Cell Diagnostics</td>
			<td>16229B</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 5-RED</td>
			<td>Advanced Cell Diagnostics</td>
			<td>16229C</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP 6-RED</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001820</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast RED-A</td>
			<td>Advanced Cell Diagnostics</td>
			<td>2001821</td>
			<td></td>
		</tr>
		<tr>
			<td>Fast RED-B</td>
			<td>Advanced Cell Diagnostics</td>
			<td>16230F</td>
			<td></td>
		</tr>
		<tr>
			<td>50X Wash Buffer</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310091</td>
			<td></td>
		</tr>
		<tr>
			<td>Negative Control Probe- Mouse DapB-1ZZ</td>
			<td>Advanced Cell Diagnostics</td>
			<td>701021</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive Control Probe- Mouse (Mm)-PPIB-1ZZ</td>
			<td>Advanced Cell Diagnostics</td>
			<td>701081</td>
			<td></td>
		</tr>
		<tr>
			<td>Control slide-mouse 3T3 cell pellet</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310045</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Other supplies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Humidifying Paper</td>
			<td>Advanced Cell Diagnostics</td>
			<td>310015</td>
			<td></td>
		</tr>
		<tr>
			<td>Washing Rack</td>
			<td>American Master Tech Scientific</td>
			<td>9837976</td>
			<td></td>
		</tr>
		<tr>
			<td>Washing Dishes</td>
			<td>American Master Tech Scientific</td>
			<td>LWS20WH</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Barrier Pen</td>
			<td>Vector Laboratory</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glass, 24 x 50 mm</td>
			<td>Fisher Scientific</td>
			<td>12--545-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>002689</td>
			<td></td>
		</tr>
		<tr>
			<td>100% ethanol (EtOH)</td>
			<td>Decon Labs</td>
			<td>2805</td>
			<td></td>
		</tr>
		<tr>
			<td>formalde solution</td>
			<td>Fisher Scientific</td>
			<td>SF94-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Hematoxylin I</td>
			<td>American Master Tech Scientific</td>
			<td>17012359</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting Medium</td>
			<td>Vector Labs</td>
			<td>H-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Fisher Scientific</td>
			<td>173942</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitative analysis of alternative pre-mrna splicing in mouse brain sections using rna in situ hybridization assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell type-specific fashion. AS expression patterns are typically analyzed by RT-PCR and RNA-seq using RNA samples isolated from a population of cells. In situ examination of AS expression patterns for a particular biological structure can be carried out by RNA in situ hybridization (ISH) using exon-specific probes. However, this particular use of ISH has been limited because alternative exons are generally too short to design exon-specific probes. In this report, the use of BaseScope, a recently developed technology that employs short antisense oligonucleotides in RNA ISH, is described to analyze AS expression patterns in mouse brain sections. Exon 23a of neurofibromatosis type 1 (Nf1) is used as an example to illustrate that short exon-exon junction probes exhibit robust hybridization signals with high specificity in RNA ISH analysis on mouse brain sections. More importantly, signals detected with exon inclusion- and skipping-specific probes can be used to reliably calculate the percent spliced in values of Nf1 exon 23a expression in different anatomical areas of a mouse brain. The experimental protocol and calculation method for AS analysis are presented. The results indicate that BaseScope provides a powerful new tool to assess AS expression patterns in situ.

BaseScope ISH was carried out using three mouse strains: CD1 wild type mice, C57BL/6J wild type mice, and Nf123aIN/23aIN mutant mice in the C57BL/6J background, in which exon 23a is included in all cell types as a result of engineered splice site mutations14,15.
As a first step, the ISH assay system was tested using company provided reagents: slides t...

Watch this Scientific Journal Video about Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay at JoVE.com

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Alternative splicing (AS) occurs in more than 90% of human genes. The expression pattern of an alternatively spliced exon is often regulated in a cell ...

quantitative-analysis-alternative-pre-mrna-splicing-mouse-brain

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Genetics

Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

8.8K Views.  Case Western Reserve University. An in situ hybridization (ISH) protocol that uses short antisense oligonucleotides to detect alternative pre-mRNA splicing patterns in mouse brain sections is described.

Video: Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative without tools to study gene function. The development and sharing of detailed protocols for the establishment of new model systems in laboratories, and for tools to carry out functional studies, is thus crucial for leveraging the power of genomics. Coleoptera (beetles) are the largest clade of insects and occupy virtually all types of habitats on the planet. In addition to providing ideal models for fundamental research, studies of beetles can have impacts on pest control as they are often pests of households, agriculture, and food industries. Detailed protocols for rearing and maintenance of D. maculatus laboratory colonies and for carrying out dsRNA-mediated interference in D. maculatus are presented. Both embryonic and parental RNAi procedures-including apparatus set up, preparation, injection, and post-injection recovery-are described. Methods are also presented for analyzing embryonic phenotypes, including viability, patterning defects in hatched larvae, and cuticle preparations for unhatched larvae. These assays, together with in situ hybridization and immunostaining for molecular markers, make D. maculatus an accessible model system for basic and applied research. They further provide useful information for establishing procedures in other emerging insect model systems.

Rearing and Double-stranded RNA-mediated Gene Knockdown in the Hide Beetle, Dermestes maculatus

Here, we present protocols for rearing an intermediate-germ beetle, Dermestes maculatus (D. maculatus) in the lab. We also share protocols for embryonic and parental RNAi and methods for analyzing embryonic phenotypes to study gene function in this species.

Advances in genomics have raised the possibility of probing biodiversity at an unprecedented scale. However, sequence alone will not be informative ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of tissues, we have developed numerous modifications and improvements to our original fluorescent in situ hybridization (FISH) protocol. To facilitate throughput and cost effectiveness, all steps, from probe generation to signal detection, are performed using exon 96-well microtiter plates. Digoxygenin (DIG)-labelled antisense RNA probes are produced using either cDNA clones or genomic DNA as templates. After tissue fixation and permeabilization, probes are hybridized to transcripts of interest and then detected using a succession of anti-DIG antibody conjugated to biotin, streptavidin conjugated to horseradish peroxidase (HRP) and fluorescently conjugated tyramide, which in the presence of HRP, produces a highly reactive intermediate that binds to electron dense regions of immediately adjacent proteins. These amplification and localization steps produce a robust and highly localized signal that facilitates both cellular and subcellular transcript localization. The protocols provided have been optimized to produce highly specific signals in a variety of tissues and developmental stages. References are also provided for additional variations that allow the simultaneous detection of multiple transcripts, or transcripts and proteins, at the same time.

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

The described RNA in situ hybridization protocol allows the detection of RNA in whole Drosophila embryos or dissected tissues. Using 96-well microtiter plates and tyramide signal amplification, transcripts can be detected at high resolution, sensitivity, and throughput, and at a relatively low cost.

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these diseases, do not exist in isolation when they colonize the vector; rather, they likely engage in interactions with resident microorganisms in the gut lumen. The vector microbiota has been demonstrated to play an important role in pathogen transmission for several vector-borne diseases. Whether resident bacteria in the gut of the Ixodes scapularis tick, the vector of several human pathogens including Borrelia burgdorferi, influence tick transmission of pathogens is not determined. We require methods for characterizing the composition of the bacteria associated with the tick gut to facilitate a better understanding of potential interspecies interactions in the tick gut. Using whole-mount in situ hybridization to visualize RNA transcripts associated with particular bacterial species allows for the collection of qualitative data regarding the abundance and distribution of the microbiota in intact tissue. This technique can be used to examine changes in the gut microbiota milieu over the course of tick feeding and can also be applied to analyze expression of tick genes. Staining of whole tick guts yield information about the gross spatial distribution of target RNA in the tissue without the need for three-dimensional reconstruction and is less affected by environmental contamination, which often confounds the sequencing-based methods frequently used to study complex microbial communities. Overall, this technique is a valuable tool that can be used to better understand vector-pathogen-microbiota interactions and their role in disease transmission.

Visualization of Microbiota in Tick Guts by Whole-mount In Situ Hybridization

Here, we present a protocol to spatially and temporally assess the presence of viable microbiota in tick guts using a modified whole-mount in situ hybridization approach.

Infectious diseases transmitted by arthropod vectors continue to pose a significant threat to human health worldwide. The pathogens causing these ...

Single Molecule Fluorescence In Situ Hybridization (smFISH) Analysis in Budding Yeast Vegetative Growth and Meiosis

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect and count individual RNA molecules. Complementary to deep sequencing-based methods, smFISH provides information about the cell-to-cell variation in transcript abundance and the subcellular localization of a given RNA. Recently, we have used smFISH to study the expression of the gene&#160;NDC80&#160;during meiosis in budding yeast, in which two transcript isoforms exist and the short transcript isoform has its entire sequence shared with the long isoform. To confidently identify each transcript isoform, we optimized known smFISH protocols and obtained high consistency and quality of smFISH data for the samples acquired during budding yeast meiosis. Here, we describe this optimized protocol, the criteria that we use to determine whether high quality of smFISH data is obtained, and some tips for implementing this protocol in&#160;other yeast strains and growth conditions.

Single Molecule Fluorescence In Situ Hybridization smFISH Analysis in Budding Yeast Vegetative Growth and Meiosis

This single molecule fluorescence in situ hybridization protocol is optimized to quantify the number of RNA molecules in budding yeast during vegetative growth and meiosis.

Single molecule fluorescence in situ hybridization (smFISH) is a powerful technique to study gene expression in single cells due to its ability to detect ...

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Using In Vitro and In-cell SHAPE to Investigate Small Molecule Induced Pre-mRNA Structural Changes

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences is desired. We herein provide a detailed in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) protocol to study the RNA structural change in the presence of an experimental drug for spinal muscular atrophy (SMA), survival of motor neuron (SMN)-C2, and in exon 7 of the pre-mRNA of the SMN2 gene. In in vitro SHAPE, an RNA sequence of 140 nucleotides containing SMN2 exon 7 is transcribed by T7 RNA polymerase, folded in the presence of SMN-C2, and subsequently modified by a mild 2&#39;-OH acylation reagent, 2-methylnicotinic acid imidazolide (NAI). This 2&#39;-OH-NAI adduct is further probed by a 32P-labeled primer extension and resolved by polyacrylamide gel electrophoresis (PAGE). Conversely, 2&#39;-OH acylation in in-cell SHAPE takes place in situ with SMN-C2 bound cellular RNA in living cells. The pre-mRNA sequence of exon 7 in the SMN2 gene, along with SHAPE-induced mutations in the primer extension, was then amplified by PCR and subject to next-generation sequencing. Comparing the two methodologies, in vitro SHAPE is a more cost-effective method and does not require computational power to visualize results. However, the in vitro SHAPE-derived RNA model sometimes deviates from the secondary structure in a cellular context, likely due to the loss of all interactions with RNA-binding proteins. In-cell SHAPE does not need a radioactive material workplace and yields a more accurate RNA secondary structure in the cellular context. Furthermore, in-cell SHAPE is usually applicable for a larger range of RNA sequences (~1,000 nucleotides) by utilizing next-generation sequencing, compared to in vitro SHAPE (~200 nucleotides) that usually relies on PAGE analysis. In case of exon 7 in SMN2 pre-mRNA, the in vitro and in-cell SHAPE derived RNA models are similar to each other.

Detailed protocols for both in vitro and in-cell selective 2&#39;-hydroxyl acylation analyzed by primer extension (SHAPE) experiments to determine the secondary structure of pre-mRNA sequences of interest in the presence of an RNA-targeting small molecule are presented in this article.

In the process of drug development of RNA-targeting small molecules, elucidating the structural changes upon their interactions with target RNA sequences ...

Use of Single Molecule Fluorescent In Situ Hybridization (SM-FISH) to Quantify and Localize mRNAs in Murine Oocytes

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), quantitative, real-time RT-PCR (RT-qPCR) and RNA sequencing. When these techniques are performed using a single oocyte or embryo, low-copy mRNAs are not reliably detected. To overcome this problem, oocytes or embryos can be pooled together for analysis; however, this often leads to high variability amongst samples. In this protocol, we describe the use of fluorescence in situ hybridization (FISH) using branched DNA chemistry. This technique identifies the spatial pattern of mRNAs in individual cells. When the technique is coupled with Spot Finding and Tracking&#160;computer software, the abundance of mRNAs in the cell can also be quantified. Using this technique, there is reduced variability within an experimental group and fewer oocytes and embryos are required to detect significant differences between experimental groups. Commercially available branched-DNA SM-FISH kits have been optimized to detect mRNAs in sectioned tissues or adherent cells on slides. However, oocytes do not effectively adhere to slides and some reagents in the kit were too harsh resulting in oocyte lysis. To prevent this lysis, several modifications were made to the FISH kit. Specifically, oocyte permeabilization and wash&#160;buffers designed for the immunofluorescence of oocytes and embryos replaced the proprietary buffers. The permeabilization, washes, and incubations with probes and amplifier were performed in 6-well plates and oocytes were placed on slides at the end of the protocol using mounting media. These modifications were able to overcome the limitations of the commercially available kit, in particular, the oocyte lysis. To accurately and reproducibly count the number of mRNAs in individual oocytes, computer software was used. Together, this protocol represents an alternative to PCR and sequencing to compare the expression of specific transcripts in single cells.

Use of Single Molecule Fluorescent In Situ Hybridization SM-FISH to Quantify and Localize mRNAs in Murine Oocytes

To reproducibly count the numbers of mRNAs in individual oocytes, single molecule RNA fluorescence in situ hybridization (RNA-FISH) was optimized for non-adherent cells. Oocytes were collected, hybridized with the transcript specific probes, and quantified using an image quantification software.

Current methods routinely used to quantify mRNA in oocytes and embryos include digital reverse-transcription polymerase chain reaction (dPCR), ...

Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated with a better outcome than alcohol- and tobacco-related OPSCC. Chromogenic in situ hybridization (CISH) of HPV viral RNA could allow the semiquantitative evaluation of viral transcripts of the oncogenic proteins E6 and E7 and an in situ visualization with a good spatial resolution. This technique allows the diagnosis of an active infection with the visualization of HPV transcription in the tumoral HPV-infected cells. An advantage of this technique is the avoidance of contamination from nonneoplastic HPV-infected cells adjacent to the tumor. Overall, its good diagnosis performances have it considered to be the gold standard for active HPV infection identification. Since E6 and E7 viral protein interaction with cell proteins pRb and p53 is mandatory for cell transformation, HPV RNA CISH is functionally relevant and acutely reflects active oncogenic HPV infection. This technique is clinically relevant as well since &#34;low&#34; or &#34;high&#34; HPV transcription levels helped the identification of two prognosis groups among HPV-related p16-positive head and neck cancer patients. Here we present the protocol for manual HPV RNA CISH performed on formalin-fixed paraffin-embedded (FFPE) slides with a kit obtained from the manufacturer. Instead of chromogenic revelation, RNA in situ hybridization may also be performed with fluorescent revelation (RNA FISH). It may also be combined with conventional immunostaining.

Human papillomavirus (HPV) RNA chromogenic in situ hybridization is considered to be one of the gold standards for active human papillomavirus infection detection within tumors. It allows the visualization of HPV E6-E7 mRNA expression with localization and semiquantitative evaluation of its signal.

Human papillomavirus (HPV) infection is a major risk factor for a subtype of oropharyngeal squamous cell carcinoma (OPSCC), which tends to be associated ...

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.