Name: Quantitative Analysis of Alternative Pre-mRNA Splicing in Mouse Brain Sections Using RNA In Situ Hybridization Assay (Video) | JoVE Journal
Uploaded: 2018-08-26T13:00:00.000+00:00
Duration: 11 min 22 s
Description: Learn RNA in situ hybridization to detect alternative pre-mRNA splicing patterns in mouse brain sections. This sensitive assay uses antisense oligonucleotides and signal amplification for robust isoform-specific detection.

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5 minutes

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>

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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.

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

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...

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

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 ...

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Learn RNA in situ hybridization to detect alternative pre-mRNA splicing patterns in mouse brain sections. This sensitive assay uses antisense oligonucleotides and signal amplification for robust isoform-specific detection.

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

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