Name: quantitative analysis of alternative pre-mrna splicing in mouse brain sections using rna in situ hybridization assay
Uploaded: 2018-08-26T13:00:00
Description: 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

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 Embedde...

<ol>
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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 ...

This method can help answer key questions in the field of alternative pre-mRNA splicing. Specifically it provides a robustly, sensitive assay system to examine alternative splicing patterns in situ in tissue slices. With this method, alternative splicing can be analyzed simultaneously in many subtypes that are contained in complex biological structures such as a mouse brain.The main advantage of this technique is that it uses shock anti-static exon junction probes in mRNA in situ hybridization to produce isoform specific hybridization signals. Building signal amplification technology increases the sensitivity of the detection to provide robust hybridization signals. The signals detected was exon inclusion escaping specific probes that are used to calculate the percentage splice in vitals at different anatomical areas of a mouse brain.In this study NF1 exon 23a is used as an example to illustrate the use of the assay system. However, the system can be adapted to analysis expression patterns of many alternative exons. To start mouse brain section D paraffinization, fill two washing dishes with 250 milliliters of xylene.And another two with 250 milliliters of 100%ethanol inside a fume hood. Insert the tissue slides into a washing rack. Next, place the washing rack in a xylene containing dish and incubate for five minutes while moving the rack up and down at least three times and repeat with the second xylene dish.Then place the rack in an ethanol containing dish and incubate for two minutes with the same up and down movements and then repeat with the second ethanol dish. To dry the slides, move the rack from the second ethanol dish and place it inside an incubator at 60 degrees Celsius for five minutes. Use a hydrophobic barrier pen to draw a 0.75 inch by 0.75 inch barrier around the tissue section and air dry for three minutes at room temperature.Turn on the hybridization oven and set the temperature at 40 degrees Celsius. After completely wetting humidifying paper with distilled water, place it in a humidity control tray. Then insert the humidity control tray into the hybridization oven.After placing the dried slides on a bench, completely cover the tissue sections on each slide with five to eight drops of hydrogen peroxide solution. Incubate for 10 minutes at room temperature. Tap the slides on an absorbent paper, one at a time to remove the hydrogen peroxide solution and insert the slides into a washing rack.Immerse the rack in a washing dish containing water while moving it up and down three to five times to wash the slides. Fill a one liter glass beaker placed on a hot plate with 700 milliliters of 1X target removal treatment and cover it with foil. Insert a thermometer through the foil cover and turn on the hot plate, set on high temperature for 10 to 15 minutes.Once the temperature of the retrieval reagent reaches 98 degrees Celsius, set the hot plate at low temperature to maintain a mild boiling condition. Next, carefully remove the foil and place the rack inside the beaker. Cover the beaker with foil and incubate for 15 minutes.Use forceps to transfer the rack from the beaker to a washing dish containing 200 milliliters of distilled water and incubate for 15 seconds. After 15 seconds place the rack in a washing dish containing 100%ethanol and incubate for three minutes. Remove the rack from the dish and dry at 60 degrees Celsius for 10 minutes.After placing the slides on a stain rack, cover the tissue sections with five drops of protease 3 per slide. Carefully place the rack in the humidity control tray and insert the tray into the hybridization oven and incubate at 40 degrees Celsius for 30 minutes. After removing the rack from the tray, tap the slides to remove excess liquid and insert them into a washing rack.Place the rack into a washing dish containing distilled water and move the rack up and down three to five times to wash the slides. To prepare for in situ hybridization, incubate the probes and AMP reagents at room temperature for 30 minutes. Insert the humidity control tray into the hybridization oven and set at 40 degrees Celsius.For probe hybridization, remove the slides from the washing rack and tap them to remove excess liquid. Place the slides on a stain rack. Use a pencil to label the slides with the probe name.Place three adjacently cut brain tissue slides to be hybridized with three different neural fiber mitosis type one probes together. Because there are very different isoform specific probes, I use adjacently cut tissue sections. It is critical that these sections are treated exactly the same in hybridization, amplification, and signal detection steps to ensure the accuracy of the percentage splice in calculation.Cover the tissue sections with four drops of probe per section, one slide at a time to prevent drying out of the sections. Then place the stain rack in the humidity control tray and insert it into the 40 degrees Celsius oven for two hours. Next, place a washing rack in a washing dish filled with 200 milliliters of 1X wash buffer.Then tap each slide, one at a time on a paper towel to remove excess liquid. And immediately insert each slide into the washing rack. Incubate at room temperature for two minutes in a dish filled with fresh wash buffer while moving the rack up and down three to five times and then repeat the wash in a new dish.For signal amplification, tap each slide to remove excess wash buffer and place them on a stain rack. Cover the tissue section on each slide with four drops of AMP zero reagent. Next, place the rack in the humidity control tray, insert the tray into the oven and incubate at 40 degrees Celsius for 30 minutes.Wash the slide twice with wash buffer as previously done in this experiment. Repeat the signal amplification step with AMP reagents one through six as described in the text protocol and wash the slides after each step. For signal detection, remove excess wash buffer from the slides by tapping and place them on a stain rack.Next, mix two microliters of fast red B with 120 microliters of fast red A in a micro centrifuge tube. Add the solution to the tissue sections on each slide to fully cover them. Place the stain rack in the humidity control tray, cover the tray and incubate for 10 minutes at room temperature.Then remove the solution and insert the slides into a washing rack. Place the rack in a washing dish containing tap water to wash the slides and repeat the wash. After the second wash, tap on an absorbent paper to remove excess water.Next, place the rack in a dish containing 50%hematoxylin staining solution. Move the rack up and down several times. And incubate for two minutes at room temperature.Transfer the rack to a washing dish containing tap water. Move the rack up and down five times and repeat the wash until the slides become clear, while tissue sections remain purple. Then transfer the rack to a dish containing 0.02%ammonia water.Move the rack up and down two to four times until the sections turn blue. Wash the slides with tap water three to five times in the staining dish. Next, place the rack inside an incubator and incubate for 15 minutes at 60 degrees Celsius to dry the slides.When completely dry, add one drop of mounting medium on each slide to cover the slice. Then, carefully place a 24 by 50 millimeter coverslip over the tissue sections on each slide. After air drying the slides for 30 minutes at room temperature, proceed with data collection.To calculate the percent spliced in or PSI value for exon 23a, the number of cells and dots in three sub-regions of cortex, and hippocampus CA3 are counted. RNA in situ hybridization assay is performed in brain sections of CD1 wild type mice using three different NF1 specific probes. Red punctate dots observed in the cortex and hippocampus sections, indicate positive signals by NF1 total, inclusion specific, and skipping specific probes.The ISH signals from the three probes are then counted and used to calculate PSI. RNA in situ hybridization assay is performed in the cortex of C57 black 6J wild type mice and C57 black 6J mutant mice in which exon 23a is included in all cell types. Red punctate dots observed in the cortex of C57 black 6J wild type mice, indicate positive signals by NF1 total, inclusion specific, and skipping specific probes.In C57 black 6J mutant mice, similar signals are obtained by NF1 total and inclusion specific probes. However, no signal is detected when skipping specific probe is used, indicating that two probes targeting exon inclusion or skipping are highly specific. Because multi-color labeling on the same tissue slides is not yet available for this procedure, different probes are used on the different tissue slides.To obtain the accurate percent splicing values, it's important to use adjacently cut slides and treating slides in the exact same way throughout this procedure. Complementing this procedure are the measures like immunohistochemistry, can be performed to answer additional questions regarding the localization and function of differentially expressed splicing isoforms.

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Unit 1

Transcription: DNA to RNA

SCIENTISTS IN ACTION

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

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

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

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

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

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

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

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

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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 ...

An Assay for Quantifying Protein-RNA Binding in Bacteria

In the initiation step of protein translation, the ribosome binds to the initiation region of the mRNA. Translation initiation can be blocked by binding ...