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 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.
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 92–94% 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 300–1,000 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.
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 “Role of alternative pre-mRNA splicing in vertebrate development”.
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
2. Sample Pretreatment
3. ISH Assay
4. Data Collection and Analysis
NOTE: Use a slide scanner to scan the images at 40X magnification.
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 that have 3T3 cells, and negative and positive ISH probes. As shown in Figure 3, no signal was detected when the negative control probe was used, while many punctate dots were detected when the positive control probe was used.
Next, ISH was carried out using mouse brain sections and Nf1-specific probes that are designed to detect Nf1 transcripts that contain exon 23a, skip exon 23a or both isoforms (Figure 2). These probes target specific exon-exon junctions. Two brain regions were selected to examine the AS expression pattern of Nf1 exon 23a: cortex and hippocampus CA3 (Figure 4). For each region, count three sub-regions, as indicated in Figure 4, recording both cell and dot numbers. For each region, approximately 400 cells were counted in total and used to calculate the dots/cell ratio. The AS expression pattern for Nf1 exon 23a is calculated as PSI.
The ISH signals obtained with the three Nf1 probes in the cortex and hippocampus of CD1 mice are shown in Figure 5A-5F and Table 3. The cortex region from C57BL/6J Nf1+/+ and Nf123aIN/23aIN mice was also analyzed (Figure 6).
The result of these experiments led to several conclusions. First, the sum of signals, shown as dots/cell, detected by exon 23a inclusion-specific and skipping-specific probes equals to that detected by the probe hybridizing to both isoforms (Table 3), which suggests that the three probes hybridized with the Nf1 transcripts with similar efficiency. Second, the PSI value of exon 23a in cortex, 10%, is consistent with the previously reported RT-PCR result, in which cortex was dissected from adult C57BL/6J mouse brain followed by total RNA isolation and RT-PCR analysis14,15. This result suggests that the BaseScope ISH can be used to quantitatively analyze AS expression patterns in addition to transcript levels in tissue sections. Third, the results obtained with the Nf123aIN/23aIN mutant brain tissues, in which all of the Nf1 transcripts contain exon 23a14,15, showed similar levels of signals when inclusion-specific and Nf1 total probes were used and no signal when skipping-specific probe was used (Figure 6), indicating that the two probes targeting exon inclusion or skipping are highly specific.
Figure 1: Equipment used in ISH. (A) Washing dish and washing rack. (B) Hybridization oven. (C) Target retrieval beaker and hot plate set. (D) Humidity control tray and stain rack. (E) Humidifying paper indicated by a red arrowhead inside the humidity control tray. Please click here to view a larger version of this figure.
Figure 2: Probes used to detect Nf1 exon 23a AS expression patterns. All of the probes contain one ZZ pair oligonucleotide. The inclusion-specific probe (red) targets the junction of exons 23a and 24, the skipping-specific probe (green) targets the junction of exons 23 and 24, and the Nf1 total probe (blue) targets the junction of exons 26 and 27. Please click here to view a larger version of this figure.
Figure 3: ISH using negative (A-B) and positive (C-D) controls on 3T3 cells. A control slide containing 3T3 cells was processed through steps 2 (sample pretreatment) and 3 (ISH procedure) in the protocol. Positive signals are shown as red punctate dots indicated by red arrows. B and D are zoomed in images of A and C, respectively. Scale bar = 20 µm (A and C); 5 µm (B and D). Please click here to view a larger version of this figure.
Figure 4: Picture of a mouse brain coronal section. The red boxes indicate the areas selected for counting of cell and signal numbers in cortex and hippocampus CA3 regions. Scale bar = 1 mm. Please click here to view a larger version of this figure.
Figure 5: RNA ISH signals detected using Nf1-specific probes and mouse brain sections. CD1 brains were processed through steps 1-4 in the protocol. Red punctate dots indicate positive signals when the three Nf1-specific probes (shown in Figure 3) are used. Cortex (A-C) and hippocampus (D-F) are shown. Nf1 total probe was used in A and D, inclusion-specific probe in B and E, and skipping-specific probe in C and F. Scale bar = 40 µm (A-C); 400 µm (D-F). Please click here to view a larger version of this figure.
Figure 6: RNA ISH signals detected in Nf1+/+ and Nf123aIN/23aIN brain sections. Both mouse strains are in the C57BL/6J background. The brains were processed through steps 1-4 in the protocol. A-C. Nf1+/+ cortex. D-F. Nf123aIN/23aIN cortex. Nf1 total probe was used in A and D, inclusion-specific probe in B and E, and skipping-specific probe in C and F. Scale bar = 40 µm. Please click here to view a larger version of this figure.
Order of incubation | Chemicals in TT Dish | Time |
1 | 250 mL of xylene | 5 min |
2 | 250 mL of xylene | 5 min |
3 | 250 mL of 100% ethanol | 2 min |
4 | 250 mL of 100% ethanol | 2 min |
Table 1: Incubation procedure of step 2.1.1.
AMP Solution | Incubation Time | Incubation Temperature |
AMP 1 | 15 min | 40 °C |
AMP 2 | 30 min | 40 °C |
AMP 3 | 30 min | 40 °C |
AMP 4 | 15 min | 40 °C |
AMP 5-RED | 30 min | Room Temperature |
AMP 6-RED | 15 min | Room Temperature |
Table 2: Signal amplification procedure of step 3.2.2.
Region | skipping | inclusion | skipping+inclusion | total | PSI |
cortex | 2.9±0.1 | 0.32±0.03 | 3.21 | 3.22 | 10 |
CA3 | 5.37±0.2 | 0.2±0.04 | 5.56 | 5.65 | 3.71 |
Table 3: RNA ISH results calculated from the image shown in Figure 5. The numbers, shown in dots/cell, were calculated using more than 400 cells that are included in the three sub-regions in cortex and CA3 (Figure 4). Standard errors are included.
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 shown to be successful for use in this ISH protocol. In this case, however, the deparaffinization step in 2.1 was changed to washing of OCT with PBS for 5 min. In addition, although it is very convenient to use the hybridization oven provided by ACD, use of a simple incubator set at 40 °C combined with a slide staining tray used for immunohistochemistry gives comparable results. The important thing is to keep the moisture in the box by including wet filter papers throughout the procedure.
There is a limitation in this procedure. Currently, it is not feasible to perform multi-color labeling on the same tissue slide. Thus, different probes can only be used on adjacently cut, different sections. To obtain accurate PSI values, it is critical to ensure that the incubation time of probe hybridization, signal amplification and detection is the same for every tissue section. In the future, implementing the use of multi-color probes on the same tissue slide, as used in the RNAscope procedures, will be highly desirable.
The RNA ISH described in this report provides a powerful new tool to quantitatively analyze AS expression patterns in situ. This technology can be applied to study many alternative exons. In a recent report, it was successfully used to examine the differential expression patterns of the four ErbB4 splicing isoforms in neurons and oligodendrocytes at cellular resolution20. As shown in this and several other reports, combination of BaseScope RNA ISH with immunochemistry will be a powerful approach to study the localization and function of the differentially expressed splicing isoforms6,20.
The authors have nothing to disclose.
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.
Equipment | |||
Hybridization Oven | Advanced Cell Diagnostics | 241000ACD | |
Humidity Control Tray (with lid) | Advanced Cell Diagnostics | 310012 | |
Stain Rack | Advanced Cell Diagnostics | 310017 | |
Hot plate | Fisher Scientific | 1160049SH | |
Imperial III General Purpose Incubator | Lab-Line | 302 | |
Slide Scanner | Leica | SCN400 | |
Name | Company | Catalog Number | コメント |
Reagents | |||
Pretreatment kit | Advanced Cell Diagnostics | 322381 | |
Hydrogen Peroxide | Advanced Cell Diagnostics | 2000899 | |
Protease III* | Advanced Cell Diagnostics | 2000901 | |
10X Target Retrieval | Advanced Cell Diagnostics | 2002555 | |
BaseScope Detection Reagent Kit | Advanced Cell Diagnostics | 332910 | |
AMP 0 | Advanced Cell Diagnostics | 2001814 | |
AMP 1 | Advanced Cell Diagnostics | 2001815 | |
AMP 2 | Advanced Cell Diagnostics | 2001816 | |
AMP 3 | Advanced Cell Diagnostics | 2001817 | |
AMP 4 | Advanced Cell Diagnostics | 16229B | |
AMP 5-RED | Advanced Cell Diagnostics | 16229C | |
AMP 6-RED | Advanced Cell Diagnostics | 2001820 | |
Fast RED-A | Advanced Cell Diagnostics | 2001821 | |
Fast RED-B | Advanced Cell Diagnostics | 16230F | |
50X Wash Buffer | Advanced Cell Diagnostics | 310091 | |
Negative Control Probe- Mouse DapB-1ZZ | Advanced Cell Diagnostics | 701021 | |
Positive Control Probe- Mouse (Mm)-PPIB-1ZZ | Advanced Cell Diagnostics | 701081 | |
Control slide-mouse 3T3 cell pellet | Advanced Cell Diagnostics | 310045 | |
Name | Company | Catalog Number | コメント |
Other supplies | |||
Humidifying Paper | Advanced Cell Diagnostics | 310015 | |
Washing Rack | American Master Tech Scientific | 9837976 | |
Washing Dishes | American Master Tech Scientific | LWS20WH | |
Hydrophobic Barrier Pen | Vector Laboratory | H-4000 | |
Glass Slides | Fisher Scientific | 12-550-15 | |
Cover Glass, 24 x 50 mm | Fisher Scientific | 12–545-F | |
Name | Company | Catalog Number | コメント |
Chemicals | |||
Ammonium hydroxide | Fisher Scientific | 002689 | |
100% ethanol (EtOH) | Decon Labs | 2805 | |
formalde solution | Fisher Scientific | SF94-4 | |
Hematoxylin I | American Master Tech Scientific | 17012359 | |
Mounting Medium | Vector Labs | H-5000 | |
Xylene | Fisher Scientific | 173942 |