The authors thank the department of Pathology of Hopital Europ&#233;en Georges Pompidou and Necker (Laurianne Chambolle, Elodie Michel, and Gis&#232;le Legall); the Histology platform of PARCC, Hopital Europ&#233;en Georges Pompidou (Corinne Lesaffre); Virginia Clark for language editing; Alexandra Elbakyan for her contribution.

The protocol follows ethical guidelines and was approved by the Ethical Committee (Comit&#233;-de-Protection-des-Personnes Ile-de-France-II, #2015-09-04).
1. Preparation of the materials
<ol>
	<li>Preparation of 1x wash buffer
	<ol>
		<li>Prepare 3 L of 1x wash buffer by adding 2.94 L of distilled water and one bottle (60 mL) of wash buffer (50x) (see the Table of Materials) to a large carboy. Mix well. 
		NOTE: The 1x wash buffer may be prepared ahead of time and stored at room temperature for up to 1 month.</li>
	</ol>
	</li>
	<li>Preparation of counterstaining reagents
	<ol>
		<li>Prepare 50% hematoxylin.
		<ol>
			<li>In a fume hood, add 100 mL of Gill&#8217;s hematoxylin I (see the Table of Materials) to 100 mL of distilled water in a staining dish. 
			NOTE: The 50% hematoxylin staining solution can be reused for up to 1 week.</li>
		</ol>
		</li>
		<li>Prepare 0.02% (w/v) ammonia water (bluing reagent).</li>
		<li>In the fume hood, add 1.43 mL of 1 N ammonium hydroxide to 250 mL of distilled water in a graduated cylinder or another container. Seal the cylinder with paraffin film. Mix its contents well for 3x&#8211;5x. 
		NOTE: For assay quantitation, it is critical to use ammonium hydroxide. The reagents may be prepared ahead of time. Ensure all containers remain covered.</li>
	</ol>
	</li>
	<li>Preparation of 1x target retrieval reagent
	<ol>
		<li>In a large beaker, add 70 mL of 10x target retrieval reagent (see the Table of Materials) to 630 mL of distilled water.</li>
		<li>Place the beaker on a heating plate with a magnetic stirrer. Cover it with aluminum foil.</li>
		<li>Boil its contents at 100 &#176;C for 10&#8211;15 min. 
		NOTE: Do not let it boil for more than 30 min.</li>
	</ol>
	</li>
	<li>Reagent equilibration
	<ol>
		<li>Ensure the hybridization oven is on and at 40 &#176;C. Place wet humidifying paper at the bottom of the tray. 
		NOTE: A hybridization oven (see the Table of Materials) is needed for steps 3.4 to 4.2.</li>
		<li>Remove the amplification reagents (AMP1&#8211;AMP6, see the Table of Materials) from the refrigerator and keep them at room temperature, at least 30 min before the relevant incubation step.</li>
		<li>Before each use, warm the target and/or control probes for at least 10 min at 40 &#176;C in the oven or in a water bath or incubator.</li>
	</ol>
	</li>
</ol>
2. Adhesion enhancing and deparaffinization in the fume hood
NOTE: Start the protocol with 3&#8211;5 &#181;m-thick histological samples mounted on unstained slides.
<ol>
	<li>In order to enhance adhesion, bake the slides at 60 &#176;C for 1 h or at 40 &#176;C overnight in an oven.</li>
	<li>Charge the slide rack with unstained histological slides. Immerse the slide rack for 5 min in fresh xylene contained in a staining dish, with occasional agitation. repeat with fresh xylene.</li>
	<li>Immerse the slide rack for 3 min in fresh 100% ethanol contained in a staining dish, with constant agitation. Repeat with fresh 100% ethanol.</li>
	<li>Let the slides dry for 2 min at room temperature. 
	NOTE: Do not reuse deparaffinization reagents for dehydration of the slides after the assay.</li>
</ol>
3. Tissue pretreatment
NOTE: These steps follow the &#8220;standard&#8221; pretreatment recommendation according to the manufacturer&#8217;s instructions for head and neck samples. The timing of sections 3.1 and 3.2 may need to be adjusted depending on the manipulated tissue.
<ol>
	<li>Blockade of peroxidase activity
	<ol>
		<li>Add 4&#8211;6 drops of hydrogen peroxide (see the Table of Materials) to each slide and incubate them for 10 min at room temperature.</li>
		<li>Wash the slides 2x for 2 min in distilled water at room temperature.</li>
	</ol>
	</li>
	<li>Breakage of RNA/tissue bounds 
	<ol>
		<li>With a claw, remove the aluminum foil from the boiling 1x target retrieval reagent (TTR1x) from section 1.3 and stop stirring. Immerse the slide rack slowly and very carefully for 15 min. Cover the beaker again with the aluminum foil. 
		NOTE: Simmering has to persist during this step. 
		CAUTION: Use the claw to manipulate the aluminum foil and the slide rack so as to avoid burn injuries. Make sure to wear proper personal protective equipment, such as gloves and a lab coat.</li>
		<li>With the claw, immediately transfer the hot slide rack to a distilled water bath and wash it for 2 min. 
		NOTE: Make sure the samples do not cool down in the TTR1x.</li>
		<li>Wash the slides in fresh 100% ethanol for 2 min.</li>
		<li>Let the slides dry at room temperature for 2 min.</li>
	</ol>
	</li>
	<li>Barrier creation
	<ol>
		<li>With a hydrophobic barrier pen (see the Table of Materials), draw a barrier around the sample. Let it dry out at for least 5 min. 
		NOTE: Allow the barrier to really dry out. If it wears out during the procedure, do not hesitate to draw it again. Avoid touching the tissue with the pen. The protocol can be paused overnight here.</li>
	</ol>
	</li>
	<li>Protease digestion
	<ol>
		<li>Place the slides on the slide rack and add ~4 drops of Protease Plus per sample (see the Table of Materials).</li>
		<li>Cover the humidity control tray with a lid and insert it into the hybridization oven for 30 min at 40 &#176;C. 
		NOTE: To prevent evaporation, make sure the turn knob is completely turned to the lock position.</li>
		<li>Remove the tray from the oven and remove the slide rack.</li>
		<li>One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with distilled water.</li>
		<li>Wash the slides 2x for 2 min in distilled water at room temperature. Agitate constantly.</li>
	</ol>
	</li>
</ol>
4. Running the assay
NOTE: Do not let sections dry out between the incubation steps.
<ol>
	<li>Hybridization of the HPV probe 
	NOTE: Ensure the probes are prewarmed to dissolve any precipitation prior to use. For this step, instead of HPV probe, peptidyl-prolyl isomerase B (PPIB) for positive control or dihydrodipicolinate reductase (DAPB) for negative control (see the Table of Materials) may also be used.
	<ol>
		<li>Tap and/or flick the slides to remove any excess liquid and place them in the slide rack. Add ~4 drops of HPV probe to entirely cover each section.</li>
		<li>Cover the tray with a lid and insert it into the oven for 2 h at 40 &#176;C. 
		NOTE: To prevent evaporation, make sure the turn nob is completely turned to the lock position.</li>
		<li>Remove the tray from the oven and remove the slide rack.</li>
		<li>One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with 1x wash buffer.</li>
		<li>Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.</li>
	</ol>
	</li>
	<li>Hybridization of AMP1, AMP2, AMP3, and AMP4 
	NOTE: These steps include hybridization in the hybridization oven and AMP1&#8211;AMP4 from the purchased kit (see the Table of Materials).
	<ol>
		<li>Tap and/or flick to remove any excess liquid from the slides and place them in the slide rack. Add ~4 drops of AMP1 to entirely cover each section.</li>
		<li>Cover the tray with a lid and insert it into the oven for 30 min at 40 &#176;C.</li>
		<li>Remove the tray from the oven and remove the slide rack.</li>
		<li>One slide at a time, quickly remove any excess liquid and place the slide in a slide rack submerged in a staining dish filled with 1x wash buffer.</li>
		<li>Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.</li>
		<li>Repeat steps 4.2.1&#8211;4.2.5, but use ~4 drops of AMP2 instead of AMP1 and incubate for 15 min at 40 &#176;C. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
		<li>Repeat steps 4.2.1&#8211;4.2.5, but use ~4 drops of AMP3 instead of AMP1 and incubate for 30 min at 40 &#176;C. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
		<li>Repeat steps 4.2.1&#8211;4.2.5, but use ~4 drops of AMP4 instead of AMP1 and incubate for 15 min at 40 &#176;C. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
	</ol>
	</li>
	<li>Hybridization of AMP5 and AMP6 
	NOTE: These steps do not include hybridization in the oven but hybridization at room temperature. AMP5 and AMP6 come from the purchased kit (see the Table of Materials).
	<ol>
		<li>Tap and/or flick the slides to remove any excess liquid and place them in the slide rack. Add ~4 drops of AMP5 to entirely cover each section.</li>
		<li>Cover the tray with a lid and incubate for 30 min at room temperature.</li>
		<li>One slide at a time, quickly remove any excess liquid and place it in a slide rack submerged in a staining dish filled with 1x wash buffer.</li>
		<li>Wash the slides in 1x wash buffer for 2 min at room temperature with constant agitation. Repeat this with fresh 1x wash buffer.</li>
		<li>Repeat steps 4.3.1&#8211;4.3.4, but use ~4 drops of AMP6 instead of AMP5 and incubate for 15 min at room temperature. Wash the slides 2x for 2 min, both times in fresh wash buffer.</li>
	</ol>
	</li>
</ol>
5. Signal detection with 3,3&#39;-diaminobenzidine
CAUTION: Diaminobenzidine (DAB) is toxic. Follow appropriate precautions and safety guidelines when disposing of and handling this chemical.
<ol>
	<li>Mix equal volumes of DAB-A and DAB-B (see the Table of Materials) in an appropriately sized tube by dispensing the same number of drops of each solution. Make ~120 &#181;L of DAB substrate per section (~2 drops of each reagent/total of 4). Mix it well for 3x&#8211;5x.</li>
	<li>Take each slide, one at a time, from the slide rack and tap and/or flick to remove the excess liquid before placing it in the slide rack.</li>
	<li>Pipette ~120 &#956;L of DAB onto each tissue section. Ensure the sections are covered, and incubate for 10 min at room temperature.</li>
	<li>Dispose the remaining DAB according to local regulation and insert the slide into a slide rack submerged in a staining dish filled with tap water.</li>
</ol>
6. Counterstaining
<ol>
	<li>Move the slide rack to a staining dish containing 50% hematoxylin staining solution let it rest for 30 s at room temperature. Note that the slides will become purple.</li>
	<li>Immediately transfer the slide rack back to a staining dish containing tap water, and wash the slides 3x&#8211;5x by moving the rack up and down.</li>
	<li>Keep repeating the washing step with fresh tap water until the slides are clear, while sections remain purple.</li>
	<li>Replace the tap water in the staining dish with 0.02% ammonia water. Move the rack up and down 2x&#8211;3x. Note that the tissue section should turn blue.</li>
	<li>Replace the ammonia water with tap water. Wash the slides 3x&#8211;5x.</li>
</ol>
7. Dehydration
<ol>
	<li>Move the slide rack to a staining dish containing 70% ethanol in the fume hood and let it rest for 2 min with occasional agitation.</li>
	<li>Move the slide rack to a first staining dish containing 100% ethanol and let it rest for 2 min with occasional agitation.</li>
	<li>Move the slide rack to a second staining dish containing 100% ethanol and let it rest for 2 min with occasional agitation.</li>
	<li>Move the slide rack to a staining dish containing xylene and let it rest for 5 min with occasional agitation.</li>
</ol>
8. Slide mounting
<ol>
	<li>Remove the slides from the slide rack and lay them flat with the sections facing up in the fume hood.</li>
	<li>Mount one slide at a time by adding 1 drop of a xylene-based mounting medium to each slide and carefully placing a 24 mm x 50 mm coverslip over the section. Avoid trapping any air bubbles.</li>
	<li>Air-dry the slides for &#8805;5 min.</li>
</ol>
9. Sample evaluation
<ol>
	<li>Examine the tissue sections under a standard brightfield microscope at 20x&#8211;40x magnification.</li>
</ol>

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CB: boards, seminars: MSD, BMS, Astrazeneca, Roche

HPV RNA CISH performed with a purchased kit is a powerful tool for the detection of viral transcripts and it indicates active HPV infection. Performed manually, the steps of the protocol are overall easy to follow, and the purchased kit is convenient. This technique allows the staining of 19 histological samples plus one control slide at once, and the assay lasts around 8 h. It is critical not to let the samples dry out between steps unless otherwise mentioned. The pretreatment condition may need to be adjusted depending...

HPV RNA CISH is a powerful tool for the detection of active HPV infection, which may prove crucial in benign or malignant lesions in various locations such as the oropharynx or the uterine cervix. The detection of an active HPV infection may support the diagnosis of an HPV-induced lesion and, thereby, influence its treatment and prognosis.
HPV is the most frequent sexually transmitted infection, and over 100 viral genotypes have been described1. Schematically, low-risk genotypes such as genotypes 6 and 11 are known to induce genital warts, recurrent respiratory papillomatosis, and other benign lesions, whereas high-risk genotypes such as genotypes 16 and also 18 are responsible for most cervical cancers and anal cancers and play a role in HNSCC oncogenesis in variable proportions as accounted for by regional epidemiological data2.
Several tools are available for the detection of HPV infection. As a high-risk HPV infection leads to the expression of viral oncogenic proteins E6 and E73, the detection of E6 and E7 transcripts is widely viewed as the gold standard for active HPV infection identification4. HPV RNA CISH can be performed on FFPE samples that are quite easily obtained from patients suffering from various HPV-related diseases. Its performance has been evaluated in squamous intraepithelial neoplasia in the cervix, the anus, and the vagina, and in invasive squamous cell carcinoma in the cervix, the anus, and the upper aerodigestive tract5: it achieves a sensitivity of over 98% among HPV DNA polymerase chain reaction (PCR)-positive cases. This is slightly better than p16 immunostaining (93%) and HPV DNA in situ hybridization (DNA ISH: 97%), which are more commonly used. In another cohort of 57 patients with squamous cell carcinoma (SCC) arising from the head and neck region, the genital region, the skin, and the urinary tract, compared to HPV DNA ISH, HPV RNA CISH achieved better sensitivity (100% versus 88%) and specificity (87% versus 74%)6.
P16 immunostaining is an indirect marker reflecting cell cycle disruption that may be caused (but not exclusively) by HPV infection4,7. This cost-effective test possesses good sensitivity and a negative predictive value and is recommended as a surrogate marker of high-risk HPV infection in oropharynx cancer (OPC) by the College of American Pathologists (CAP) and by the Union for International Cancer Control (UICC)8.
Though this paper solely focuses on the detection of HPV in HNSCC, HPV RNA CISH is clinically relevant in various other conditions that involve HPV infection. For instance, this technique may improve the accuracy of the diagnosis of low-grade squamous intraepithelial lesions of the cervix (LSIL, formerly known as cervical intraepithelial neoplasia, grade 1 [CIN1]) for morphologically ambiguous cases9. Regarding oropharyngeal SCC, HPV RNA CISH allows the identification of HPV-related SCC, labeled as distinct from HPV-unrelated oropharyngeal SCC in the recent eighth edition of the TNM Classification of Head and Neck Cancer (of the Union for International Cancer Control [UICC])10. Since HPV-related SCC exhibits a better prognosis with longer survival and enhanced radiotherapy and chemotherapy sensitivity than HPV-unrelated SCC11,12,13, the detection of HPV infection may impact patient management14,15. Besides, HPV RNA CISH can be used for the diagnosis of HPV-related multiphenotypic sinonasal carcinoma with a higher signal than HPV DNA CISH16. Several multivariate analyses suggest that the detection of E6 and E7 transcripts is correlated with a better prognosis in oropharyngeal SCC overall7,15,17,18 and in the subgroup of p16-positive oropharyngeal SCC19,20.
Here we present the protocol for manual HPV RNA CISH performed on FFPE slides with a kit obtained from the manufacturer.&#160;

<table>
	<tbody>
		<tr>
			<td>Hematoxylin solution, Gill No. 1</td>
			<td>Merck</td>
			<td>GHS132</td>
			<td></td>
		</tr>
		<tr>
			<td>HybEZ Oven (110v)</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>321710</td>
			<td></td>
		</tr>
		<tr>
			<td>HybEZ slide rack</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>300104</td>
			<td></td>
		</tr>
		<tr>
			<td>ImmEdge Hydrophobic Barrier Pen</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>310018</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope 2.5 HD Detection Reagents-BROWN</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>322310</td>
			<td>This kit includes amplification reagents AMP1, AMP2, AMP3, AMP4, AMP5 and AMP6, and detection reagents DAB-A and DAB-B</td>
		</tr>
		<tr>
			<td>RNAscope 3-Plex Negative Control Probe</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>320871</td>
			<td>DAPB</td>
		</tr>
		<tr>
			<td>RNAscope 3-Plex Positive Control Probe</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>320861</td>
			<td>PPIB</td>
		</tr>
		<tr>
			<td>RNAscope H202 &#38; Protease Plus Reagent</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>322330</td>
			<td>Hydrogen Peroxyde x2 and Protease Plus x 1</td>
		</tr>
		<tr>
			<td>RNAscope Probe- HPV16/18</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>311121</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Target Retrieval Reagents</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>322000</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Wash Buffer Reagents</td>
			<td>Advanced Cell Diagnostics Inc.</td>
			<td>310091</td>
			<td>Wash Buffer 50X x4</td>
		</tr>
	</tbody>
</table>

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.

As described here, in head and neck squamous cell cancer, a case may be considered positive in the presence of brown punctiform staining in the cytoplasm or in the nuclei of tumor cells. In most studies, the signal is considered as either &#8220;positive&#8221; or &#8220;not detected&#8221;14. Methods of semiquantification of the signal have been reported but lack standardization between teams. For example, in some studies, the signals were scored as 1+ with 1&#8211;3 dots per tumor cell, 2+ with ...

Watch this Scientific Journal Video about Chromogenic In Situ Hybridization as a Tool for HPV-Related Head and Neck Cancer Diagnosis at JoVE.com

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

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 ...

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Research

JoVE Journal

Genetics

10.2K Views.  Paris Descartes University. 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.

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

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization (mFISH) and Spectral Karyotyping (SKY) in Irradiated Mice

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially compromised, can easily be identified by conventional chromosome staining techniques. However, detection of stable aberrations, which involve exchange or translocation of genetic materials without considerable modification in the chromosome morphology, requires sophisticated chromosome painting techniques that rely on in situ hybridization of fluorescently labeled DNA probes, a chromosome painting technique popularly known as fluorescence in situ hybridization (FISH). FISH probes can be specific for whole chromosome/s or precise sub-region on chromosome/s. The method not only allows visualization of stable aberrations, but it can also allow detection of the chromosome/s or specific DNA sequence/s involved in a particular aberration formation. A variety of chromosome painting techniques are available in cytogenetics; here two highly sensitive methods, multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY), are discussed to identify inter-chromosomal stable aberrations that form in the bone marrow cells of mice after exposure to total body irradiation. Although both techniques rely on fluorescent labeled DNA probes, the method of detection and the process of image acquisition of the fluorescent signals are different. These two techniques have been used in various research areas, such as radiation biology, cancer cytogenetics, retrospective radiation biodosimetry, clinical cytogenetics, evolutionary cytogenetics, and comparative cytogenetics.

Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice

The present protocol describes the usefulness of multiple fluorescence in situ hybridization (mFISH) and spectral karyotyping (SKY) in identifying inter-chromosomal stable aberrations in the bone marrow cells of mice after exposure to total body irradiation.

Ionizing radiation (IR) induces numerous stable and unstable chromosomal aberrations. Unstable aberrations, where chromosome morphology is substantially ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

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 ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Canalostomy As a Surgical Approach to Local Drug Delivery into the Inner Ears of Adult and Neonatal Mice

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) has been shown to be a useful approach to local drug delivery into the inner ear. The goal of this article is to describe, in detail, the surgical techniques involved in canalostomy in both adult and neonatal mice. As indicated by fast-green dye and adeno-associated virus serotype 8 with the green fluorescent protein gene, the canalostomy facilitated broad distribution of injected reagents in the cochlea and vestibular end-organs with minimal damage to hearing and vestibular function. The surgery was successfully implemented in both adult and neonatal mice; indeed, multiple surgeries could be performed if required. In conclusion, canalostomy is an effective and safe approach to drug delivery into the inner ears of adult and neonatal mice and may be used to treat human inner ear diseases in the future.

Here we describe canalostomy procedure which allows local drug delivery into the inner ears of adult and neonatal mice through the semicircular canal with minimum damage to hearing and vestibular function. This method can be used to inoculate viral vectors, pharmaceuticals, and small molecules into the mouse inner ear.

Local delivery of therapeutic drugs into the inner ear is a promising therapy for inner ear diseases. Injection through semicircular canals (canalostomy) ...

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 ...

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.

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.

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 ...

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 ...