Name: detection of inter-chromosomal stable aberrations by multiple fluorescence in situ hybridization (mfish) and spectral karyotyping (sky) in irradiated mice
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Description: Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

This study was supported by Arkansas Space Grant Consortium and National Space Biomedical Research Institute through National Aeronautics and Space Administration, grants NNX15AK32A (RP) and RE03701 (MH-J), and P20 GM109005 (MH-J), and the US Veterans Administration (MH-J). We thank Christopher Fettes, Program Coordinator for the Department of Environmental and Occupational Health at the University of Arkansas for Medical Sciences, for editorial assistance in preparation of the manuscript.

All animal studies were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The animal protocol was approved by the Institutional Animal Care and Use Committee of the University of Arkansas for Medical Sciences. All animals were housed under standard air-conditioned animal facility at 20 &#177; 2 &#176;C with 10 - 15 hourly cycles of fresh air and free access to standard rodent food and water. Upon arrival, the mice were h...

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Several critical steps determine the success of mFISH and SKY. The first and most critical step is to optimize the colchicine treatment for in vivo mitotic arrest of the bone marrow mononuclear cells. The colchicine concentration and treatment time individually or in concert determine the mitotic index as well as chromosome condensation-two important prerequisites for effective chromosome painting. A high colchicine concentration or longer treatment time leads to highly condensed chromosomes, incompatible for pr...

The two most reliable methods of identifying radiation-induced inter-chromosomal stable aberrations are multiple fluorescence in situ hybridization (mFISH), which allows the painting of two or more chromosomes simultaneously, and spectral karyotyping (SKY), which imparts a distinct color to each homologous chromosome pair in the genome. Unlike unstable aberrations, stable aberrations are persistent in nature and may be propagated for several generations in irradiated populations1, and are regarded as critical molecular "signatures" of radiation-induced cytogenetic lesions2. Studies by various groups have shown that stable aberrations are associated with the pathogenesis and development of a number of diseases, including cancer3. Before the era of chromosome painting (also referred as molecular cytogenetics), the conventional G-banding technique was the only method for detecting stable chromosomal aberrations. However, chromosome banding is a challenge to cytogeneticists because the resolution is limited, reproducibility is uncertain, it is a labor-intensive procedure, and it requires highly skilled and experienced cytogeneticists for reliable data interpretation4. Moreover, the classic banding technique does not allow detection of complex chromosomal rearrangements, which involve the interaction of three or more breaks distributed among two or more chromosomes, a common outcome of radiation damage. Complex aberrations may persist in individuals many years after radiation exposure, making them useful for retrospective biodosimetry5. Therefore, an alternate approach was required to overcome the limitations of conventional banding techniques to detect stable chromosomal rearrangements. 
In the late 1960s, the pioneering work of Gall and Pardue (1969) on molecular hybridization using nucleic acid probes labeled with radioactive material commenced a new era in the field of cytogenetics, which allowed detection of a specific DNA sequence on chromosomes6. However, the use of radioactive probes for molecular hybridization had several drawbacks: radioactive probes are relatively unstable, probe activity depends on radioactive decay of the isotope used, hybridization takes a longer time, the resolution is limited, the probes are relatively costly, and the radioactive materials are a health hazardous. Thus, it became necessary to develop and design non-radioactive probes. The introduction of fluorescent tagged nucleic acid probes in the 1980s and 1990s overcame the limitations of radioactive probes and greatly enhanced the safety, sensitivity, and specificity of the hybridization technique7-10. Fluorescent probes give rise to extremely bright signals when observed under fluorescence microscopes equipped with the appropriate excitation and emission filters. Any loss, gain, or rearrangement of fluorescent labeled chromosome/s or a part of the chromosome is easily identifiable with this FISH technique.
Analysis of chromosomal aberrations by FISH painting has led to marked progress in cytogenetic research over the years. Designing fluorescent labeled probes for specific applications ranging from locus-specific probes to whole-chromosome painting probes has advanced the field significantly; this has also facilitated the detection of submicroscopic ("cryptic") rearrangement, which was not possible by conventional chromosome banding. Chromosome painting by mFISH and SKY have proven to be valuable tools for the identification of simple and complex inter-chromosomal rearrangements. The basic principles for both techniques are similar, but the method of detection and discrimination of fluorescent signal after in situ hybridization and the process of image acquisition are different. In mFISH, separate images of each of the four fluorochromes are captured by using narrow bandpass microscope filters; dedicated software is then used to combine the images. While in SKY, image acquirement is based on a combination of epifluorescence microscopy, charge-coupled device imaging, and Fourier spectroscopy, which allows the measurement of the entire emission spectrum with a single exposure at all image points. In both mFISH and SKY, monochrome images are captured independently, then merged, and finally, unique pseudo-colors are assigned to the chromosomes in monochromatic images based on the specific dye attached to each fluorochrome probe.
The contribution of mFISH and SKY analysis in the radiation biology field is remarkable, particularly for retrospective dose estimation of human exposure to IR (radiation biodosimetry)11-14, radiation carcinogenesis risk assessment15, as well as detection and risk estimation of radiotherapy-related secondary cancer16. A recent study on mice has shown that a FISH-based chromosome painting technique is also an important tool for evaluating the efficacy of radiation countermeasure17. In the present study, the effect of total body radiation exposure on the induction of stable chromosomal aberrations in the bone marrow cells of mice has been demonstrated using mFISH and SKY techniques.

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			<td height="20" style="height:20px;width:451px;">Formamide</td>
			<td style="width:176px;">Sigma-Aldrich</td>
			<td style="width:108px;">221198-100ML</td>
			<td style="width:155px;"></td>
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			<td height="20" style="height:20px;">SSC Buffer 20&#215; Concentrate</td>
			<td>Sigma-Aldrich</td>
			<td>S6639-1L</td>
			<td></td>
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		<tr height="20">
			<td height="20" style="height:20px;">SKY Laboratory Reagent for Mouse</td>
			<td>Applied Spectral Imaging</td>
			<td>FPRPR0030/M40</td>
			<td></td>
		</tr>
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			<td height="20" style="height:20px;">CAD - Concentrated Antibody Detection Kit</td>
			<td>Applied Spectral Imaging</td>
			<td>FPRPR0033</td>
			<td></td>
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			<td height="20" style="height:20px;">Single Paints Customized - 3 Colors; Mouse chromosome 1: Red, Mouse chromosome 2: Green, Mouse chromosome 3: Aqua</td>
			<td>Applied Spectral Imaging</td>
			<td>FPRPR0182/10</td>
			<td></td>
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			<td height="20" style="height:20px;">Glass coverslips</td>
			<td>Fisher Scientific</td>
			<td>12-545B</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tween 20</td>
			<td>Fisher Scientific</td>
			<td>BP337-100</td>
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			<td height="20" style="height:20px;">Hydrochloric acid, 37%, Acros Organics</td>
			<td>Fisher Scientific</td>
			<td>AC45055-0025&#160;</td>
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			<td height="20" style="height:20px;">Fisherbrand Glass Staining Dishes&#160; with Screw Cap</td>
			<td>Fisher Scientific</td>
			<td>08-816</td>
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		<tr height="20">
			<td height="20" style="height:20px;">KaryoMAX Potassium Chloride Solution&#160;</td>
			<td>Life Technologies</td>
			<td>10575-090</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Fisherbrand Superfrost Plus Microscope Slides</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
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			<td height="20" style="height:20px;">Colcemid powder</td>
			<td>Fisher Scientific</td>
			<td>50-464-757&#160;</td>
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		<tr height="20">
			<td height="20" style="height:20px;">Histopaque-1083&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>10831</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Shepherd Mark I, model 25 137Cs irradiator</td>
			<td>J. L. Shepherd &#38; Associates</td>
			<td>Model 484B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Syringe 1 ml</td>
			<td>BD Biosciences</td>
			<td>647911</td>
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			<td height="20" style="height:20px;">Ethyl Alcohol, 200 Proof</td>
			<td>Fisher Scientific</td>
			<td>MEX02761</td>
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			<td height="20" style="height:20px;">PBS, (1X PBS Liq.), w/o Calcium and Magnesium</td>
			<td>Fisher Scientific</td>
			<td>ICN1860454</td>
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			<td height="20" style="height:20px;">Fetal Bovine Serum</td>
			<td>Fisher Scientific</td>
			<td>10-437-010</td>
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			<td height="20" style="height:20px;">Methanol</td>
			<td>Fisher Scientific</td>
			<td>A454SK-4</td>
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			<td height="20" style="height:20px;">Glacial acetic acid</td>
			<td>Fisher Scientific</td>
			<td>AC295320010</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Zeiss Microscope</td>
			<td>Zeiss</td>
			<td>AXIO Imager.Z2</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Total body irradiation induces numerous chromosomal aberrations in the bone marrow cells of irradiated mice. The current protocol is optimized for in vivo mitotic arrest of bone marrow cells after radiation exposure, harvest of bone marrow cells from the hind legs of irradiated mice, isolation of bone marrow mononuclear cells by density gradient centrifugation, preparation of metaphase cell spreads, and subsequent detection of radiation-induced stable chromosomal aberrations by m...

Watch this Scientific Journal Video about Detection of Inter-chromosomal Stable Aberrations by Multiple Fluorescence In Situ Hybridization mFISH and Spectral Karyotyping SKY in Irradiated Mice at JoVE

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.

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 ...

The overall goal of this molecular cytogenic method is to observe interchromosomal stable aberrations in the bone marrow cells of mice exposed to total body irradiation. This method can help answer the key question in the radiation biology field such as the risk of inducing stable chromosomal aberrations in the bone marrow cells of mice exposed to total body radiation. The main advantage of this technique is that it allows guided visualization of radiation induced stable genetic damages in the bone marrow cells of mice that can be propagated through many cell generations.After isolating bone marrow from mouse femur and tibia bones and making a single cell suspension according to the text protocol, carefully overlay the cell suspension on an equal volume of bone marrow mononuclear cell separation medium. Centrifuge the gradient at 400 times G at room temperature for 30 minutes. Then, carefully collect the buffy coat without disturbing the remaining layers and transfer the solution into a new 15 milliliter centrifuge tube.To prepare metaphase cell spreads, add 10 milliliters of PBS to the tube containing the buffy coat. Then centrifuge the tube at 400 times G at room temperature for five minutes. Next, carefully remove the supernatant and gently tap the tube to break up the cell pellet.Then add 10 milliliters of PBS and centrifuge the suspension again. Remove the supernatant without disturbing the cell pellet. Break up the pellet and add drop by drop four milliliters of pre-warmed hypotonic 0.075 molar potassium chloride solution with gentle, constant shaking.Incubate the cell suspension at 37 degrees Celsius for 20 minutes in a water bath. Then, add an equal volume of fixative to the tube and mix gently by inverting the tube. Centrifuge the tube, and remove the supernatant.Then, break up the cell pellet and add fresh fixative. Repeat the spinning and the addition of fixative five more times. After resuspending the cells in 400 to 600 microliters of fixative, drop 30 microliters of fixed cells onto precleaned and wet slides tilted at a 45 degree angle and allow the slides to air dry completely overnight.To carry out mouse chromosome painting by mFISH place the slide containing the metaphase cell spreads in 2X SSC for two minutes at room temperature. Then, dehydrate the slide by serial ethanol washing in 70%80%and 100%ethanol for two minutes in each wash. Preheat 40 milliliters of denaturation solution in a glass coplin jar to 70 degrees Celsius for 30 minutes.Then transfer the slide into the prewarmed solution and incubate the sample for one to 1 1/2 minutes to denature the chromosomes. Immediately use ice cold 70%ethanol to quench the slide for two minutes to stop the denaturation process and prevent denatured chromosomes from reannealing. Then dehydrate the slide using an ethanol series beginning with 80%ethanol for two minutes.Next, place the slide in 100%ethanol for two minutes. Then completely dry the slide at room temperature. To denature the probe, briefly centrifuge the probe mixture supplied by the manufacturer, then transfer 10 microliters into a 500 microliter snap cap centrifuge tube and incubate the tube in a water bath at 80 degrees Celsius for seven minutes.Now place the tube containing the denatured probe into a water bath at 37 degrees Celsius for 10 minutes. Then apply the probe mixture onto a slide with denatured chromosomes. Carefully cover the area with an 18 millimeter by 18 millimeter glass coverslip and eliminate any visible air bubbles by very gently pressing the coverslip to the slide.Use rubber cement to seal all four sides of the coverslip. And incubate the slide in the dark in a humidified chamber at 37 degrees Celsius for 12 to 16 hours. Following hybridization, carefully remove the rubber cement and coverslip and place the slide in prewarmed 0.4X SSC at 74 degrees Celsius for five minutes.Then transfer the slide into washing solution three for two minutes. Add 20 microliters of antifade mounting medium with DAPI counterstain onto the slide and cover it with a glass coverslip. Use lab tissue paper to gently press on the coverslip to remove any air bubbles and excess mounting solution.Then use nail polish to seal the edges of the coverslip. View the slides using a fluorescent microscope equipped with the appropriate filters. For spectral karyotyping of mouse chromosomes, after the probes are hybridized onto the denatured chromosomes and the samples are washed according to the text protocol, apply 80 microliters of SY5 staining reagent to the slide.Place a 24 by 60 millimeter plastic coverslip on top of the sample, and incubate it in the dark in a humidified chamber at 37 degrees Celsius for 40 minutes. Dip the slide into a glass coplin jar containing prewarmed washing solution three and incubate it in a water bath at 45 degrees Celsius with shaking for two minutes. Repeat the wash three times.Apply 80 microliters of SY5.5 staining reagent to the sample, cover it with a 24 by 60 millimeter plastic coverslip, and incubate it in the dark in a humidified chamber at 37 degrees Celsius for 40 minutes. Use prewarmed washing solution three to wash the slide three times then hold the slide in a tilted position against a paper towel to drain the excess fluid. Add 20 microliters of antifade DAPI reagent to the sample and carefully place a glass coverslip on top without introducing any air bubbles.Use nail polish to seal the edges and observe the sample with an epifluorescence microscope equipped for capturing sky images. This figure shows representative metaphase cell spreads with normal and aberrant mouse chromosome pairs one, two, and three. Here is a stable aberration involving chromosome one where a part of chromosome one has been integrated into a non-painted DAPI chromosome while another part has formed an acentric fragment.In this example, a part of chromosome two has been integrated into a non-painted chromosome. This stable aberration involves chromosome three. Stable aberrations involving all three painted chromosomes are seen in this metaphase spread.Shown here is an example of spectro karyotyping of a normal female mouse. This panel represents a stable aberration involving chromosomes four and 12. Finally, the stable chromosomal aberration in this panel involves chromosome seven and 10.Once mastered, this technique can be done within 48 to 72 hours if it is done properly. While attempting this procedure, it is important to remember that only proliferating cells should be used. Following this procedure and other methods like in band can be used to answer additional questions like whether inter-chromosomal stable aberrations are present in the irradiated cells.After its development, this technique paved the way for the researchers in the field of molecular cytogenetics to explore the association between genetic aberrations with the various diseases. After watching this video you should have a clearer idea how to determine inter-chromosomal stable aberrations. Don't forget that working with colchicine can be extremely dangerous because it is carcinogen and precautions such as wearing gloves should always be taken while performing this experiment.

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Research

JoVE Journal

Genetics

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and transmitted from one generation to the next through the female oocyte is of fundamental importance. Because of the genetic tractability, and the elegant, ordered simplicity by which oocyte development proceeds, Drosophila oogenesis has become an invaluable system for mitochondrial study. An EdU (5-ethynyl-2&#180;-deoxyuridine) labeling method was utilized to detect mitochondrial DNA (mtDNA) replication in Drosophila ovaries. This method is superior to the BrdU (5-bromo-2'-deoxyuridine) labeling method in that it allows for good structural preservation and efficient fluorescent dye penetration of whole-mount tissues.
Here we describe a detailed protocol for labeling replicating mitochondrial DNA in Drosophila adult ovaries with EdU. Some technical solutions are offered to improve the viability of the ovaries, maintain their health during preparation, and ensure high-quality imaging. Visualization of newly synthesized mtDNA in the ovaries not only reveals the striking temporal and spatial pattern of mtDNA replication through oogenesis, but also allows for simple quantification of mtDNA replication under various genetic and pharmacological perturbations.

In Situ Labeling of Mitochondrial DNA Replication in Drosophila Adult Ovaries by EdU Staining

Drosophila oogenesis continues to be exceptionally useful in the study of mitochondrial proliferation and inheritance. This manuscript describes a detailed protocol used to label the replicating mitochondrial DNA (mtDNA) in Drosophila adult ovaries with 5-ethynyl-2&#180;-deoxyuridine (EdU), which facilitates uncovering mechanisms associated with mitochondrial inheritance that were previously debatable.

The mitochondrial genome is inherited exclusively through the maternal line. Understanding of how the mitochondrion and its genome are proliferated and ...

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. To assess the impact of PM on nuclear genetic integrity, structural chromosomal aberrations are scored in the metaphase spreads of mouse RAW264.7 macrophage cells. PM is collected from ambient air with a high volume total suspended particles sampler. The collected material is solubilized and filtered to retain the water-soluble, fine portion. The particles are characterized for chemical composition by nuclear magnetic resonance (NMR) spectroscopy. Different concentrations of particle suspension are added onto an in vitro culture of RAW264.7 mouse macrophages for a total exposure time of 72 hr, along with untreated control cells. At the end of exposure, the culture is treated with colcemid to arrest cells in metaphase. Cells are then harvested, treated with hypotonic solution, fixed in acetomethanol, dropped onto glass slides and finally stained with Giemsa solution. Slides are examined to assess the structural chromosomal aberrations (CAs) in metaphase spreads at 1,000X magnification using a bright-field microscope. 50 to 100 metaphase spread are scored for each treatment group. This technique is adapted for the detection of structural chromosomal aberrations (CAs), such as chromatid-type breaks, chromatid-type exchanges, acentric fragments, dicentric and ring chromosomes, double minutes, endoreduplication, and Robertsonian translocations in vitro after exposure to PM. It is a powerful method to associate a well-established cytogenetic endpoint to epigenetic alterations.

Analysis of the Ambient Particulate Matter-induced Chromosomal Aberrations Using an In Vitro System

This protocol describes techniques for the quantification and characterization of chromosomal aberrations in vitro in RAW264.7 mouse macrophages after treatment with ambient air particulate matter.

Exposure to particulate matter (PM) is a major world health concern, which may damage various cellular components, including the nuclear genetic material. ...

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 ...

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 ...

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including biological such as T-DNA or transposon, chemical such as ethyl methanesulfonate (EMS), or physical such as ionization radiation. The type of mutation observed varies depending on the mutagen used. For ionization radiation induced mutants, mutations include deletion, duplication, or rearrangement. While T-DNA or transposon-based mutagenesis is limited to species that are susceptible to transformation, chemical or physical mutagenesis can be applied to a broad range of species. However, the characterization of mutations derived from chemical or physical mutagenesis traditionally relies on a map-based cloning approach, which is labor intensive and time consuming. Here, we show that a high-density genome array-based comparative genomic hybridization (aCGH) platform can be applied to efficiently detect and characterize copy number variations (CNVs) in mutants derived from fast neutron bombardment (FNB) mutagenesis in Medicago truncatula, a legume species. Whole genome sequence analysis shows that there are more than 50,000 genes or gene models in M. truncatula. At present, FNB-induced mutants in M. truncatula are derived from more than 150,000 M1 lines, representing invaluable genetic resources for functional studies of genes in the genome. The aCGH platform described here is an efficient tool for characterizing FNB-induced mutants in M. truncatula.

An Array-based Comparative Genomic Hybridization Platform for Efficient Detection of Copy Number Variations in Fast Neutron-induced Medicago truncatula Mutants

This protocol provides experimental steps and information about reagents, equipment, and analysis tools for researchers who are interested in carrying out whole genome array-based comparative genomic hybridization (CGH) analysis of copy number variations in plants.

Mutants are invaluable genetic resources for gene function studies. To generate mutant collections, three types of mutagens can be utilized, including ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene expression, but whether looping is responsible for or a result of alterations in gene expression is still unknown. Until recently, how chromatin looping affects the regulation of gene activity and cellular function has been relatively ambiguous, and limitations in existing methods to manipulate these structures prevented in-depth exploration of these interactions. To resolve this uncertainty, we engineered a method for selective and reversible chromatin loop re-organization using CRISPR-dCas9 (CLOuD9). The dynamism of the CLOuD9 system has been demonstrated by successful localization of CLOuD9 constructs to target genomic loci to modulate local chromatin conformation. Importantly, the ability to reverse the induced contact and restore the endogenous chromatin conformation has also been confirmed. Modulation of gene expression with this method establishes the capacity to regulate cellular gene expression and underscores the great potential for applications of this technology in creating stable de novo chromatin loops that markedly affect gene expression in the contexts of cancer and development.

Chromatin looping plays a significant role in gene regulation; however, there have been no technological advances that allow for selective and reversible modification of chromatin loops. Here we describe a powerful system for chromatin loop re-organization using CRISPR-dCas9 (CLOuD9), demonstrated to selectively and reversibly modulate gene expression at targeted loci.

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

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 ...