Name: detection of viral rna by fluorescence in situ hybridization (fish)
Uploaded: 2012-05-05T14:00:00
Duration: 10 min 16 s
Description: Watch this Scientific Journal Video about Detection of Viral RNA by Fluorescence in situ Hybridization FISH at JoVE.com

The authors thank past and present members of the lab for contributions to the development of the methodology outlined here and to Alan Cochrane for advice. L.A. is a recipient of a Canadian Institutes of Health Research (CIHR) Doctoral Fellowship and AJM is supported by a Fraser, Monat and MacPherson Career Award. This work is supported by a grant from the CIHR (grant #MOP-56974).

1. Probe Preparation
<ol>
	<li>Digest 1 &mu;g of the in vitro transcription vector, pKS(+)pol 236nt 9 with Kpn1 enzyme at 37 &deg;C for 1 hour and run the linearized DNA product on an agarose gel. The fragment should be approximately 2500 bp.</li>
	<li>Cut the fragment out of the gel and purify using the Roche Micro Elute Gel Extraction Kit (all reagents used are listed in Table 1). Perform the final elution in 30 &mu;l elution buffer. Run 5 &mu;l of the product on an agarose gel to verify the purification.</li>
	<li>Mix an in vitro transcription reaction (see recipe below) using the Roche DIG RNA labeling kit and incubate at 37 &deg;C for 2 hours.</li>
</ol>
In vitro transcription reaction:
<table border="1">
	<tbody>
		<tr>
			<td>Linearized DNA</td>
			<td>23 &mu;l</td>
		</tr>
		<tr>
			<td>1X DIG RNA labeling mix (Roche)</td>
			<td>4 &mu;l</td>
		</tr>
		<tr>
			<td>1X Transcription buffer</td>
			<td>8 &mu;l</td>
		</tr>
		<tr>
			<td>20U RNase OUT</td>
			<td>1 &mu;l</td>
		</tr>
		<tr>
			<td>80U T7 RNA polymerase</td>
			<td>4 &mu;l</td>
		</tr>
		<tr>
			<td>Total</td>
			<td>40 &mu;l</td>
		</tr>
	</tbody>
</table>
<ol start="4">
	<li>Add 228 U of DNase I and incubate at 37 &deg;C for 15 min.</li>
	<li>Stop the reaction by adding EDTA pH 8.0 to a final concentration of 20 mM.</li>
	<li>Purify the reaction by using the Quick Spin Columns from Roche as specified by the manufacturer.</li>
	<li>Purify the probe by precipitation with 1/10 volume DEPC-treated 3M NaOAc pH 5.2 and 2.5 volumes of ice-cold 95% ethanol. Mix and incubate at -80 &deg;C for 30 min to overnight.</li>
	<li>Centrifuge the probe for 15 min at 4 &deg;C at 15,000 x g.</li>
	<li>Remove the supernatant and wash the pellet in ice-cold 70% ethanol. Centrifuge again for 5 min at 4 &deg;C at 15,000 x g.</li>
	<li>Remove the supernatant and dry the pellet. Resuspend the pellet in 50 &mu;l water (DNase/RNase free) containing 10 U Invitrogen RNase OUT.</li>
	<li>Estimate the concentration of probe by spectrophotometry (measure RNA at 260 nm), and dilute to a concentration of 5 ng/&mu;l. Store in aliquots at -20 &deg;C for short-term use, or -80 &deg;C for long-term storage. It is recommended to perform inter assay comparisons so conserve an aliquot for this.</li>
</ol>
2. Cell Fixation
<ol>
	<li>It is initially important to seed adherent cells onto untreated, sterile glass coverslips so the final confluency upon harvesting is approximately 70-80%. For non-adherent cells, grow as normal and when ready to collect, incubate them with coverslips that have been treated with 0.01% w/v poly-L-lysine (Sigma-Aldrich, Inc.) for 1 hour. For a typical 12-well polystyrene tissue culture plate (3.8 cm2), 150,000 cells (e.g. HeLa) are plated at 24 hours prior to transfection. Non-adherent cells can be infected or transfected as usual and allowed to adhere to glass cover slips before fixation 3.</li>
	<li>Discard the media and wash the cells with 1X phosphate-buffered saline prepared in double distilled water (DPBS) for 1 min. Diethylpyrocarbonate (DEPC, Sigma-Aldrich, Inc.)-treated 1X PBS may also be used, however untreated PBS cannot as it may contain RNase enzymes.</li>
	<li>Discard the PBS and add 4% paraformaldehyde, enough to cover the cells completely. Incubate for 15-20 min.</li>
	<li>Discard the paraformaldeyhyde and wash cells with 1X DPBS for 1 min.</li>
	<li>Discard the DPBS and add 0.1 M glycine (dissolved in 1X DPBS). Incubate for 10 min.</li>
	<li>Discard the glycine and wash the cells with 1X DPBS for 1 min.</li>
	<li>Discard the DPBS and add 0.2% Triton-X (dissolved in 1X DPBS). Incubate for 5-10 min. If staining for nucleolar or nuclear envelope proteins, permeabilize with Triton X-100 for no more than 5 min or the protein localization will become diffuse.</li>
	<li>Discard Triton X-100 and wash once in 1X DPBS for 1 min.</li>
	<li>For long-term storage, replace PBS with 70% ethanol and store at 4 &deg;C for up to four months. Alternatively, wash once more with 1X DPBS and proceed to FISH.</li>
</ol>
3. Fluorescence in situ Hybridization (FISH)
<ol>
	<li>If the coverslips were stored in ethanol, replace the ethanol with 1X DPBS and allow the cells to rehydrate for 30 min. Rehydration can be done overnight if the coverslips are stored at 4 &deg;C.</li>
	<li>Wash the coverslips with 1X DPBS for 1 min.</li>
	<li>Prepare slides to incubate the coverslips on, taking care to clean and label them well.</li>
	<li>For an 18mm coverslip, add 50 &mu;l of DNase solution (25 units/coverslip, commercially available) to the slide. Add the coverslip, being sure to place it cell side down, and incubate it for 15 min at room temperature.</li>
	<li>Wash the coverslips with 1X DPBS for 1 min.</li>
	<li>Add 50 &mu;l hybridization mix (see recipe below) per coverslip onto a slide and incubate the coverslip cell side down for 16-18 h at 42 &deg;C. The incubation should be performed in a tray containing a 50% formamide, 2X SSPE mix (12.5 ml deionized formamide, 2.5 ml 20X SSPE, 10 ml water).</li>
</ol>
Hybridization Mix (for one coverslip, 50 &mu;l):
<table border="1">
	<tbody>
		<tr>
			<td>Amount of stock</td>
			<td>Material (at final concentration)</td>
		</tr>
		<tr>
			<td>25 &mu;l</td>
			<td>Formamide, 50% (deionized; any source)</td>
		</tr>
		<tr>
			<td>5 &mu;l (of 10 mg/ml)</td>
			<td>tRNA, 1 mg/ml (Sigma-Aldrich, Inc)</td>
		</tr>
		<tr>
			<td>5 &mu;l (of 20X)</td>
			<td>SSPE, 2X</td>
		</tr>
		<tr>
			<td>5 &mu;l (of 50X)</td>
			<td>Denharts, 5X</td>
		</tr>
		<tr>
			<td>0.125 &mu;l (of 5 units)</td>
			<td>RNase OUT</td>
		</tr>
		<tr>
			<td>5 &mu;l (25 ng of 5 ng/&mu;l)</td>
			<td>Probe</td>
		</tr>
		<tr>
			<td>5 &mu;l</td>
			<td>H2O DEPC, to make the final volume 50 &mu;l</td>
		</tr>
	</tbody>
</table>
<ol start="7">
	<li>Incubate coverslips cell side down in 50 &mu;l 50% formamide (diluted in 1X DPBS) for 15 min at 42 &deg;C.</li>
	<li>Wash the coverslips twice in 2X SSPE by incubating them cell side down in 50 &mu;l 2X SSPE (20X SSPE diluted in 1X DPBS) for 5 min each at 42 &deg;C.</li>
	<li>Wash the coverslips in 1X DPBS for 1 min.</li>
</ol>
4. Immunofluorescence Staining
<ol>
	<li>Block the coverslips by placing them cell side down in 50 &mu;l 1X Roche blocking solution (10X Roche blocking solution diluted in 1X DPBS) for 30 min.</li>
	<li>Incubate the coverslips cell side down in 50 &mu;l primary antibody solution for 1 hour at 37 &deg;C. Anti-DIG antibody should be diluted according to manufacturer's direction in 1X blocking solution. If other antibodies are being used to stain proteins, they may be added at the correct concentration provided that all antibodies used are derived from different host species.</li>
	<li>Wash the coverslips for 10 min in 1X DPBS.</li>
	<li>Incubate the coverslips cell side down in 50 &mu;l secondary antibody solution for 1 hour at 37 &deg;C. Alexa Fluor-conjugated antibodies (Invitrogen) can be used to generate a range of colors and are used at 1:500 in 1X blocking solution, 1X DPBS.</li>
	<li>Wash the coverslips twice for 10 min each in 1X DPBS.</li>
	<li>Dry the coverslips cell side up on Whatman paper. Be sure to cover the coverslips to avoid exposure to light and bleaching.</li>
	<li>Once all liquid is evaporated, mount the coverslips onto fresh slides using 8 &mu;l ImmunoMount (Thermo Scientific, Inc.). Gently tap down the coverslip to eliminate any air bubbles.</li>
	<li>Apply nail polish to the edges of the coverslip to secure it in place.</li>
	<li>The visualization of RNA and proteins by microscopy techniques is also critical to the success of this technique. Settings on the microscope used can help or hinder visualization so it is key that the settings on the microscope be set correctly and be consistent from one sample to another in a given experiment. For detailed microscopy settings, optics and antibody combinations please see references1,10.</li>
</ol>
5. Representative Results
An example of viral RNA staining can be seen in Figure 2. The viral RNA for HIV-1 is seen throughout the cytoplasm displayed diffusely for the most part, although small cytoplasmic punctae are not uncommon. The specificity can be seen by comparing positive cells to surrounding cells that display no fluorescence. As mentioned in the introduction, HIV-1 that lacks the regulatory Rev protein produces RNA which is retained in the nucleus: this can be visualized as a bright signal in the nucleus, and a lack of RNA fluorescence signal in the cytoplasm. Overexpression of cellular proteins is also capable of shifting the distribution of the HIV-1 viral RNA. In Figure 2, the overexpression of proteins involved in endosomal vesicle trafficking (e.g., Rab7-interacting lysosomal protein (RILP)11 or N-terminally deleted RILP (RILPN)11, and proteins that interfere with dynein motor function [e.g. p50/dynamitin1,12], interfere with the normal steady-state localization of the viral genomic RNA as determined by FISH analyses. RILP expression leads to re-localization of the viral genomic RNA to the microtubule organization center (MTOC) 13; RILPN disperses late endosomes within the cytoplasm due to the inability to bind to p150Glued of the dynein motor complex 14 and p50/dynamitin blocks the large subunit of dynein motor and releases late endosomes but also the viral genomic RNA and HIV-1 structural proteins to the cell periphery1 (Figure 2). The manipulation of the steady-state localization of the viral genomic RNA, a key contributor to the infectiousness of virus particles, would be key to the eventual development of therapeutics. In our hands, viral RNA appears in the cell as early as 3 hours post-transfection and infection10 and becomes readily observable by this FISH technique by 12 hours.
<img alt="Figure 1" src="/files/ftp_upload/4002/4002fig1.jpg" /> 
Figure 1. Flow chart of the protocol for FISH/IF co-analyses. Cells are grown on coverslips and then fixed with paraformaldehyde. Treatments of glycine and Triton-X are performed before the cells are either used for FISH/IF or stored in 70% ethanol for storage. Cells dehydrated for storage are rehydrated in 1X DPBS (DEPC-treated PBS) before continuing on to FISH/IF. Cells are treated with DNase I and left overnight to hybridize with probe. Once hybridization is complete, the cells are washed with formamide, as well as with SSPE, and a final 1X DPBS rinse. Blocking solution is applied to the cells, followed by incubation with the primary antibodies. After washing, secondary antibodies are applied. Two final washes in 1X DPBS complete the staining procedure where upon the coverslips are dried and mounted on slides for imaging.
<img alt="Figure 2" src="/files/ftp_upload/4002/4002fig2.jpg" /> 
Figure 2. Representative results using FISH/IF co-analyses. A) HIV-1 is transfected into HeLa cells and in some cases with constructs that cause the overexpression of cellular proteins (RILP, p50/Dynamitin and RILP&Delta;N (an amino-terminal deletion mutant)). FISH/IF co-analyses was performed: RNA is identified in green with either G3BP or LAMP1 (red) to differentiate. B) HIV-1 is expressed in HeLa and collected at different time points. Viral RNA expression is tracked in green while the cellular protein, hnRNP A1, is stained in red. Size bars are 10 &mu;m. Images modified from references 1 (select figures from panel A; RILP, p50/Dynamitin, and RILP deltaN) and 17 (panel B).

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No conflicts of interest declared.

FISH/IF co-analyses is a reliable method to visualize viral RNA in cells which has now been refined 9,15,16. Over the course of several years, we have developed a refined method of staining for RNA. This technique can be used on a wide array of cell types provided the probe is specific to the target RNA17. By labeling the probe with DIG, we are capable of visualizing the RNA by simple staining. When staining for RNA and other proteins are combined, FISH/IF co-analyses become a powerful tool to observe cellular structures and protein/RNA localization.
The specificity of the RNA detection is quite high. This is illustrated in Figure 2. In some of the original work that focused on viral genomic RNA localization, FISH established that a lack of Rev trapped viral RNA in the nucleus18. Since this, abundant new information on viral genomic RNA localization has been obtained using the technique outlined here. By overexpressing cellular proteins, specific populations -and not all- of the viral RNA can be forced to localize to different regions of the cell. RILP overexpression, which resembles the phenotype obtained when hnRNP A2 is depleted by siRNA in HIV-1-expressing cells13, causes the RNA to visibly accumulate at the MTOC. The viral genomic RNA can be pushed to the cell periphery by disabling the minus-end motor protein, dynein: p50/Dynamitin overexpression or knockdown of the dynein heavy chain1 results in the release of viral genomic RNA from intracellular domains to the cellular periphery (Figure 2). A mutant of RILP, RILP&Delta;N, which no longer binds the dynein motor, disperses endosomes (tagged by LAMP1) into the cytoplasm because they are no longer actively localized at juxtanuclear domains. These results point to the notion of a plastic population of HIV-1 genomic RNA and in particular, that different pools of HIV-1 genomic RNA exist with sometimes identifiable roles in the viral replication cycle. These same notions have already been identified for the protein encoded by this mRNA, Gag 19.
There are limitations to the uses of FISH/IF co-analyses that are related to antibody choice and availability. The optimization of the best combination of primary and secondary antibodies, and their concentrations, will take time to optimize (see Table 1 &amp; 2). Antibodies made from hybridomas or produced by major companies can have concentrations which vary anywhere from 1:2 to 1:2000, but be sure to follow the guidelines provided by the manufacturer for best results. The host in which the antibody is produced must also be taken under consideration. Mixing sheep with goat antibodies should also be avoided in primary and secondary antibodies as this combination results in high background due to cross-species recognition (data not shown). For Alexa-Fluor secondary antibodies, the concentration can be used at 1:500 for almost any antibody in the set. The other drawback to FISH/IF co-analyses as described in this report is it captures the RNA at its location at steady-state, after cells have been fixed and permeabilized using paraformaldehyde and detergent (Figure 1). 
However, RNA imaging in live cells has been achieved through a variety of means 20-22, mostly using variations of viral RNA genomes that are tagged by fluorescent proteins such as GFP. However, additional means to identify RNA localization in live cells continue to surface. These new methods involve the tagging RNA by means of Spinach 23, SNAP 24, or MTRIP 2. These techniques also suffer from a few drawbacks including the requirement to permeabilize cells before adding substrates or that a moiety must be engineered to tag the mRNA in order detect the mRNA by microscopy. Indeed, the major advantage of FISH analyses outlined in this report lies in the fact that the native RNA is unaltered leading to the most physiologically relevant results.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>18mm coverslips</td>
 <td>VWR</td>
 <td>48380 046</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>16% paraformaldehyde</td>
 <td>J.T. Baker</td>
 <td>S898-07</td>
 <td>To make 4%: Dissolve 20g in 500mL 1XDPBS at 60&deg;C with 5 drops NaOH. pH to 7.2 off the heat and store at -20 &deg;C. Do not exceed 70&deg;C!</td>
 </tr>
 <tr>
 <td>Triton-X</td>
 <td>OmniPur</td>
 <td>9400</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Micro Elute Gel Extraction Kit</td>
 <td>Roche</td>
 <td>D6294-02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DIG RNA Labelling Mix</td>
 <td>Roche</td>
 <td>11277073910</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Transcription T7 RNA Polymerase</td>
 <td>Invitrogen</td>
 <td>18033-019</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Quick Spin Columns</td>
 <td>Roche</td>
 <td>11814427001</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DNase I</td>
 <td>Invitrogen</td>
 <td>18047-019</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>1X DPBS</td>
 <td>Gibco</td>
 <td>14190-250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formamide</td>
 <td>EMD</td>
 <td>FX0420-8</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>tRNA</td>
 <td>Invitrogen</td>
 <td>15401-021</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RNase OUT</td>
 <td>Invitrogen</td>
 <td>10777-019</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Fluorescent Antibody Enhancer Set for DIG Detection #4 (blocking solution)</td>
 <td>Roche</td>
 <td>1768506</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Alexa Fluor secondary antibodies</td>
 <td>Invitrogen</td>
 <td>See Table 2</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Hybridization Oven</td>
 <td>Boekel Scientific</td>
 <td>Model 24100</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Microslides</td>
 <td>VWR</td>
 <td>48300-047</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Immunomount</td>
 <td>Thermoscientific</td>
 <td>9990402</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Table 1. Identification of specific reagents and equipment 
<table border="1">
 <tbody>
 <tr>
 <td>Species of anti-DIG antibody (dilution; company, catalogue number)</td>
 <td>Colours</td>
 <td>Alexa Fluor used (1:500)</td>
 </tr>
 <tr>
 <td rowspan="3">Mouse (1:500;Sigma, B7405)</td>
 <td>green</td>
 <td>Alexa Fluor 488 donkey anti-mouse (Invitrogen, A21202)</td>
 </tr>
 <tr>
 <td>red</td>
 <td>Alexa Fluor 594 donkey anti-mouse (Invitrogen, A21203)</td>
 </tr>
 <tr>
 <td>blue</td>
 <td>Alexa Fluor 647 donkey anti-mouse (Invitrogen, A31571)</td>
 </tr>
 <tr>
 <td rowspan="3">Sheep (1:200;Roche, 11376623)</td>
 <td>green</td>
 <td>Alexa Fluor 488 donkey anti-sheep (Invitrogen, A11015)</td>
 </tr>
 <tr>
 <td>red</td>
 <td>Alexa Fluor 594 donkey anti-sheep (Invitrogen, A11016)</td>
 </tr>
 <tr>
 <td>blue</td>
 <td>Alexa Fluor 647 donkey anti-sheep (Invitrogen, A21448)</td>
 </tr>
</tbody>
</table>
Table 2. Combinations of secondary antibodies and anti-DIG listed with catalogue numbers and concentrations.

detection of viral rna by fluorescence in situ hybridization (fish)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Watch this Scientific Journal Video about Detection of Viral RNA by Fluorescence in situ Hybridization FISH at JoVE.com

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

detection-of-viral-rna-by-fluorescence-in-situ-hybridization-fish

Research

JoVE Journal

Biology

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell level to detect the spatial dynamics of RNA localization with simultaneous insight into the localization of proteins, epigenetic modifications and other details which can be highlighted by immunofluorescence. X-chromosome inactivation is a paradigm for long non-coding RNA (lncRNA)-mediated gene silencing. X-inactive specific transcript (Xist) lncRNA accumulation (called an Xist cloud) on one of the two X-chromosomes in mammalian females is a critical step to initiate X-chromosome inactivation. Xist RNA directly or indirectly interacts with various chromatin-modifying enzymes and introduces distinct epigenetic landscapes to the inactive X-chromosome (Xi). One known epigenetic hallmark of the Xi is the Histone H3 trimethyl-lysine 27 (H3K27me3) modification. Here, we describe a simple and quick immuno-FISH protocol for detecting Xist RNA using RNA FISH with multiple oligonucleotide probes coupled with immunofluorescence of H3K27me3 to examine the localization of Xist RNA and associated epigenetic modifications. Using oligonucleotide probes results in a shorter incubation time and more sensitive detection of Xist RNA compared to in vitro transcribed RNA probes (riboprobes). This protocol provides a powerful tool for understanding the dynamics of lncRNAs and its associated epigenetic modification, chromatin structure, nuclear organization and transcriptional regulation.

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...