This work was supported by the Office of Naval Research via US Naval Research Laboratory core funds.

1. LNA probes &amp; experimental design
<ol>
	<li>Design LNA probes that are the reverse complement of your transcribed sRNA/mRNA or have them custom designed at <a href="http://www.exiqon.com">www.exiqon.com</a>. If using the custom design option, you will know the sequence, but not the LNA spiking pattern. The addition of each LNA residue into a DNA or RNA oligonucleotide results in an increase in Tm of between two and 10 &deg;C for a LNA-RNA hybridization duplex14. Ideally, the designed LNA probes should be between 20-25 nucleotides in length (longer probes are more difficult to synthesize) and have a Tm of between 85-90 &deg;C for RNA hybridization.</li>
	<li>After designing the sequence, use BLAST <a href="http://blast.ncbi.nlm.nih.gov/Blast.cgi">http://blast.ncbi.nlm.nih.gov/Blast.cgi</a> or compare the probe sequence to your local database to ensure the probe is specific only to your sRNA/mRNA of interest.</li>
	<li>When ordering the LNA probe, add a biotin-TEG modification to the 5' end of the LNA oligonucleotide. The biotinylation is necessary for post-hybridization staining with a streptavidin-dye conjugate. It is possible to add this modification to the 3' end if necessary, but the addition to the 5' end is less expensive and should result in higher yields.</li>
	<li>Three negative controls should be included in the method: (i) a 'no LNA' control in which a LNA probe is not added during the hybridization step, (ii) a 'no dye' control in which the LNA probe is hybridized to the target sRNA but the hybridization event is not detected due to the absence of the fluorescent stain, and (iii) a 'non-expressed sRNA/mRNA' control that utilizes a LNA probe that targets a non-existent or non-expressed sRNA in order to monitor non-specific hybridization.</li>
	<li>The specific LNA probe here is complementary to the sRNA CsrB and has the sequence 5'-biotin-TEG-gTccAttTccCgtCctTagCagC-3' (LNA monomers in uppercase letters).</li>
	<li>Following receipt of lyophilized LNA, resuspend with nuclease-free water to 50 &mu;g/mL and store in 10-20 &mu;L aliquots at -20 &deg;C.</li>
</ol>
Solutions
<ul>
	<li>8% paraformaldehye (pfa), 10% acetic acid in PBS: Mix 1 mL 32% pfa, 400 &mu;L acetic acid, 400 &mu;L 10X PBS, pH = 7.4, and 2.2 mL nuclease-free water. For frozen aliquots, freeze in single use aliquots at -20&deg;C without acetic acid.</li>
	<li>60% dextran sulfate solution: add 6.0 g dextran sulfate to a 50 mL conical tube and add water to a final volume of 10 mL. Heat this mixture to 65 &deg;C in a water bath until the dextran sulfate is dissolved (could take 1-2 hr). Additional water may need to be added throughout the solubilization process to maintain a 10 mL volume. Aliquot the 60% dextran sulfate solution and store at -20 &deg;C until use.</li>
</ul>
2. Fixation
<ol>
	<li>Harvest 1x108 cells of bioluminescent Vibrio campbellii or your bacteria of interest and transfer them into a 1.5 mL microcentrifuge tube. This quantity of cells provides enough starting material for four flow-FISH samples (one specific LNA probe sample and three negative controls). Additional 1.5 mL microcentrifuge tube aliquots should be collected if other sRNA/mRNA species need to be tested.</li>
	<li>Pellet the bacterial cells by centrifugation at 2300 x g for five minutes. Discard the supernatant by pipetting and resuspend the cells in 400 &mu;L of 1X PBS.</li>
	<li>To this mixture, add 400 &mu;L of an 8% paraformaldehye (pfa) and 10% acetic acid in 1X phosphate buffered saline (1X PBS) solution, mix well, and incubate for 10 minutes at 25 &deg;C mixing once after five minutes. All incubations, unless otherwise noted, are performed in a heated tube holder. The pfa solution should be prepared fresh or used from frozen aliquots after the addition of acetic acid. Paraformaldehyde is a crosslinking fixative that helps to preserve cell morphology and retain intracellular RNA.</li>
	<li>Pellet the cells from this mixture by centrifugation at 4500 x g for two minutes and remove the supernatant by pipetting. All future steps that require centrifugation should use the same settings (7K, 2 min). It is important to note that following centrifugation the supernatants should be removed by pipetting and not decanting.</li>
	<li>Wash the cells twice with 400 &mu;L 1X PBS and resuspend the final cell pellet in 400 &mu;L 1X PBS. The cells are now fixed and can be stored at 4 &deg;C in 400 &mu;L 1X PBS for up to seven days.</li>
</ol>
3. Diethyl pyrocarbonate treatment
<ol>
	<li>Prepare 400 &mu;L of a 0.1% solution of diethyl pyrocarbonate (DEPC) in 1X PBS (400 &mu;L of this solution is needed for every sample to be tested so scale accordingly). The DEPC solution should be prepared fresh from a DEPC stock. DEPC treatment inactivates RNases by carbethoxylation and decreases the background signal.</li>
	<li>Add 400 &mu;L of the 0.1% DEPC solution to each fixed bacterial cell sample and mix well by pipetting. Incubate this mixture at 25&deg;C for 12 minutes. Wash the cells twice with 400 &mu;L 1X PBS, pellet the cells (7K, 2 min) and remove the supernatant.</li>
</ol>
4. Permeabilization
<ol>
	<li>Add 1.0 mg of lysozyme to 1 mL Tris-EDTA buffer (10 mM Tris, 1 mM EDTA, pH 8.0) for a 1.0 mg/mL solution. This solution should be prepared fresh. Add 800 &mu;L of the 1 mg/mL lysozyme solution to the cells, mix thoroughly by pipetting and incubate at 25 &deg;C for 30 minutes with occasional mixing. Pellet the cells (7K, 2 min) and remove the supernatant. Lysozyme acts as a permeabilization reagent by hydrolyzing the peptidoglycan present in bacterial cell walls.</li>
	<li>Prepare a 3.0 &mu;g/mL solution of proteinase K in TE buffer. This solution should be prepared fresh from a 20 mg/mL proteinase K stock that is stored at -20 &deg;C. Proteinase K is a serine protease that digests a variety of proteins and helps the accessibility of the probes and reagents to the RNA.</li>
	<li>Add 800 &mu;L of the 3.0 &mu;g/mL proteinase K solution to the cell pellet and mix thoroughly by pipetting. Incubate at 25 &deg;C for 15 minutes with vortexing halfway through the incubation.</li>
	<li>Pellet the cells (7K, 2 min), remove the supernatant and wash the cells once with 400 &mu;L 1X PBS. After removing the wash supernatant, resuspend the cells in 400 &mu;L 1X PBS and add 100 &mu;L of the resuspension into four new 1.5 mL microcentrifuge tubes. Pellet the cells (7K, 2 min) and remove the supernatant.</li>
</ol>
5. Hybridization
<ol>
	<li>Prepare 100 &mu;L of hybridization buffer (HB) for each sample. The HB is made up of 50% formamide by volume, 10% dextran sulfate by mass (add 16.7 &mu;L of the 60% dextran sulfate solution for every 100 &mu;L), 1X Denhardt's solution, 50 mM sodium phosphate pH 7.0, 2X sodium citrate (SSC), 20 &mu;g of sheared salmon sperm DNA (SSSD) and 20 &mu;g yeast tRNA.</li>
	<li>Each of the components of the HB has a different purpose. Formamide lowers the melting temperature of nucleic acid duplexes thereby helping with denaturation of the RNA and minimizing cell damage. Dextran sulfate is used as a volume-excluding polymer to concentrate the probe and increase the hybridization rate. Denhardts solution, yeast tRNA and SSSD serve as a blocking agents to reduce non-specific binding.</li>
	<li>In each of the cell resuspension-containing 1.5 mL microcentrifuge tubes, add 95 &mu;L HB and 5.0 &mu;L of the sRNA/mRNA-specific LNA [20 pmol of specific LNA final concentration] or water (for the no LNA negative control). For example:
	<ol class="lalpha">
		<li>Tube 1 - 95 &mu;L HB + 5.0 &mu;L sRNA/mRNA-specific probe</li>
		<li>Tube 2 - 95 &mu;L HB + 5.0 &mu;L sRNA/mRNA-specific probe ('no dye' negative control)</li>
		<li>Tube 3 - 95 &mu;L HB + 5.0 &mu;L sRNA/mRNA probe ('non-expressed sRNA/mRNA' negative control)</li>
		<li>Tube 4 - 95 &mu;L HB + 5.0 &mu;L water ('no LNA' negative control)</li>
	</ol>
	</li>
</ol>
<ol start="4">
	<li>Incubate all four samples at 60 &deg;C for 60 minutes. The hybridization temperature depends on the LNA probe(s) Tm and should be set at approximately 30 &deg;C below the predicted Tm for RNA annealing. Both the elevated hybridization temperature and formamide in the HB ensures the denaturation of secondary structure of the sRNA or mRNA of interest.</li>
</ol>
6. Post-hybridization washes
<ol>
	<li>Following hybridization, add 1.0 mL 0.1X SSC with 0.1% tween-20 (SSCT) to the hybridization mixture. This is necessary to allow the cells to be removed from the viscous hybridization solution. Pellet the cells (7K, 2 min) and remove the supernatant.</li>
	<li>Add 200 &mu;L of 50% formamide, 2X SSC, 0.1% tween-20 to the cell pellets, mix thoroughly and incubate for 30 minutes at 65 &deg;C in a heating block.</li>
	<li>Add 1 mL 0.1X SSCT to the mixture, pellet the cells (7K, 2 min) and remove the supernatant.</li>
	<li>Add 500 &mu;L 0.1X SSCT to resuspend the cells and incubate for 40 minutes at 65 &deg;C in a heat block. Pellet the cells (7K, 2 min) and remove the supernatant.</li>
</ol>
7. Blocking and staining
<ol>
	<li>Prepare the blocking buffer (BB) and the streptavidin-dye staining solution. Prepare a 1X BB solution from a 5X stock (commercially available, see Reagents). For every set of four tubes, prepare 1.2 mL 1X BB.</li>
	<li>Add 200 &mu;L 1X BB to the cell pellets, resuspend and incubate for 30 minutes at 25&deg;C.</li>
	<li>A streptavidin-conjugated dye is used to detect the biotinylated LNA probes. While blocking, prepare a solution of 2 &mu;g/mL DyLight 488 streptavidin or your desired dye-streptavidin conjugate in 1X BB for 30 min (for three samples, make 300 &mu;L).</li>
	<li>After incubation with 1X BB, pellet the cells (7K, 2 min) and remove the supernatant. Add 50 &mu;L of the streptavidin-dye solution to the cell pellets, mix thoroughly, and incubate for 12 minutes at 25 &deg;C with constant mixing on a vortex or in a thermomixer. For the 'no dye' control, add 50 &mu;L 1X blocking buffer only. For the rest of the protocol, protect the tubes from light using aluminum foil.</li>
	<li>Wash the cells once with 500 &mu;L 0.1X SSCT buffer and once with 400 &mu;L 1X PBS with 0.1% tween-20 (PBST). Pellet the cells (7K, 2 min) and remove the supernatant.</li>
	<li>Following the initial streptavidin-conjugate staining, the signal is amplified by introducing a biotinylated anti-streptavidin antibody to the streptavidin-dye conjugate and then staining a second time with the streptavidin-dye thus increasing the signal output per hybridized LNA probe (Figure 2).</li>
	<li>Add 100 &mu;L of 1 &mu;g/mL biotinylated anti-streptavidin antibody in 1X PBS, resuspend the cells, and incubate at 25 &deg;C for 30 minutes with occasional mixing. Pellet the cells (7K, 2 min), remove the supernatant and wash twice with 400 &mu;L 1X PBST.</li>
	<li>Add 50 &mu;L of the 2 &mu;g/mL streptavidin-dye solution to the cells, mix thoroughly, and incubate for 12 minutes at 25&deg;C with constant mixing on a vortex or in a thermomixer. For the 'no dye' control, add 50 &mu;L 1X blocking buffer only.</li>
	<li>Wash the cells once with 500 &mu;L 0.1X SSCT buffer and once with 400 &mu;L 1X PBST.</li>
	<li>Resuspend the cells in 200 &mu;L 1X PBST and store at 4 &deg;C until ready for flow cytometry analysis. For smaller bacterial cells set the flow rate to slow to decrease the core size. In addition, for flow cytometers with forward scatter threshold cutoffs, the cutoff must be set as low as possible to ensure the small bacterial cells are detected.</li>
	<li>For DyLight 488 detection, the flow cytometer should be equipped with a 488 nm laser and standard emission filters for FITC. At least 2x104 events should be collected. For compatibility with flow cytometers equipped with different lasers, other streptavidin-conjugated fluorophores can be used for this protocol.</li>
</ol>
8. Representative results
An example of cells that are fixed and permeabilized effectively is shown in the flow cytometry dot plot in Figure 3A. When using the conditions described above for permeabilization, the cell population is small and homogenous indicating few cell aggregates. When higher (5 mg/mL) concentrations of lysozyme are used, cells are more likely to aggregate and show increased forward scatter values, indicative of larger particles (Figure 3B). An example of flow cytometry data from a successful LNA flow-FISH experiment is shown in Figure 4. The histogram demonstrates the specific detection of a target sRNA along with three negative controls. The 'no dye' negative control produces the least fluorescence, followed by the 'no LNA' and 'non-expressed sRNA' negative controls. Finally, the sample with the sRNA-specific LNA probe produces the greatest fluorescence. Once the method and LNA probes are validated in this manner, additional experiments can be designed to monitor changes in the sRNA signal over time, in response to mutations or varied culture conditions, etc.
<img alt="Figure 1." src="/files/ftp_upload/3655/3655fig1.jpg" /> 
Figure 1. Depiction of the overall scheme of a LNA flow-FISH experiment for the detection of bacterial sRNA.
<img alt="Figure 2." src="/files/ftp_upload/3655/3655fig2.jpg" /> 
Figure 2. Staining and amplification of the LNA flow-FISH signal. a) Hybridization of the biotinylated LNA probe. b) Staining with the fluorescent DyLight 488 streptavidin conjugate. c) Binding of the biotinylated anti-streptavidin antibody to the DyLight 488 streptavidin. The antibody can bind streptavidin through its antigen-binding site or can be bound by streptavidin through its biotin residues. d) Amplification of the signal by further staining with the fluorescent DyLight 488 streptavidin conjugate.
<img alt="Figure 3." src="/files/ftp_upload/3655/3655fig3.jpg" /> 
Figure 3. Flow cytometry dot plots showing forward versus side scatter results for a) 1 mg/mL lysozyme, 3 &mu;g/mL proteinase K permeabilization and b) 5 mg/mL lysozyme, 3 &mu;g/ml proteinase K permeabilization.
<img alt="Figure 4." src="/files/ftp_upload/3655/3655fig4.jpg" /> 
Figure 4. Histogram analysis of the LNA flow-FISH results. The fluorescent signal generated from each sample is shown by the four traces: black - sRNA-specific LNA probe; red - 'non-expressed sRNA' negative control; blue - 'no LNA' negative control; purple - 'no dye' negative control.

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The authors have nothing to disclose.

The LNA flow-FISH method presented here was used to detect the expression of a sRNA from the Gram-negative marine bacterium Vibrio campbellii whose expression has previously been confirmed via microarray-based expression profiling and reverse transcription polymerase chain reaction16. To date, we have used this method to monitor the expression of a variety of RNAs (e.g. trans-encoded sRNA, sRNA that modulate protein activity, riboswitches, and mRNA). As such, we are confident that the method is adaptable and can be used to detect any sRNA or mRNA target and can be modified for use in any bacterial species or cell type. If modification of this protocol is necessary for other cell types, the most important variable to consider manipulating is the permeabilization step. For example, when modifying the permeabilization conditions described here, changes in the concentrations of lysozyme and proteinase K should be tested. We found that doubling or tripling the amount of lysozyme and proteinase K resulted in major changes in the LNA flow-FISH results. Furthermore, when testing additional permeabilization conditions, the cells should be analyzed for clumping, cell loss, and the best obtainable signal to background fluorescence ratio. The extent of cell clumping can be tested for by microscopy and/or flow cytometry. By flow cytometry, cell clumps are present as tails in a forward vs. side scatter dot plot and the correct permeabilization method will minimize this tailing effect. This method is also amenable to multiplex sRNA detection without major modifications to the described protocol as additional LNA probes labeled with other haptens such as digoxigenin could be added during the hybridization step and then detected with an anti-digoxigenin antibody and secondary antibody-fluorophore conjugate. Currently, the only limitation that we have encountered with this method is its inability to generate sufficient signal from weakly expressed sRNA species and efforts are underway to determine the detection threshold (in absolute copy number per cell).
Overall, the LNA flow-FISH method provides the opportunity to measure bacterial sRNA or mRNA expression at the single cell-level in a high throughput manner to provide insights on the presence and relative abundance of an sRNA species and the degree to which its expression varies in a population.

<table border="1">
	<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments </td>
 </tr>
 <tr>
 <td>10X Phosphate buffered saline (PBS), pH 7.4</td>
 <td>Applied Biosystems/Ambion</td>
 <td>AM9625</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nuclease-free water </td>
 <td>Applied Biosystems/Ambion</td>
 <td>AM9932</td>
 <td>(not DEPC treated)</td>
 </tr>
 <tr>
 <td>32% paraformaldehyde</td>
 <td>Electron Microscopy Sciences</td>
 <td>RT 15714</td>
 <td>Ethanol free</td>
 </tr>
 <tr>
 <td>Acetic acid</td>
 <td>Acros Organics</td>
 <td>327290010</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Diethyl pyrocarbonate (DEPC)</td>
 <td>Sigma Aldrich</td>
 <td>D5758</td>
 <td>Keep desiccated </td>
 </tr>
 <tr>
 <td>Lysozyme</td>
 <td>Sigma Aldrich</td>
 <td>L2879</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Proteinase K (20 mg/mL)</td>
 <td>Applied Biosystems/Ambion</td>
 <td>AM2546</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Formamide</td>
 <td>Applied Biosystems/Ambion</td>
 <td>AM9342</td>
 <td>Deionized, aliquot and freeze</td>
 </tr>
 <tr>
 <td>Dextran sulfate MW &gt; 500,000</td>
 <td>Sigma Aldrich</td>
 <td>D8906</td>
 <td>Aliquot 60% solution and freeze</td>
 </tr>
 <tr>
 <td>Denhardt's solution 50X concentrate</td>
 <td>Sigma Aldrich</td>
 <td>D2532</td>
 <td>Aliquot and freeze</td>
 </tr>
 <tr>
 <td>Sodium citrate buffer 20X</td>
 <td>Applied Biosystems/Ambion</td>
 <td>AM9770</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Salmon sperm DNA (sheared) 10 mg/mL</td>
 <td>Applied Biosystems/Ambion</td>
 <td>AM9680</td>
 <td>Aliquot and freeze</td>
 </tr>
 <tr>
 <td>Yeast tRNA (10 mg/mL)</td>
 <td>Life Technologies/Invitrogen</td>
 <td>15401-011</td>
 <td>Aliquot and freeze</td>
 </tr>
 <tr>
 <td>Tween-20 </td>
 <td>Sigma Aldrich</td>
 <td>P9416</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>5X in situ hybridization blocking solution</td>
 <td>Vector Laboratories</td>
 <td>MB-1220</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DyLight 488 streptavidin </td>
 <td>Vector Laboratories</td>
 <td>SA-5488-1</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Biotinylated anti-streptavidin</td>
 <td>Vector Laboratories</td>
 <td>BA-0500</td>
 <td>Aliquot and freeze</td>
 </tr>
	 </tbody>
 </table>

locked nucleic acid flow cytometry-fluorescence in situ hybridization (lna flow-fish): a method for bacterial small rna detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular environment. In order to increase throughput, FISH can be combined with flow cytometry (flow-FISH) to enable the detection of targeted nucleic acid sequences in thousands of individual cells. As a result, flow-FISH offers a distinct advantage over lysate/ensemble-based nucleic acid detection methods because each cell is treated as an independent observation, thereby permitting stronger statistical and variance analyses. These attributes have prompted the use of FISH and flow-FISH methods in a number of different applications and the utility of these methods has been successfully demonstrated in telomere length determination1,2, cellular identification and gene expression3,4, monitoring viral multiplication in infected cells5, and bacterial community analysis and enumeration6.
Traditionally, the specificity of FISH and flow-FISH methods has been imparted by DNA oligonucleotide probes. Recently however, the replacement of DNA oligonucleotide probes with nucleic acid analogs as FISH and flow-FISH probes has increased both the sensitivity and specificity of each technique due to the higher melting temperatures (Tm) of these analogs for natural nucleic acids7,8. Locked nucleic acid (LNA) probes are a type of nucleic acid analog that contain LNA nucleotides spiked throughout a DNA or RNA sequence9,10. When coupled with flow-FISH, LNA probes have previously been shown to outperform conventional DNA probes7,11 and have been successfully used to detect eukaryotic mRNA12 and viral RNA in mammalian cells5.
Here we expand this capability and describe a LNA flow-FISH method which permits the specific detection of RNA in bacterial cells (Figure 1). Specifically, we are interested in the detection of small non-coding regulatory RNA (sRNA) which have garnered considerable interest in the past few years as they have been found to serve as key regulatory elements in many critical cellular processes13. However, there are limited tools to study sRNAs and the challenges of detecting sRNA in bacterial cells is due in part to the relatively small size (typically 50-300 nucleotides in length) and low abundance of sRNA molecules as well as the general difficulty in working with smaller biological cells with varying cellular membranes. In this method, we describe fixation and permeabilzation conditions that preserve the structure of bacterial cells and permit the penetration of LNA probes as well as signal amplification steps which enable the specific detection of low abundance sRNA (Figure 2).

Watch this Scientific Journal Video about Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection at JoVE.com

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

A novel high-throughput method is described that enables the detection and relative quantitation of small RNA and mRNA expression from single bacterial cells using locked nucleic acid probes and flow cytometry-fluorescence in situ hybridization.

Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization (LNA flow-FISH): a Method for Bacterial Small RNA Detection

Fluorescence in situ hybridization (FISH) is a powerful technique that is used to detect and localize specific nucleic acid sequences in the cellular ...

locked-nucleic-acid-flow-cytometry-fluorescence-situ-hybridization

Research

JoVE Journal

Immunology and Infection

25.1K ビュー.  Naval Research Laboratory. 小説ハイスループット法は、ロックされた核酸プローブとフローサイトメトリー、蛍光を用いて、単一の細菌細胞からの検出と小さなRNAとmRNA発現の相対定量を可能にすることが記載されているその場でハイブリダイゼーション。

ビデオ: Locked Nucleic Acid Flow Cytometry-fluorescence in situ Hybridization LNA flow-FISH: a Method for Bacterial Small RNA Detection

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in situ hybridization (FISH) for rapid culture-independent detection of Salmonella spp. Cell-charged tapes can also be placed face-down on selective agar for solid-phase enrichment prior to detection. Alternatively, low-volume liquid enrichments (liquid surface miniculture) can be performed on the surface of the tape in non-selective broth, followed by FISH and analysis via flow cytometry. To begin, sterile adhesive tape is brought into contact with fresh produce, gentle pressure is applied, and the tape is removed, physically extracting microbes present on these surfaces. Tapes are mounted sticky-side up onto glass microscope slides and the sampled cells are fixed with 10% formalin (30 min) and dehydrated using a graded ethanol series (50, 80, and 95%; 3 min each concentration). Next, cell-charged tapes are spotted with buffer containing a Salmonella-targeted DNA probe cocktail and hybridized for 15 - 30 min at 55&deg;C, followed by a brief rinse in a washing buffer to remove unbound probe. Adherent, FISH-labeled cells are then counterstained with the DNA dye 4',6-diamidino-2-phenylindole (DAPI) and results are viewed using fluorescence microscopy. For solid-phase enrichment, cell-charged tapes are placed face-down on a suitable selective agar surface and incubated to allow in situ growth of Salmonella microcolonies, followed by FISH and microscopy as described above. For liquid surface miniculture, cell-charged tapes are placed sticky side up and a silicone perfusion chamber is applied so that the tape and microscope slide form the bottom of a water-tight chamber into which a small volume (&le; 500 &mu;L) of Trypticase Soy Broth (TSB) is introduced. The inlet ports are sealed and the chambers are incubated at 35 - 37&deg;C, allowing growth-based amplification of tape-extracted microbes. Following incubation, inlet ports are unsealed, cells are detached and mixed with vigorous back and forth pipetting, harvested via centrifugation and fixed in 10% neutral buffered formalin. Finally, samples are hybridized and examined via flow cytometry to reveal the presence of Salmonella spp. As described here, our "tape-FISH" approach can provide simple and rapid sampling and detection of Salmonella on tomato surfaces. We have also used this approach for sampling other types of fresh produce, including spinach and jalape&ntilde;o peppers.

Combination of Adhesive-tape-based Sampling and Fluorescence in situ Hybridization for Rapid Detection of Salmonella on Fresh Produce

This protocol describes a simple adhesive-tape-based approach for sampling of tomato and other fresh produce surfaces, followed by rapid whole cell detection of Salmonella using fluorescence in situ hybridization (FISH).

This protocol describes a simple approach for adhesive-tape-based sampling of tomato and other fresh produce surfaces, followed by on-tape fluorescence in ...

Transfection and Mutagenesis of Target Genes in Mosquito Cells by Locked Nucleic Acid-modified Oligonucleotides

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new infections each year. Despite decades of research, there is still no vaccine against malaria, highlighting the need for novel control strategies. One innovative approach is the use of genetically modified mosquitoes to effectively control malaria parasite transmission. Deliberate alterations of cell signaling pathways in the mosquito, via targeted mutagenesis, have been found to regulate parasite development 1. From these studies, we can begin to identify potential gene targets for transformation. Targeted mutagenesis has traditionally relied upon the homologous recombination between a target gene and a large DNA molecule. However, the construction and use of such complex DNA molecules for generation of stably transformed cell lines is costly, time consuming and often inefficient. Therefore, a strategy using locked nucleic acid-modified oligonucleotides (LNA-ONs) provides a useful alternative for introducing artificial single nucleotide substitutions into episomal and chromosomal DNA gene targets (reviewed in 2). LNA-ON-mediated targeted mutagenesis has been used to introduce point mutations into genes of interest in cultured cells of both yeast and mice 3,4. We show here that LNA-ONs can be used to introduce a single nucleotide change in a transfected episomal target that results in a switch from blue fluorescent protein (BFP) expression to green fluorescent protein (GFP) expression in both Anopheles gambiae and Anopheles stephensi cells. This conversion demonstrates for the first time that effective mutagenesis of target genes in mosquito cells can be mediated by LNA-ONs and suggests that this technique may be applicable to mutagenesis of chromosomal targets in vitro and in vivo.

Oligonucleotides can be used to site specifically substitute a single nucleotide of transfected target genes in both Anopheles gambiae and Anopheles stephensi cells.

Plasmodium parasites, the causative agent of malaria, are transmitted through the bites of infected Anopheles mosquitoes resulting in over 250 million new ...

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. Although the natural occurrence of both diseases&#39; is now relatively rare, the possibility of terrorist groups using these pathogens as a bioweapon is real. Because of the disease&#39;s inherent communicability, rapid clinical course, and high mortality rate, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management.
Recombinant reporter phage may provide a rapid and specific approach for the detection of Y. pestis and B. anthracis. The Centers for Disease Control and Prevention currently use the classical phage lysis assays for the confirmed identification of these bacterial pathogens 2-4. These assays take advantage of naturally occurring phage which are specific and lytic for their bacterial hosts. After overnight growth of the cultivated bacterium in the presence of the specific phage, the formation of plaques (bacterial lysis) provides a positive identification of the bacterial target. Although these assays are robust, they suffer from three shortcomings: 1) they are laboratory based; 2) they require bacterial isolation and cultivation from the suspected sample, and 3) they take 24-36 h to complete. To address these issues, recombinant &#34;light-tagged&#34; reporter phage were genetically engineered by integrating the Vibrio harveyi luxAB genes into the genome of Y. pestis and B. anthracis specific phage 5-8. The resulting luxAB reporter phage were able to detect their specific target by rapidly (within minutes) and sensitively conferring a bioluminescent phenotype to recipient cells. Importantly, detection was obtained either with cultivated recipient cells or with mock-infected clinical specimens 7.
For demonstration purposes, here we describe the method for the phage-mediated detection of a known Y. pestis isolate using a luxAB reporter phage constructed from the CDC plague diagnostic phage &#934;A1122 6,7 (Figure 1). A similar method, with minor modifications (e.g. change in growth temperature and media), may be used for the detection of B. anthracis isolates using the B. anthracis reporter phage W&#946;::luxAB 8. The method describes the phage-mediated transduction of a biolumescent phenotype to cultivated Y. pestis cells which are subsequently measured using a microplate luminometer. The major advantages of this method over the traditional phage lysis assays is the ease of use, the rapid results, and the ability to test multiple samples simultaneously in a 96-well microtiter plate format.
<img alt="Figure 1" src="/files/ftp_upload/2740/2740fig1.jpg" /> 
Figure 1. Detection schematic. The phage are mixed with the sample, the phage infects the cell, luxAB are expressed, and the cell bioluminesces. Sample processing is not necessary; the phage and cells are mixed and subsequently measured for light.

&#039;Bioluminescent&#039; Reporter Phage for the Detection of Category A Bacterial Pathogens

A simple method for the identification of priority bacterial pathogens is to use genetically engineered reporter phage. These reporter phage, which are specific to their particular host species, are capable of rapidly transducing a bioluminescent signal response to host cells. Herein, we describe the use of reporter phage for the detection of Yersinia pestis.

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes. Investigation of transcripts and gene regulators that are expressed in tissues in which the mosquito host and pathogen interact, and in organs involved in reproduction are of great interest for strategies to reduce mosquito-borne disease transmission and disrupt egg development. A number of tools have been employed to study and validate the temporal and tissue-specific regulation of gene expression. Here, we describe protocols that have been developed to obtain spatial information, which enhances our understanding of where specific genes are expressed and their products accumulate. The protocol described has been used to validate expression and determine accumulation patterns of transcripts in tissues related to mosquito-borne pathogen transmission, such as female salivary glands, as well as subcellular compartments of ovaries and embryos, which relate to mosquito reproduction and development. 
 The following procedures represent an optimized methodology that improves the efficiency of various steps in the protocol without loss of target-specific hybridization signals. Guidelines for RNA probe preparation, dissection of soft tissues and the general procedure for fixation and hybridization are described in Part A, while steps specific for the collection, fixation, pre-hybridization and hybridization of mosquito embryos are detailed in Part B.

Hybridization in situ of Salivary Glands, Ovaries, and Embryos of Vector Mosquitoes

Temporal and spatial gene expression analyses have a crucial role in functional genomics. Whole-mount hybridization in situ is useful for determining the localization of transcripts within tissues and subcellular compartments. Here we outline a hybridization in situ protocol with modifications for specific target tissues in mosquitoes.

Mosquitoes are vectors for a diverse set of pathogens including arboviruses, protozoan parasites and nematodes.  Investigation of transcripts and gene ...

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. One important application of this method is the development of high-quality physical maps useful for improving the genome assemblies for various organisms. The natural banding pattern of polytene and mitotic chromosomes provides guidance for the precise ordering and orientation of the genomic supercontigs. Among the three mosquito genera, namely Anopheles, Aedes, and Culex, a well-established chromosome-based mapping technique has been developed only for Anopheles, whose members possess readable polytene chromosomes 1. As a result of genome mapping efforts, 88% of the An. gambiae genome has been placed to precise chromosome positions 2,3 . Two other mosquito genera, Aedes and Culex, have poorly polytenized chromosomes because of significant overrepresentation of transposable elements in their genomes 4, 5, 6. Only 31 and 9% of the genomic supercontings have been assigned without order or orientation to chromosomes of Ae. aegypti 7 and Cx. quinquefasciatus 8, respectively. Mitotic chromosome preparation for these two species had previously been limited to brain ganglia and cell lines. However, chromosome slides prepared from the brain ganglia of mosquitoes usually contain low numbers of metaphase plates 9. Also, although a FISH technique has been developed for mitotic chromosomes from a cell line of Ae. aegypti 10, the accumulation of multiple chromosomal rearrangements in cell line chromosomes 11 makes them useless for genome mapping. Here we describe a simple, robust technique for obtaining high-quality mitotic chromosome preparations from imaginal discs (IDs) of 4th instar larvae which can be used for all three genera of mosquitoes. A standard FISH protocol 12 is optimized for using BAC clones of genomic DNA as a probe on mitotic chromosomes of Ae. aegypti and Cx. quinquefasciatus, and for utilizing an intergenic spacer (IGS) region of ribosomal DNA (rDNA) as a probe on An. gambiae chromosomes. In addition to physical mapping, the developed technique can be applied to population cytogenetics and chromosome taxonomy/systematics of mosquitoes and other insect groups.

Fluorescent in situ Hybridization on Mitotic Chromosomes of Mosquitoes

Among the three mosquito genera, namely Anopheles, Aedes, and Culex, physical genome mapping techniques were established only for Anopheles, whose members possess readable polytene chromosomes. For the genera of Aedes and Culex, however, cytogenetic mapping remains challenging because of the poor quality of polytene chromosomes. Here we present a universal protocol for obtaining high-quality preparations of mitotic chromosomes and an optimized FISH protocol for all three genera of mosquitoes.

Fluorescent in situ hybridization (FISH) is a technique routinely used by many laboratories to determine the chromosomal position of DNA and RNA probes. ...

Bacterial Artificial Chromosomes: A Functional Genomics Tool for the Study of Positive-strand RNA Viruses

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose genomes have the same polarity as cellular mRNA. The cDNA-based reverse genetics system is a seminal method that enables direct manipulation of the viral genomic RNA, thereby generating recombinant viruses for molecular and genetic studies of both viral RNA elements and gene products in viral replication and pathogenesis. It also provides a valuable platform that allows the development of genetically defined vaccines and viral vectors for the delivery of foreign genes. For many positive-strand RNA viruses such as Japanese encephalitis virus (JEV), however, the cloned cDNAs are unstable, posing a major obstacle to the construction and propagation of the functional cDNA. Here, the present report describes the strategic considerations in creating and amplifying a genetically stable full-length infectious JEV cDNA as a bacterial artificial chromosome (BAC) using the following general experimental procedures: viral RNA isolation, cDNA synthesis, cDNA subcloning and modification, assembly of a full-length cDNA, cDNA linearization, in vitro RNA synthesis, and virus recovery. This protocol provides a general methodology applicable to cloning full-length cDNA for a range of positive-strand RNA viruses, particularly those with a genome of &gt;10 kb in length, into a BAC vector, from which infectious RNAs can be transcribed in vitro with a bacteriophage RNA polymerase.

Here, a protocol is presented for creating an infectious bacterial artificial chromosome containing a full-length cDNA of the positive-strand genomic RNA of Japanese encephalitis virus. This protocol can be used to construct a functional cDNA of other positive-strand RNA viruses, making it a powerful genomic tool for studying virus biology.

Reverse genetics, an approach to rescue infectious virus entirely from a cloned cDNA, has revolutionized the field of positive-strand RNA viruses, whose ...

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. 
After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Here, we present a protocol for the rapid virus nucleic acid extraction from the virus-inactivated whole blood. The extraction is performed directly in the blood collection tubes and requires no equipment or electricity. The method is not dependent on laboratory facilities and can be used anywhere (e.g., in field hospitals).

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for ...

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

Modeling Hepatitis B Virus Infection in Non-Hepatic 293T-NE-3NRs Cells

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV models are limited to hepatoma cell lines (such as HepG2 and Huh7) overexpressing a functional HBV receptor, sodium taurocholate co-transporting polypeptide (NTCP). Here, we used 293T-NE-3NRs (293T&#160;overexpressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and HepG2-NE (HepG2 overexpressing NTCP) as model cell lines.&#160;HBV infection in these cell lines was performed either by using concentrated HBV virus particles from HepG2.2.15 or co-culturing HepG2.2.15 with the target cell lines. HBcAg immunofluorescence for HBcAg was performed to confirm HBV infection. The two methods presented here will help us study HBV infection in non-hepatic cell lines.&#160;&#160;

This manuscript describes a detailed protocol for Hepatitis B virus (HBV) infection in novel engineered 293T cells (293T-NE-3NRs, expressing human NTCP, HNF4&#945;, RXR&#945; and PPAR&#945;) and traditional hepatic cells (HepG2-NE, expressing human NTCP).

HBV mainly infects human hepatocytes, but it has also been found to infect extrahepatic tissues such as kidney and testis. Nonetheless, cell-based HBV ...

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia coli and Salmonella Typhimurium, could repress the expression of outer membrane proteins. To further investigate the regulation function of micC in Salmonella Enteritidis, we cloned the micC gene in the Salmonella Enteritidis strain 50336, and then constructed the mutant 50336&#916;micC by the &#955; Red-based recombination system and the complemented mutant 50336&#916;micC/pmicC carrying recombinant plasmid pBR322 expressing micC. qRT-PCR results demonstrated that transcription of ompD in 50336&#916;micC was 1.3-fold higher than that in the wild type strain, while the transcription of ompA and ompC in 50336&#916;micC were 2.2-fold and 3-fold higher than those in the wild type strain. These indicated that micC represses the expression of ompA and ompC. In the following study, the pathogenicity of 50336&#916;micC was detected by both infecting 6-week-old Balb/c mice and 1-day-old chickens. The result showed that the LD50 of the wild type strain 50336, the mutants 50336&#916;micC and 50336&#916;micC/pmicC for 6-week-old Balb/c mice were 12.59 CFU, 5.01 CFU, and 19.95 CFU, respectively. The LD50 of the strains for 1-day-old chickens were 1.13 x 109 CFU, 1.55 x 108 CFU, and 2.54 x 108 CFU, respectively. It indicated that deletion of micC enhanced virulence of S. Enteritidis in mice and chickens by regulating expression of outer membrane proteins.

A Non-Coding Small RNA MicC Contributes to Virulence in Outer Membrane Proteins in Salmonella Enteritidis

An &#955;-Red-mediated recombination system was used to create a deletion mutant of a small non-coding RNA micC.

A non-coding small RNA (sRNA) is a new factor to regulate gene expression at the post-transcriptional level. A kind of sRNA MicC, known in Escherichia ...