Name: visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization
Uploaded: 2020-12-23T14:00:00
Description: Watch this Scientific Journal Video about Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization at JoVE.com

This work was supported by the Ministry of Science and Higher Education for research on SARS-CoV-2, and by grants from the National Science Center (grants UMO2017/27/B/NZ6/02488 to K.P. and UMO-2018/30/E/NZ1/00874 to A.K.-P.).

1. Buffer preparation
<ol>
	<li>For 500 mL of 2x PHEM buffer, combine 18.14 g of piperazine-N,N&#8242;-bis(2-ethanesulfonic acid) (PIPES), 6.5 g of 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES), 3.8 g of ethylene glycol tetraacetic acid (EGTA), and 0.99 g of magnesium sulfate (MgSO4). Make the volume up to ~400 mL with distilled water (dH2O), stir, and adjust the pH to 7.0 using 10 M potassium hydroxide (KOH) or sodium hydroxide (NaOH). Make the final volume up to 500 mL, an...

<ol>
	<li>Choi, H. M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Third-generation+in+situ+hybridization+chain+reaction:+multiplexed,+quantitative,+sensitive,+versatile,+robust.">Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust.</a> Development. 145, (2018).</li><li>Milewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Replication+of+severe+acute+respiratory+syndrome+coronavirus+2+in+human+respiratory+epithelium.">Replication of severe acute respiratory syndrome coronavirus 2 in human respiratory epithelium.</a> Journal of Virology. 94, (2020).</li><li>Banach, B. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+airway+epithelial+cell+culture+to+identify+new+respiratory+viruses:+coronavirus+NL63+as+a+model.">Human airway epithelial cell culture to identify new respiratory viruses: coronavirus NL63 as a model.</a> Journal of Virological Methods. 156 (1-2), 19-26 (2009).</li><li>Milewska, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entry+of+human+coronavirus+NL63+into+the+cell.">Entry of human coronavirus NL63 into the cell.</a> Journal of Virology. 92, (2018).</li><li>El Baba, R., Herbein, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Management+of+epigenomic+networks+entailed+in+coronavirus+infections+and+COVID-19.">Management of epigenomic networks entailed in coronavirus infections and COVID-19.</a> Clinical Epigenetics. 12, 118 (2020).</li><li>Pinto, B. G. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ACE2+expression+is+increased+in+the+lungs+of+patients+with+comorbidities+associated+with+severe+COVID-19.">ACE2 expression is increased in the lungs of patients with comorbidities associated with severe COVID-19.</a> Journal of Infectious Diseases. 222 (4), 556-563 (2020).</li><li>Verdecchia, P., Cavallini, C., Spanevello, A., Angeli, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pivotal+link+between+ACE2+deficiency+and+SARS-CoV-2+infection.">The pivotal link between ACE2 deficiency and SARS-CoV-2 infection.</a> European Journal of Internal Medicine. 76, 14-20 (2020).</li><li>Gordon, D. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+SARS-CoV-2+protein+interaction+map+reveals+targets+for+drug+repurposing.">A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.</a> Nature. 583, 459-468 (2020).</li><li>Ferron, F., Decroly, E., Selisko, B., Canard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+viral+RNA+capping+machinery+as+a+target+for+antiviral+drugs.">The viral RNA capping machinery as a target for antiviral drugs.</a> Antiviral Research. 96 (1), 21-31 (2012).</li><li>Mehta, S., Jeffrey, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+receptors+and+signaling:+epigenetic+factors+in+the+regulation+of+innate+immunity.">Beyond receptors and signaling: epigenetic factors in the regulation of innate immunity.</a> Immunology and Cell Biology. 93 (3), 233-244 (2015).</li><li>Poppe, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+NF-kappaB-dependent+and+-independent+transcriptome+and+chromatin+landscapes+of+human+coronavirus+229E-infected+cells.">The NF-kappaB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells.</a> PLoS Pathogens. 13, 1006286 (2017).</li><li>Schafer, A., Baric, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+landscape+during+coronavirus+infection.">Epigenetic landscape during coronavirus infection.</a> Pathogens. 6 (1), 8 (2017).</li><li>Carletti, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Viral+priming+of+cell+intrinsic+innate+antiviral+signaling+by+the+unfolded+protein+response.">Viral priming of cell intrinsic innate antiviral signaling by the unfolded protein response.</a> Nature Communications. 10, 3889 (2019).</li><li>Heil, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-specific+recognition+of+single-stranded+RNA+via+toll-like+receptor+7+and+8.">Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8.</a> Science. 303 (5663), 1526-1529 (2004).</li><li>Sze, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Host+restriction+factor+SAMHD1+limits+human+T+cell+leukemia+virus+type+1+infection+of+monocytes+via+STING-mediated+apoptosis.">Host restriction factor SAMHD1 limits human T cell leukemia virus type 1 infection of monocytes via STING-mediated apoptosis.</a> Cell Host and Microbe. 14 (4), 422-434 (2013).</li><li>Kikkert, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+evasion+by+human+respiratory+RNA+viruses.">Innate immune evasion by human respiratory RNA viruses.</a> Journal of Innate Immunity. 12, 4-20 (2020).</li><li>Kennedy, E. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Posttranscriptional+m(6)A+editing+of+HIV-1+mRNAs+enhances+viral+gene+expression.">Posttranscriptional m(6)A editing of HIV-1 mRNAs enhances viral gene expression.</a> Cell Host and Microbe. 22 (6), 830 (2017).</li><li>Fulcher, M. L., Gabriel, S., Burns, K. A., Yankaskas, J. R., Randell, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Well-differentiated+human+airway+epithelial+cell+cultures.">Well-differentiated human airway epithelial cell cultures.</a> Human Cell Culture Protocols in Methods in Molecular Medicine. 107, 183-206 (2005).</li><li>Milewska, A., Owczarek, K., Szczepanski, A., Pyrc, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+coronavirus+entry+into+cells.">Visualizing coronavirus entry into cells.</a> Methods in Molecular Biology. 2203, 241-261 (2020).</li></ol>

Immuno-RNA-FISH is a reliable method for double-staining of RNA and cellular proteins. Here, a modified immuno-RNA-FISH protocol has been described that allows detection of SARS-CoV-2 RNA and cellular proteins in cell lines and HAE cultures. This protocol can be adapted for use in different cell models including cell monolayers or specific tissues. The method relies on the concept of an HCR initiated by appropriate probe localization. Next, the use of split-initiator probes to begin amplification of the signal by fluores...

SARS-CoV-2 is a novel human betacoronavirus that emerged at the end of 2019, causing an unprecedented pandemic a few months later. Because the virus is new to science, much of its biology and its impact on host cells remain unknown. Therefore, mapping the virus-cell and -tissue tropism during infection is important if its basic biological characteristics and its effects on the host are to be understood. Several techniques are used to examine virus-host interplay including biochemical, biological, and physical assays. In situ hybridization is a common method that employs labeled complementary DNA, RNA, or modified nucleic acid probes, which localize to specific DNA or RNA sequences in a cell or tissue.
A new RNA fluorescent in situ hybridization (RNA-FISH) method has been developed that incorporates modifications to increase sensitivity by amplifying the signal-to-noise ratio via an HCR1. HCR allows the study of RNA localization at a single-cell level. Owing to its high specificity, sensitivity, and resolution, this method is useful not only for basic science studies, but also for applicatory projects, e.g., diagnostics. Recently, the feasibility of this method was demonstrated for detecting SARS-CoV-2 RNA localized to ciliated cells within fully differentiated 3D human airway epithelium (HAE) cultures2. HAE cultures constitute one of the most advanced tools used to study viral infection in the context of the &#34;natural infection&#34; microenvironment3,4.
Several reports on human coronaviruses (HCoV), including SARS-CoV-2, highlight the importance of epigenetic modifications with respect to HCoV infection and pathophysiology [reviewed in 5], e.g., the methylation pattern of the gene encoding the angiotensin-converting enzyme 2 (ACE-2) receptor6,7. Interestingly, mass-spectrometric screening identified several epigenetic factors that interact with the SARS-CoV-2 proteome8. More specifically, nonstructural protein 5 (NSP5) binds to the epigenetic regulator, histone deacetylase 2, and the catalytically inactive NSP5 (C145A) interacts with tRNA methyltransferase 1 (24). Additionally, NSP16 methyltransferase activity is blocked by the methyltransferase inhibitor, sinefungin9. However, the exact role of these epigenetic factors in COVID-19 remains unclear. Replication of HCoV takes place in the cytoplasm of the infected cell, and triggers inflammatory responses that are regulated by epigenetic modifications10.
For instance, HCoV-229E fine-tunes nuclear factor-kappa B signaling and profoundly reprograms the host cellular chromatin landscape by increasing acetylation of H3K36 and H4K5 in certain regions11. The Middle East respiratory syndrome-related coronavirus infection increases levels of H3K27me3 and depletes H3K4me3 at the promoter regions of subsets of specific interferon-sensitive genes12. Additionally, viral RNA triggers cell immune responses, as demonstrated for flaviviruses13, retroviruses14,15, and coronaviruses16. The epigenetic markers on viral RNA may play a role in recognition by cellular sensors, as shown for m7A methylation of human immunodeficiency virus-1 RNA17. However, questions remain: What is the impact of SARS-CoV-2 RNA on the immune response, and are epigenetic marks involved?
Here, an optimized RNA-FISH method coupled with immunofluorescence analysis of cell lines and 3D tissues (fully differentiated HAE) has been described. Although cytological methods, such as FISH and immunofluorescence, are used widely, this new-generation in situ hybridization method based on HCR has never been used for virus detection (except in a recent publication)2. In general, immunostaining and FISH require the following steps: permeabilization to enable penetration of probes or antibodies; fixation in which cellular material is fixed and preserved; detection in which antibodies or nucleic acid probes are applied; and finally, mounting of the samples for visualization.
Although existing protocols share these general features, they vary markedly with respect to the parameters involved. Here, an optimized, simple, immuno-RNA-FISH protocol has been described to detect SARS-CoV-2 RNA in HAE cultures and Vero cells. The technique comprises the following steps: (1) fixation of cells with paraformaldehyde; (2) permeabilization with detergent or methanol (MeOH); (3) rehydration through a graded series of MeOH solutions (HAE cultures only); (4) detection; (5) amplification using HCR technology to detect SARS-CoV-2 RNA; (6) immunostaining; and (7) imaging under a confocal microscope.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Equipment</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Confocal Microscope LSM 880</td>
			<td >ZEISS </td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Grant Bio, Mini Rocker- Shaker</td>
			<td >Fisher Scientific</td>
			<td>12965501</td>
			<td></td>
		</tr>
		<tr >
			<td >Incubator Galaxy170R</td>
			<td >New Brunswick</td>
			<td>CO170R-230-1000</td>
			<td></td>
		</tr>
		<tr >
			<td >Thermomixer Comfort</td>
			<td >Eppendorf</td>
			<td>5355 000 011</td>
			<td></td>
		</tr>
		<tr >
			<td>Materials</td>
			<td></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >15 mm x 15 mm NO. 1 coverslips</td>
			<td >LabSolute</td>
			<td >7695022</td>
			<td ></td>
		</tr>
		<tr >
			<td >1.5 mL tubes</td>
			<td >FL-MEDICAL</td>
			<td >5.350.023.053</td>
			<td ></td>
		</tr>
		<tr >
			<td >12-well plate</td>
			<td >TTP</td>
			<td >92412</td>
			<td ></td>
		</tr>
		<tr >
			<td >Conical centrifuge tube</td>
			<td >Sarstedt</td>
			<td >5.332.547.254</td>
			<td ></td>
		</tr>
		<tr >
			<td >parafilm</td>
			<td >Sigma</td>
			<td >P7793-1EA</td>
			<td ></td>
		</tr>
		<tr >
			<td >serological pipets</td>
			<td >VWR Collection</td>
			<td >612-5523P, 612-5827P</td>
			<td ></td>
		</tr>
		<tr >
			<td >slide glass</td>
			<td >PTH CHEMLAND</td>
			<td >04-296.202.03</td>
			<td ></td>
		</tr>
		<tr >
			<td >Transwell ThinCerts</td>
			<td >Grainer bio-one</td>
			<td >665641</td>
			<td ></td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Alexa fluorophore 488-conjugated secondary antibodies </td>
			<td>Invitrogen</td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >&#946;5-tubulin </td>
			<td>Santa Cruz Biotechnology</td>
			<td > sc-134234</td>
			<td ></td>
		</tr>
		<tr >
			<td >DAPI</td>
			<td >Thermo Scientific</td>
			<td >D1306</td>
			<td ></td>
		</tr>
		<tr >
			<td >Disodium phosphate</td>
			<td >Sigma</td>
			<td >S51136-500G</td>
			<td ></td>
		</tr>
		<tr >
			<td >EGTA</td>
			<td >BioShop</td>
			<td >EGT101.25</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR Amplification Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BAM01522</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HCR amplifier B1-h1 Alexa Fluor 647</td>
			<td >Molecular Instruments, Inc.</td>
			<td >S013922</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR amplifier B1-h2 Alexa Fluor 647</td>
			<td >Molecular Instruments, Inc.</td>
			<td >S012522</td>
			<td ></td>
		</tr>
		<tr >
			<td >HCR Probe Hybridization Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BPH03821</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HCR probe set for SARS-CoV-2 Ncapsid</td>
			<td >Molecular Instruments, Inc.</td>
			<td >PRE134</td>
			<td></td>
		</tr>
		<tr >
			<td >HCR Probe Wash Buffer</td>
			<td >Molecular Instruments, Inc.</td>
			<td >BPW01522</td>
			<td >Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information</td>
		</tr>
		<tr >
			<td >HEPES</td>
			<td >BioShop</td>
			<td >HEP001.100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Magnesium sulfate heptahydrate</td>
			<td >Sigma</td>
			<td >63138-250G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Methanol</td>
			<td >Sigma</td>
			<td >32213-1L-M</td>
			<td ></td>
		</tr>
		<tr >
			<td >Monopotassium phosphate</td>
			<td >Sigma</td>
			<td >P5655-100G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Paraformaldehyde</td>
			<td >Sigma</td>
			<td >P6148-1KG</td>
			<td ></td>
		</tr>
		<tr >
			<td >PIPES</td>
			<td >BioShop</td>
			<td >PIP666.100</td>
			<td ></td>
		</tr>
		<tr >
			<td >Potassium Chloride</td>
			<td >Sigma</td>
			<td >P5405-250G</td>
			<td ></td>
		</tr>
		<tr >
			<td >Prolong Diamond Antifade Mounting Medium</td>
			<td >Invitrogen</td>
			<td >P36970</td>
			<td ></td>
		</tr>
		<tr >
			<td >Sodium Chloride</td>
			<td >BioShop</td>
			<td >SOD001.5</td>
			<td ></td>
		</tr>
		<tr >
			<td >Trisodium Citrate 2-hydrate</td>
			<td >POCH</td>
			<td > 6132-04-3</td>
			<td ></td>
		</tr>
		<tr >
			<td >Tween-20</td>
			<td >BioShop</td>
			<td >TWN580.500</td>
			<td ></td>
		</tr>
		<tr >
			<td ></td>
			<td >Software</td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Fluorescence Spectraviewer</td>
			<td ></td>
			<td></td>
			<td>Modeling spectral parameters</td>
		</tr>
		<tr >
			<td >ImageJ Fiji</td>
			<td ></td>
			<td ></td>
			<td >Acquiring and processing z-stack images</td>
		</tr>
	</tbody>
</table>

visualization of sars-cov-2 using immuno rna-fluorescence in situ hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in cell line&#160;and three-dimensional (3D) cultures of human airway epithelium. The method allows highly specific and sensitive visualization of viral RNA by relying on HCR initiated by probe localization. Split-initiator probes help amplify the signal by fluorescently labeled amplifiers, resulting in negligible background fluorescence in confocal microscopy. Labeling amplifiers with different fluorescent dyes facilitates the simultaneous recognition of various targets. This, in turn, allows the mapping of the infection in tissues to better understand viral pathogenesis and replication at the single-cell level. Coupling this method with immunofluorescence may facilitate better understanding of host-virus interactions, including alternation of the host epigenome and immune response pathways. Owing to sensitive and specific HCR technology, this protocol can also be used as a diagnostic tool. It is also important to remember that the technique may be modified easily to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future.

The immuno-RNA-FISH protocol described in this manuscript was carried out using two cellular systems: a Vero cell line and a 3D HAE culture. The major steps for both cellular models are shown in Table 2. The RNA-FISH protocol for visualization of SARS-CoV-2 in HAE cultures includes steps that are typical for tissue samples, i.e., permeabilization with 100% MeOH and rehydration through a graded series of MeOH-PBS and 0.1% Tween solutions. Immunofluorescence was performed a...

Watch this Scientific Journal Video about Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization at JoVE.com

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization (Video) | JoVE

Here, we describe a simple method that combines RNA fluorescence in situ&#160;hybridization (RNA-FISH) with immunofluorescence to visualize severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA. This protocol may increase understanding of the molecular characteristics of SARS-CoV-2 RNA-host interactions at a single-cell level.

Visualization of SARS-CoV-2 using Immuno RNA-Fluorescence In Situ Hybridization

This manuscript provides a protocol for in situ hybridization chain reaction (HCR) coupled with immunofluorescence to visualize severe acute respiratory ...

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