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Immunology and Infection

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

Published: December 23rd, 2020

DOI:

10.3791/62067

1Malopolska Centre of Biotechnology, Jagiellonian University, 2Microbiology Department Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, 3Department of Cardiac, Vascular and Endovascular Surgery and Transplantology, The Medical University of Silesia in Katowice, 4Silesian Centre for Heart Diseases

Here, we describe a simple method that combines RNA fluorescence in situ 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.

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 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.

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 ....

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1. Buffer preparation

  1. For 500 mL of 2x PHEM buffer, combine 18.14 g of piperazine-N,N′-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.......

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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.......

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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.......

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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.).

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Name Company Catalog Number Comments
Equipment
Confocal Microscope LSM 880 ZEISS
Grant Bio, Mini Rocker- Shaker Fisher Scientific 12965501
Incubator Galaxy170R New Brunswick CO170R-230-1000
Thermomixer Comfort Eppendorf 5355 000 011
Materials
15 mm x 15 mm NO. 1 coverslips LabSolute 7695022
1.5 mL tubes FL-MEDICAL 5.350.023.053
12-well plate TTP 92412
Conical centrifuge tube Sarstedt 5.332.547.254
parafilm Sigma P7793-1EA
serological pipets VWR Collection 612-5523P, 612-5827P
slide glass PTH CHEMLAND 04-296.202.03
Transwell ThinCerts Grainer bio-one 665641
Reagents
Alexa fluorophore 488-conjugated secondary antibodies Invitrogen
β5-tubulin Santa Cruz Biotechnology sc-134234
DAPI Thermo Scientific D1306
Disodium phosphate Sigma S51136-500G
EGTA BioShop EGT101.25
HCR Amplification Buffer Molecular Instruments, Inc. BAM01522 Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HCR amplifier B1-h1 Alexa Fluor 647 Molecular Instruments, Inc. S013922
HCR amplifier B1-h2 Alexa Fluor 647 Molecular Instruments, Inc. S012522
HCR Probe Hybridization Buffer Molecular Instruments, Inc. BPH03821 Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HCR probe set for SARS-CoV-2 Ncapsid Molecular Instruments, Inc. PRE134
HCR Probe Wash Buffer Molecular Instruments, Inc. BPW01522 Buffer can be also prepared doi:10.1242/dev.165753: Supplementary information
HEPES BioShop HEP001.100
Magnesium sulfate heptahydrate Sigma 63138-250G
Methanol Sigma 32213-1L-M
Monopotassium phosphate Sigma P5655-100G
Paraformaldehyde Sigma P6148-1KG
PIPES BioShop PIP666.100
Potassium Chloride Sigma P5405-250G
Prolong Diamond Antifade Mounting Medium Invitrogen P36970
Sodium Chloride BioShop SOD001.5
Trisodium Citrate 2-hydrate POCH 6132-04-3
Tween-20 BioShop TWN580.500
Software
Fluorescence Spectraviewer Modeling spectral parameters
ImageJ Fiji Acquiring and processing z-stack images

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