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

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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, and then split into 50 mL aliquots. Store at -20 °C until required.
   NOTE: The buffer will not be clear until the pH reaches 7.0.
2. P....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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