This protocol made it possible to visualize SARS-CoV-2 RNA in cell lines and in fully differentiated, three-dimensional human airway epithelium cultures, providing a tool to better understand viral pathogenesis and replication at the single-cell level. Owing to its high specificity, sensitivity, and resolution, this method is useful not only for basic science studies of SARS-coronavirus-2 but also for applicatory projects, such as diagnostics. Demonstrating the procedure will be Agnieszka Suder, a PhD student from my laboratory.
After fixing and permeabilizing the cells as described in the text manuscript, remove the 1X PBS solution from the wells, and add at least 300 microliters of amplification buffer to each well. Incubate the samples for 30 minutes at room temperature. Prepare each HCR hairpin by snap-cooling the desired volume in separate tubes.
To prepare 300 microliters of amplification solution, use 18 picomoles of each hairpin. Transfer the hairpin solution into tubes, and incubate them at 95 degrees Celsius for 90 seconds. Then cool to room temperature for 30 minutes in the dark.
Prepare a hairpin mixture by adding the snap-cooled H1 and H2 hairpins to the amplification buffer. Place drops of 30 to 50 microliters of hairpin mixture onto Parafilm. Place the coverslips with cells onto the drops of hairpin mixture, and incubate the samples overnight in the dark at room temperature.
To mount the slides, place two 10-microliter drops of mounting medium on a slide, ensuring that the drops are separated sufficiently to allow two coverslips to be placed on a single slide. Remove excess liquid by tapping the coverslips on a clean towel. Then place them in antifade mounting medium with the cells facing down.
Place the mounted samples on a dry, flat surface in the dark, and let them cure. After fixing and permeabilizing the HAE cells as described in the text manuscript, preamplify the samples by incubating them with amplification buffer for 30 minutes at room temperature. Use 30 picomoles of each hairpin to prepare 500 microliters of amplification solution.
Transfer the hairpin solution to the tubes, and incubate them at 95 degrees Celsius for 90 seconds. Then cool them to room temperature for 30 minutes in the dark. Prepare the hairpin solution by adding all snap-cooled hairpins to 500 microliters of amplification buffer at room temperature.
Remove the preamplification solution, and add the complete hairpin solution. Then incubate the samples overnight at room temperature in the dark. Place the cut-out membrane from the Transwell inserts onto 10 microliters of antifade mounting medium with the cells facing up, and add extra mounting medium to the membrane.
Then cover the membranes with coverslips. This immuno-RNA-FISH protocol was carried out in two cellular systems, a Vero cell line and a 3D human airway epithelium culture. Localization of SARS-coronavirus-2 subgenomic RNA in infected and mock-inoculated Vero cells is shown here.
Viral RNA can be seen in red. SARS-coronavirus-2 subgenomic RNA localization in infected and mock-inoculated human airway epithelial cells is shown here. The permeabilization protocol was optimized in Vero cells.
Permeabilization with detergent results in a clear, specific signal for SARS-coronavirus-2 subgenomic RNA, whereas using ethanol results in a blurry and unfocused image. When mounting the slides with Vero cells, it's important to place a coverslip with the cells facing down. When mounting the HAE slides, the membrane needs to be placed with the cells facing up.
This technique can be easily modified to enable detection of any RNA, including non-coding RNAs and RNA viruses that may emerge in the future. Coupled with immunofluorescence, it can be used to study the protein-RNA interactions at the single-cell level.
