Extraction of Viral RNA from Surface-Bound Virions Using a Silica Spin Column

Begin with an epithelial cell monolayer that has surface-bound virus particles.
Add a denaturing salt buffer and mix it uniformly.
The buffer disrupts host cell membranes and disassembles the viral coat proteins, releasing both host and viral contents, including RNA. It also inactivates the RNases to prevent RNA degradation.
Using an inverted microscope, confirm that all cells are lysed.
Add ethanol and transfer the suspension to a silica-based spin column.
Ethanol reduces RNA solubility, allowing ionic interactions between RNA and the silica membrane.
The unbound proteins, DNA, and other impurities flow through and are discarded.
Wash the column with a wash buffer to remove leftover proteins.
Next, wash with ethanol-containing buffer to remove contaminants.
Place the column in a fresh tube and centrifuge to eliminate residual ethanol.
Add nuclease-free water and centrifuge again to collect the purified RNA.
The recovered host and viral RNA are now ready for downstream analysis.
To extract viral RNA from JUNV Candid 1 particles, use a commercial RNA isolation kit according to the manufacturer's protocol for purification of RNA from animal cells using spin technology as follows. Lyse the cells as directed by adding 350 microliters of lysis buffer, RLT, directly to each well.
Mix the buffer several times to ensure cell lysis. Then use an inverted microscope to ensure lysis by the absence of cells. At this point, the virus has been neutralized, so the remaining steps can be performed outside of the Class 2 biosafety cabinet, provided kit tubes were not contaminated.
Transfer the lysate to the kit column, and follow the rest of the protocol as described. Include the optional Step 9 from the manufacturer's protocol, which is an additional centrifugation of the spin column to eliminate any possible carryover of buffer RPE. For the final step, use 30 microliters of nuclease-free double-distilled water to elute the purified RNA from the spin column.
Store the RNA at negative 80 degrees Celsius, or use directly in the qRT-PCR assay.