Obtaining Viral RNA for Real-Time Quantitative Polymerase Chain Reaction (qPCR) Analysis

Begin with a serially diluted, synthesized viral RNA standard solution in the culture medium.

These dilutions serve as reference standards to quantify viral RNA in the samples.

Now, take the supernatant containing the virus from the virus-infected cell culture.

This culture medium contains an inhibitor that binds to RNA-degrading enzymes and prevents the breakdown of viral RNA.

Add the processing buffer containing the proteinase K enzyme.

Briefly centrifuge the solution for even mixing.

Place the PCR plate containing the samples in the thermocycler.

First, incubate at a moderate temperature to activate proteinase K.

This enzyme digests the viral proteins that encapsulate the genome, releasing viral RNA into the solution.

Next, incubate at a higher temperature to inactivate the enzyme and denature the RNA.

Finally, store the processed samples under cold conditions until real-time quantitative PCR analysis.

First, mix 199 microliters of processing buffer with one microliter of nuclease free proteinase K. Using cell culture medium, serially dilute the prepared Dengue virus three prime UTR RNA one to ten to obtain RNA standards at concentrations ranging from 5,000,000 to 5,000 copies per microliter by repeating pipetting and vortex. Transfer 5 microliters of the culture supernatant of Dengue virus-infected cells and the Dengue virus 3 prime UTR RNA standard to either A2 PCR strips or the wells of a 96-well PCR plate.

Mix in five microliters of the solution containing processing buffer and proteinase K. Centrifuge briefly at 200 times G for five seconds. Incubate the samples in a thermocycler at 25 degrees Celsius for ten minutes and then at 75 degrees Celsius for five minutes.