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 microlit