The general outline of this kind of experiments, without going into much detail is as follow:
1: prepare several dilutions of your drug, let's say from 10^-4 to 10^4 nM.
2: plate cells in 96 well plates, consider at least three wells per dilution, plus positive and negative controls.
3: infect cells, usually 1 day after plating
4: add drug 24h post-infection
5: after 24-72h (depending on the virus you are studying), evaluate the number of infected cells compared to non-treated controls
6: plot sigmoid dose-response curves to obtain EC50 and other parameters.
The specific methods will, of course, depend on the virus studied. A pubmed search will help you find papers that describe similar analyses and specific assays for your virus.
Dear everyone,
I am doing the research on a drug which has ability to inhibits the replication of my virus.
First experiment: I perform the experiment to determine the toxicity of drug on the virus.
Second experiment: I seed cells in 96-well plate micro-titration (20,000cells/well in 100uL of MEM), after 24 hrs, I add drug with various concentrations (50uL, making two fold dilution), and after 48hrs, I infect cells with 100TCID50 of virus (50uL) . The total volume of one well is 200uL and I daily observe the Cytopathic effect(CPE) to record the data.
I don't have enough the money to perform the Real time PCR assay to quantify the amount of viral genomic RNA to confirm the reduction of virus production in the presence of drug.
But I can do the RT-PCR and Agarose gel electrophoresis.
Can you advise me to do the suitable methods to know more about my result. And I don't know whether CPE analysis only can interpret accurately the antiviral effect of drug on the virus?
I am in need of your helps.
Thank you so much.
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