Dear all, I am studying A GENE influence viral replication and translation OR NOT. After knocking out THAT GENE by CRISPR, I infected the cells (both WT and KO) with virus at MOI of 0.02 and 0.2 . 3 days later, I collected the viral supernatant for TCID50 and extracted total RNA of supernatant and cell pellet (Mixture) by TRIzol. TCID50 showed that virus titer increased in KO groups, however, qPCR showed that virus copies decreased in KO groups. I am so confused which result was convincing, thank you all for kindly help.
With TCID50 you dectect infectious viruses with qPCR you detect viral genomes; if there is damaged or incomplete viral particles released in the supernatant you will detect them by PCR but they will not be infectious
@Didier Poncet Thank you for your answer, but i am confused which one is more convincing, qpcr or tcid50? i found that plaque assay and tcid50 often appear in the papers, while qpcr is used in our lab.
Both give different (but overlapping) informations. if you consider that there is no technical problems (for example viral genomes or mRNA released in the supernatant from damaged cells or from damaged particle because of a bad samples handling ) then the TICD/qPCR ratio gives you the ratio of infectious particle /physical particle. if it is lower in one condition then it means that the viral particles produced have a problem ...
Didier Poncet Thanks again for your kindly help, and I have learned a lot from it! So i would take TCDI50 as consideration, for it represents infectious particles and shows the viral infectivity.
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