I performed a virus infection on HeLa cels and isolated RNA and whole cell lysates continuously from 0 hour to 24 hour post infection and performed a semiquantitative RTPCR and Western blotting with the respective samples. To my surprise, I see a very defined reduction in the transcript levels of a particular transcript from 12hour onwards up to 24 hours in the infected samples compared to mockinfected.
But the western blotting data shows a steady level of this protein from 0 hour to 24hour in infected vs mock. What would be the possible reasons for such a result?
And what other experiments will I have to plan for the same?
The protein you are looking at might be very stable and therefore its level goes down much slower then RNA level. Indeed the lack of direct correlation between mRNA and protein level was shown in the past
S.P. Gygi et al. “Correlation between protein and mRNA abundance in yeast,” Mol Cell Biol, 19:1720–30, 1999.
For Hela cells specifically see references here
http://book.bionumbers.org/how-fast-do-rnas-and-proteins-degrade/
As for the 'next experiment' it really depends what do you want to study... Are you interested in the protein? RNA? The interaction of the virus and cells?
Dear Nisha, usually when there is a dissociated between mRNA and protein levels, people think about miRNA regulation of the expression of your gene of interest. Look with http://www.ensembl.org whether there is (are) putative binding sites for miRNAs (or other RNAi molecule) in your gene. If yes, monitor the expression of this miRNA in your samples.
Dear Nisha,
I agree with Yoram. You may want to check on the half-life od your protein in the literature or perform a cycloheximide incubations for 24-48-72-96h and check on the WB when your protein starts to vanish.
Best,
Kamil
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