Dear all,
Recently I am performing RT-PCRs and Western blots of a mammalian cell line in which I overexpressed two vectors (wild-type and mutated) of equal promoter and sequence length. However, in both RT-PCR and Western blot I observed very different expression levels between both vectors, despite always transfecting the cells with the same amount of DNA. Could these results be acceptable for publication? Could I assume that the mutation is affecting the gene regulation of its vector in a different way? Or should I normalize this effect in some way in order to explore changes caused in other genes/proteins?
Thank you
Jaime
Hi there,
First of all you have to repeat the experiment in order to be able to validate the hypothesis that mutation has a significant effect at RNA/protein levels. If you want to normalize the data you definitely need an internal control (from a housekeeping gene both for RNA and protein levels).
Hi there,
First of all you have to repeat the experiment in order to be able to validate the hypothesis that mutation has a significant effect at RNA/protein levels. If you want to normalize the data you definitely need an internal control (from a housekeeping gene both for RNA and protein levels).
Thank you very much Dominique Liger ! I will follow your advice. =)
We do have cases where a single amino acid change/difference between two otherwise identical open reading frames under the same promoter control can result in dramatic differences in protein levels, when recombinantly expressed in E. coli. I don't know if your mutation(s) were created in the coding sequence, and if the same thing could also occur in mammalian cells. In any case, normalization with a housekeeping gene would be important, as suggested by @Dominique.
Agree with both of the above answers
Normalisation and then repetition with the same sample sets and then another biological set of replicates would allow you to place faith in what could well be a real effect
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