I am working on different cancer cell lines and looking at the expression of protein. How do I go about it?
Hi Ebidor! If you are interested in measuring levels of a protein across different cancer cell lines, it may be best if you use Western blot or immunofluorescence or something that measure protein levels, rather than mRNA expression. qRT-PCR measure mRNA levels, and although they sometimes can reflect protein levels in a particular cell line, they do not always. This is because mRNA and proteins are regulated differently - proteins are regulated post-ranscriptionally and post-translationally, and if you consider auto-regulatory loops, etc, it paints a pretty complex picture. Therefore, it is best to measure protein levels directly rather than measuring mRNA expression via qRT-PCR, and using it as a surrogate to inform protein levels in your cell lines. You can use mRNA levels in addition to protein levels to support your point, but measuring protein levels directly is important.
Hi Ebidor! If you are interested in measuring levels of a protein across different cancer cell lines, it may be best if you use Western blot or immunofluorescence or something that measure protein levels, rather than mRNA expression. qRT-PCR measure mRNA levels, and although they sometimes can reflect protein levels in a particular cell line, they do not always. This is because mRNA and proteins are regulated differently - proteins are regulated post-ranscriptionally and post-translationally, and if you consider auto-regulatory loops, etc, it paints a pretty complex picture. Therefore, it is best to measure protein levels directly rather than measuring mRNA expression via qRT-PCR, and using it as a surrogate to inform protein levels in your cell lines. You can use mRNA levels in addition to protein levels to support your point, but measuring protein levels directly is important.
Hi Ebidor, I too would recommend western blotting for comparison but the levels of a particular protein in different cell lines may be in context of that specific cell line and may or may not be helpful to derive conclusions
I would strongly recommend western blot technique rather than PCR.
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