HI
I wanted to to know which data has better significance while comparing the results of RT PCR and Western blotting.
Western blotting detects protein - which is usually the endpoint of gene expression activity and a direct molecular readout of equivalent phenotype. Hence I would say that Westerns will have better significance as RNA although made - may not get translated to equivalent higher levels of protein. On the other hand, PCR has muchhhh higher sensitivity and hence can detect even weak changes, which may not be evident with a western. Secondly, many times it is not possible to find antibody to all proteins that are made by the cell, especially when it comes to multiple isoforms.
So bottom line - is that depending on your application/question, Both have significance and use. But if you have a good antibody for your protein and are only specifically interested in that protein output changes, then clearly Western is the way to go.
Agree with Subhojit, it's mainly about whether you want protein or RNA data.
Spare yourself the pain and do RT-PCRs instead of western blots if you do not need to actually detect the proteins. It is much easier and robust....
Thank you guys. One more thing to clarify, is it possible to have difference in the expression level between protein and mRNA for the same target?
Hi Yelungka: Yes, unfortunately. It does happen, that's why you still need to do western blots sometimes. Reviewers can ask for it.
On the plus side, there is this study done:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4459080/
- Badges
- Science method