Functional Validation Of A 5' Utr Mutation

Question

[Biomed]

Hi, I would like to functionally validate a -not seen before- 3 bp deletion I found on a 5' UTR of a knoen disease causing gene. I looked at the UCSC browsers tracks for conservation and regulation and it seems like this region is both well conserved and has a lot of regulation signals associated with it. Looking at gene expression by RT-PCR looks like the best option but what other next steps would be also valuable on the path to functionally validate this mutation?

Thanks

Answer 1

[JC]

To complete the validation I will suggest an in-vivo experiment using transfected cell-lines (the expression can be quantified using RT-PCR or fusion your 5'UTR to a reporter like GFP), testing: 1) the 5' UTR with the 3bp deletion, 2) the wild-type 5' UTR, 3) a vector control for background.

But depending on the journal, a simple RT-PCR in normal and disease samples can be enough to validate the mutation.