I have recently found a mutation that leads to a premature stopcodon. I now want to know if this mutation could result in nonsense-mediated mRNA decay. Are there ways to do this?
Perform individual knockdowns of UPF1 as well as Smg6 and or Smg5/Smg7.
If your transcript gets stabilzed under these conditions compared to a control knockdown (measure the levels by qPCR) then your trasncript is an NMD substrate.
Introduction of premature stop codon may lead to mRNA or protein instability. If you have cells containing the mutation, you should quantify mRNA levels by qRT-PCR and protein levels by western blot, and compare those to appropriate control cells. If you don't have mutated cells, you can use an expression system: clone the normal and mutated cDNA into an expression vector, transfect cells (HeLa) with these constructs and perform qRT-PCR and wb.
Perform individual knockdowns of UPF1 as well as Smg6 and or Smg5/Smg7.
If your transcript gets stabilzed under these conditions compared to a control knockdown (measure the levels by qPCR) then your trasncript is an NMD substrate.
Dear Marc-David,
Thanks for the suggestions. I read something on the UPF1 and SMG variants, which seems a plausible explanation to determine NMD. However, you mention knockdowns, but I am not familiar with such an approach. Do you perhaps have a running protocol or ideas how to perform such experiments? Moreover, as both proteins have become more studied over the last years, are there perhaps commercial kids available?
Kind regards,
René
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