Hello everyone,
I'm helping an undergrad student who tried to knockout the DUSP2 gene in the PDAC cell line (Mia Paca-2). The RT-qPCR result showed a 60% reduction of gene expression, but the western blot only shows a reduction of 30% or less. He's already isolated more than 50 clones. Does anyone have any idea what might be the problem? Or how to discuss it.
Thanks in advance
First, RNA and protein levels are not always correlated 1:1, there are different aspects that control the steady state level of either such as synthesis rate and degradation that may result in a variable effect of reducing the amount of the RNA on the protein levels.
I assume your cells have a heterozygote edit, did you check what kind of change you got?
Is it a deletion or just an indel?
Is it possible that the change you introduce will effect the PCR (mutation in primer binding site) but not the ORF?
First check whether the deletion of DUSP2 is lethal for cells or not. If it is, then maximum you will get is heterozygote. Another probable problem may be multiple alleles of DUSP2 in MiaPaCa cells (common for cancer cells). My suggestion is use NHEJ inhibitor like SCR-7, this compound will increase the efficiency and try another cell line as well to check your guide RNA efficiency.
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