Hello all, I have transfected my cells with plasmid containing my shRNA sequences of my gene of interest and I'm looking this effect on 3 other genes, that when my gene is knockdown what happens to other genes. I did the rt-PCR and found that when my gene knocks down 2 genes are also knockdown but 1 other is overexpressed.
Now, I want to find out how we can do other experiment to confirm and validate that my gene is regulating or not regulating the transcription of the other genes.
RNAseq data could be a solution. You could also try overexpressing or knocking out your gene of interest. The problem with RNAi is that it could have off-target knockdown.
Hi Robert. I have overexpressed my gene of interest and other gene also highly overexpresses. So, now I have knockdown my gene of interest and that gene does not go down. I have think of another experiment which proves my gene transcriptionally regulate the other gene.
Have you investigated what transcription factor binding sites are in the promoter for your second gene? Is it known what proteins your gene of interest interacts with?
I know which proteins my gene of interest interacts with, but I dont know what transcription factor binding sites are in the promoter region for the second gene I’ll look into this.
Easiest strategy is to check if there are any direct interactions between the genes. checking their position in pathways they are involved in and/or if in different pathways, whether there are any crosstalk between the pathways. This will give you an insight of any possible direct/indirect interactions.
conducting a complement strategy, i.e. overexpression the gene that you knocked down, should support your KD results in case what you're seeing is true (and not false) and there really exists a genetic interaction between the genes. If not, it might suggest 2 possibilities: either there is a dose-dependent interaction, or there are no direct interactions at all!
Hello, thank you for your reply. Initially I overexpressed my gene and by rt-pcr I looked at the gene expression of the other gene, the results were that if my gene is overexpressed the other gene is also overexpressed. Now I have knockdown my gene and this other gene goes down but is not fully knockdown. I want to find out does my gene of interest transcriptionally regulate this other gene or not, would it be possible with reporter gene assay etc?
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