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Higher mRNA level after RNAi?


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#1 wingdx

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Posted 12 February 2012 - 11:58 AM

I'm rather new to this technique and am trying to test the function of a gene using RNAi in vivo. I’ve fed my insect with dsRNAs at 500ng/ul and found expected decrease in enzymatic activities of the gene. What confuses me is when I tested the mRNA level using rt-PCR. There was actually higher amount of mRNA of the gene. I've read some references and most of them said that the expected results should be reduced mRNA levels, which is in conflict with the enzymatic activity I'm observing. I was wondering if it is possible that the cells are trying to compensate the product and are upregulating the expression of the gene?
Thanks.

#2 wincel

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Posted 17 February 2012 - 01:43 PM

There is the phenomenon to activating RNAs in mammalian cells, mostly promoter targeting RNAs. That way your mRNA can go up despite dsRNA. And if your gene is not transcriptionally regulated but posttranscriptionally that mean you see no change in protein.
Another possible explaination is that several genes at least in mammals are regulated by some antisense-transcript. Addition of an dsRNA can target both and if the antisense-targeting effect is stronger your mRNA can go up too.
But the enzyme dropping while RNA goes up is odd.
And last but not least but not very likely it can happen that your gene has some feedback regulation with the protein level dropping inducing an increased promoter activity.

Do you know the half life of your gene? So you can make sure you measure the effect of the protein produced after your RNAi kicks in? And are you sure your RT-PCR is working correctly, how about different splicing variants and you measure a variant which is coming up but not active as enzyme? Is your enzyme active as is or does it need some modification to become active? Worst case: your RNAi is affecting the housekeeping gene you normalize to. Have you excluded this by testing several housekeeping genes in parallel?
You could do some Northern Blot to see if your RT-PCR is giving correct data and what the length of the transcripts is.
And I suppose you normalize your enzyme activity to something. If it is a gene, make sure it is not affected by your treatment as well.

Edited by wincel, 17 February 2012 - 01:51 PM.





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