Increased transcripts after RNAi - (Jun/02/2006 )

Hi friends,

I was doing a RNAi experiment this week.
All the preparations went well just the result is a bit desolate.
I had 4 samples:

1) 34 ul (100 worms in RPMI) + 10 ul dsRNA target gene (250 pmol/ml) in elution buffer + 56 ul RPMI
2) 34 ul (100 worms in RPMI) + 100 ul ds RNA control gene (250 pmol/ml) in elution buffer + 16 ul RPMI
3) 34 ul (100 worms in RPMI) + 66 ul RPMI
4) 34 ul (100 worms in RPMI) + 116 ul Elution buffer

I isolated RNA from the worms and did the RealTime-PCR. Then I calculated the transcript quantity against an endogenous control.
What came out was that the target gene transcript quantity was 6x higher in 1) and 7x higher in 3) than in 2) and 4).
This is quite strange because one would expect lower quantites in 1) and approx. the same high quantities in 2), 3), and 4).
I ask myself if the results of 2) and 4) are somehow influenced by the elution buffer which contains Tris-EDTA.
The worms looked quite ok and were at least not killed by the Elution buffer.

Do you have an idea what could have gone wrong?

Thanks

the results dont make sense like u pointed out. I dont have any idea as to what could have gone wrong. It would be nice of u to share the results when u repeat the above exp., as I am quite curious know what happens.

Is RPMI creating the strange result, as u seem to have increased gene transcript when u added more RPMI comapred to elution buffer.

It happens so often in our lab that the target gene dsRNA treated samples show an increased transcript number. It is not really clear yet why.
We thought maybe the primer probes (Taqman) for the RT PCR bind to dsDNA that has not been properly digested after dsRNA synthesis and has been transferred through the whole experiment into the RT PCR reaction mix. That`s why we synthesize the dsRNA now from a DNA fragment of the target gene upstream of the region where the RT PCR primers bind.
It helped for some samples but not for many.

Hi scolix,

I repeated the experiment.

1) 40 ul (100 worms in RPMI) + 250 pmol dsRNA (target gene) in elution buffer (10 ul) + 34 ul elution buffer + 16 ul RPMI
2) 40 ul (100 worms in RPMI) + 250 pmol dsRNA (control gene) in elution buffer (44 ul) + 16 ul RPMI
3) 40 ul (100 worms in RPMI) + 44 ul elution buffer + 16 ul RPMI

All samples had the same volume of RPMI and elution buffer.
Instead of a decrease of the transcript levels of the target gene I saw again a significant increase compared to the controls 2) and 3), which were very similar to each other.
As I am trying to silence a gene which has several alternatively spliced isoforms - depending on which tissue you look at - I wonder if the increase of transcription has anything to do with that.
Is it possible that by silencing only half the isoforms the transcription of the other isoforms is induced strongly in response to that?

Any help is very welcome! Thanks

P.s.: Inreases in transcript levels were observed several times in our lab, but nobody has an idea what`s the mechanism behind it.

This phenomenon has been reported many times here. Make sure that the results are reproducible and there is no off-target. Probably this is a novel mechanism of gene regulation.

Do you have some links for me?
It is difficult to convince my supervisor of this idea.
Well, on the other hand, we wouldn`t know how to change the proceduce of the experiment.
Seems fine so far. And the controls look good as well.

Look at some papers dealing with interferon response related to RNAi. Mayb this phenomenon is better explained there.

Will do. Thanks.

I don`t work with mammalian samples. If I understand it right, interferon is not an issue then?

No clue why we get such results.
one possible explanation as reported for miRNA mechanism of action is that the duplex of siRNA and mRNA are is stable and is not degraded. To get around this problem, its better to design primers such that the amplicon spans the siRNA target region.
The other reason which may sound vague but your scramble control is good, so try to change the scramble siRNA and check out your results once again.
Third thing is when you design siRNA region, check out the target region is not concealed in mRNA folding/sec. str (use RNA fold program).