negative control siRNA - (Sep/01/2005 )

Hi,

I find the negative control siRNA also knock down my gene expression as same as gene specific siRNA. Anyone has the same experience?

Also, i find a lot of papers do not present the data of siRNA knock down by RT-PCR analysis. Is RT-PCR is not accurate method to analyze the effect of knock down?

Thanks.

Weimin

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How do you analysis the expression level change? RT-PCR,do you mean real time PCR, if it is just PCR it may not be proper to quantify the expression change!

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yes, i use real time pcr to detect the expression change. this negative control works well with GAPDH knock down. BTW, the GAPDH and my gene kncok down were performed at same condition.

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If you are sure your realtime PCR result is reliable, you should check the sequence of your control siRNA against the mRNA sequence and also the promoter sequence of the gene, because partial match can sometimes induce RNAi and targeting promoter by siRNA can silence gene at transcriptional level.

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Yes, I agree with bioforum, otherwise your control siRNA may also induce off-target effects or interferon response. I know that Dharmacon has validated control siRNA (ie reduced off-target effects as measured by gene array).

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I believe that there are only 2 good controls for siRNA experiments.

The use of three different siRNA's directed against the same gene. Finding the same results with three independend sequences makes it very unlikely that the observed phenotype is due to a-specific knock-down.

A better way is to rescue the phenotype of an siRNA with overexpression of the concerned gene from an expressionvector with a mutations in the cds which mutates the siRNA bindingssite without altering the protein sequence.

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Of course you're right!! However, using several siRNAs against a single target will prevent you from false effects but won't protect you against an artifactual effect of your control siRNA. For instance if your control siRNA increase the expression of protein X due to interferon response or any other off-target effect and your silenced target protein does nothing on protein X then you may conclude that the silencing of your target protein has induced down regulation of protein X which is not the case. Thus, I would advise you to use several different controls, I mean commercially avaible control siRNA, empty transfection... You may also use the same siRNA as your target siRNA in which you introduce intended mutations. I think there's typically no perfect control in silencing experiments so use as many controls as possible at least at the beginning. Good Luck.

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"Also, i find a lot of papers do not present the data of siRNA knock down by RT-PCR analysis. Is RT-PCR is not accurate method to analyze the effect of knock down? "

I was told that quantitative real time PCR is not a reliable way to assay knockdown unless the PCR primers are chosen specifically to span the RISC cleavage site. If the primers do not include the region that is cleaved, you can still get amplification and won't see an effect, therefore western blot is a better assay.

Curious if anyone has observed or tested this. Do people have a preferential assay for showing knockdown--quantitative PCR vs Western blot vs flow cytometry, etc.? If you use PCR, do you check that the primers span the cleavage site?

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Like I mentioned above, the only control for this is to rescue the phenotype of an siRNA with overexpression of the concerned gene from an expressionvector with a mutations in the cds which mutates the siRNA bindingssite without altering the protein sequence.



Transfections with empty vectors or mutated siRNAs are not good as controls for the siRNA, but they are important controls for the effect of the transfection/infection and should fot that reason always be included.

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I guess this is the common "OFF-TARGET effect" which are published by many papers.
There are commerical companies which provide a modified RNAi Oligos (improved version) which has less off-target effect.

You can try to change your standard siRNA to a modified RNAi oligos to see if it helps!

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