I recommend Silencer Select siRNA from Thermo Fisher. They already have pre validated sequence for DAB1 for rat and the knockown efficiency are pretty high. They also have positive and negative controls to help you with your experiment.

https://www.thermofisher.com/br/en/home/life-science/rnai/synthetic-rnai-analysis/ambion-silencer-select-sirnas.html?icid=cvc-sirna-analysis-c3t2

Hello Ivayla,

at best you try three different siRNA sequences and determine the silencing efficacy by RT-qPCR to select the best sequence. Validated sequences provide a "theoretic" good silencing efficacy but your biologic samples cannot read the validation certificate and must not follow them. I used myselfe a validated sequence and the silencing effect was 20%. Then I selelcted another sequence from

http://dharmacon.gelifesciences.com/design-center/?redirect=true

and it worked x fold better.

Finally the validated ones are a good start but you have to test them to ensure a good effect. At least testing two other sequences in addition will provide one very good sequence :-)

There has been an interesting publication, where an identical screen in the same lab with the same people under  absolutely identical conditions an RNAi screen was performed, once using the silencer select and then the Dharmacon library (Hasson et al, Nature, 2013 Dec 12;504(7479):291-5). Each screen yielded a few hundred hits – but there was only an overlap of below 25 individual genes – which is pretty close to what you would expect if the hits were randomly chosen. This translates into: either one of the libraries were bad, or both. Reproducibility of RNAi results using different siRNAs against the same gene is surprisingly lousy owing to divergent Knock Down efficiency on the one hand and off-target effects on the other.

Because off target effects appear to blur the results RNAi experiments so badly we now collaborate with a company siTOOLs Biotech offering complex siRNA pools, called siPOOLs, that consist of as many as 30 selected siRNAs all targeting your gene – with a maximally diverse off-target pattern, so that at commonly used concentrations the off target effects are simply diluted out – at the same time, all siRNAs in the pool act on the common on target – leading to enhanced and robust KD efficiency. The idea is simple, the company produces customized siPOOLs on demand, the prizing is decent and our experience with these reagents has been very convincing.

So – this would be my recommendation – it takes a few days until the pool is generated, but in the end you safe a lot of time and money. Also comes with pos. and neg. - control. The web site is somewhat weak, (but the blog is fun reading) – and it gives you some information on the principle. The paper on siPOOLs has been published in NAR  in 2014.

Hope that info was helpful

I have taken a view on the siPOOL publication (http://nar.oxfordjournals.org/content/42/12/8049.long) and the results are very sound.

Worth mentioning is that if I want to silence a certain transcript I am interested in the expectation is that other genes are affected due to missing feedback regulation, repression and enhancement or isn´t it?

To say, as shown in Fig. 4 A, that only a few fluctuations in the transcriptome with siPOOL are better than those with a single sequence is for me a bit to superficial. In addition  Scyl1 is a kinase-like protein and the inhibition of a kinase or phosphase, to have the direct opposite enzmye, will in most cases affect the transcription of many genes I guess, as we see it also for very specific tyrosine kinase inhibitors.

I agree that many of the deregulated genes of the single siRNA in 4 A will disappeare if you perform a qPCR analysis and the overlap will be similar to those of the siPool 15 or 60 sequence.

In fact most experiments use siRNA to investigate how silencing of a certain gene affects a pathway of interest and the effect will be more or less the same if you use a single siRNA or a siPOOL. In addition the list price of a siPOOL is also superior as the silencing effect.

In turn if you investigate a transcript without known effects it will be a very good approach to reject 80% of the false positive regulations form the beginning and  reduced affected genes after RNA-seq. In addition it can ease the filtering of RNA-seq data for specific affected genes but it it is not mentioned in the article if the deregulated ones by siPOOL are those interacting with the silencing target.

What I wonder about is that the fold change of the deregulated genes reside more or less on the level of the single siRNA. The effect on other genes is not improved by a more specific silencing?

I´m not sure I understand the points you make - anyway, I don´t think that our discussion relates to the question initially brought up be Ivayla. Maybe you should discuss your questions with the siTOOLs people, I´m sure they appreciate your feedback.

I just wanted to mention that every company selling RNAi assays only emphasize their product specificity.

At least the experimenter has to consider what is important, the effect of silencing a gene and the effect on a known pathway or investigating a gene function from scratch or on the whole transcriptome.

When you have to investigate only one gene it makes sense to use siPool because if several siRNAs fail you end with the same or even a higher price for one gene. With siPool you get 30 siRNA sequences anyway and a good silencing efficacy as they have written.

At least investigating effects on the transcriptome after silencing a gene are interesting for me so I am very glad about your input and added my considerations :-)