Basic questions about siRNA - (Dec/15/2005 )

Hi,

I am going to be doing an siRNA transfection (oligos) into a cell line and had a question about how to assess the efficiency of the knockdown. I would like to screen first via Taqman and then western. Here's my question: I already have a good taqman probe against the gene and wanted to know if I can use that same probe or do I need to design a new one targeting where the mRNA cleavage occurs. I am assuming that the whole mRNA gets degraded, so where the probe sits is irrelevant--Right?

Second very basic question: Why do companies cell annealed and unannealed snese and antisense siRNAs--one has to anneal them prior to transfection--so why not just sell the annealed set--Am I missing something?

As you can tell--I'm very new to this!

Thanks in advance

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Hi!

You do not need to design a new probe for Taqman. mRNA´s 'usually' consits of 5´cap and a poly-A-tail which, among other things, protect them from degradation.
siRNA mediated degradation is an endonucleolytic cleavage 'cutting' the mRNA somewhere at the siRNA target site (I think it is between nucleotide 10-11..., not sure right now).
This results in unprotected 5´and 3´ends and thus enhanced degradation of your target mRNA and prevents translation... .
So in the end the whole mRNA should be degraded.

Good luck,
Cheers

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Bomber has answered your first question.

Regarding the second one, most if not all companies sell annealed siRNA. If you ask for single stranded siRNA, you will get it. Sometimes you will need single stranded for example, for labeling one of them.

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Thanks guys!

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Hi Bomber,

I have posted a question about RNAi in plant and qRT-PCR detection. PCRMAN points me to your previsous message. I want to repost my questions here and think you may have some suggestions.

Thanks.

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Is there anyone can share some tips or experience of using qRT-PCR to detect RNAi effects in plant? For example, which region should I design primer/probe? If I have a RNAi vector using full length cDNA for both sense and antisense, can I still detect the gene suppression with qRT-PCR?

Thanks.

Jeff

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pcrman,

Thanks for your reply. I am still not sure if I have found the right answer. First, I think siRNA and RNAi have different mechanism for knockdown mRNA. Second, is it possible that the primer/probe also binds to the sense and antisense region from vector? Is it possible that the qPCR detects the region from vector in addition to the gene from plant? Those questions have bothered me for a while but I haven't found any clear answer or good reference paper yet.

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First, siRNA and RNAi are not the same type of thing. siRNA is an RNA oligo that can silence gene expression at both transcriptional and post-transcriptional level, the so called RNAi phenomenon.

So you use vector to transcribe both sense and antisense strand, which presumably will form long dsRNA which will be further processed by dicer into short dsRNA. I don't know much about what is going on in plants, but I guess you will be able to detect both sense and antisense expression because not all transcripts will form dsRNA and be processed by dicer. In addition, if the expression is strong, the RNAi machinery may be exhausted after some time and thus a large portion of the transcripts from the vector will be expressed in the cytoplasm.

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PCRMAN,

Thanks for correction on the RNAi and siRNA term.

Assuming what you were saying is right, it would be really hard to have a qRT-PCR assay for detecting the decrease of target gene expression, especially fulll length cDNA was used in the vector. Really hope I can find a good paper/reference about this.

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