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I don't work at the wet lab and don't know all the details about the knockdown techniques.
My question is:
How lncRNA knockdown is done?
For example - you have lncRNA that is functional in the nucleus. How is it possible to do knockdown using iRNA if interference happens only in the cytoplasm?
Sorry if there is biology related misconception in my question.

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3 Answers

up vote 2 down vote accepted

Knockdown of lncRNA in mammals is not done via RNAi. Instead, one transfers antisense DNA oligos which bind to the RNA. This triggers the action of the RNase H enzyme, which degrades RNA-DNA duplexes. It degrades the lncRNA.

UPDATE: For reference, I learned about this from a seminar, and it is not very well documented, but after some literature search I found this paper to quote:

Although we (Fig. 3A) and others (42, 49) have used siRNA to knock down NEAT1 lncRNA and although the knockdown of strictly nuclear RNAs has been well described (53, 54), we remained concerned that because NEAT1 is mostly a nuclear RNA (55), it may not be very efficiently targeted by the RNAi machinery. Nuclear RNAs, however, are proficiently targeted using complementary antisense (AS) oligodeoxynucleotides that recruit nuclear RNase H activity to degrade the RNA (56). Hence, we also employed complementary AS oligodeoxynucleotides to knock down NEAT1 lncRNA in HIV-1 NL4-3-infected Jurkat cells (Fig. 4C, left). The AS approach reduced NEAT1 (Fig. 4C, left) and increased HIV-1 p24 production (Fig. 4C, right), providing results consistent with those from siRNA-mediated knockdown of NEAT1 (Fig. 4A and B).

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Reference or it didn't happen :) –  Pgibas May 16 '13 at 13:42
    
I added a reference –  Drosophila May 16 '13 at 18:15
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Nuclear RNAi happens.. check these articles:

http://www.nature.com/nrg/journal/v14/n2/execsumm/nrg3355.html

http://nar.oxfordjournals.org/content/early/2011/11/07/nar.gkr891.full

Also there is some evidence that Ago2 binds to lncRNAs.

However, you can employ other techniques to knock down lncRNAs for e.g. ribozymes.

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This is controversial, but most groups in the field have concluded that RNAi does not work in the nucleus despite the presence of Ago. When lncRNA have been KD'd with RNAi, they are usually shown to be present in both the nucleus and cytoplasm, and the KD is thought to occur in the cytoplasm, not nucleus. Of course as always in biology it is possible that nuclear RNAi does occur in some cell types or under some special conditions. Gapmer oligonucleotides acting via RNAseH are usually an excellent way to KD a nuclear lncRNA, but I would add that you should be prepared to test say 6 or more different oligos, as the efficacy is unpredictable, and in contrast to RNAi, there are no great generally available algorithms for antisense oligo design.

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