I am trying to show lncRNA (long noncoding RNA) depletion at the RNA level after using pool of siRNAs or single siRNAs. I designed different primers spanning the locus as well as using Taqman assay, but nothing worked. Primers are OK, Ct around 27-30. Positive control for transfection worked nicely giving 90% of knock down. Any ideas?
thanks
Hi Lovorka
Sometimes, knockdown of lincRNAs is not efficient since those molecules are mainly nuclear and the RNAi machinery is not working properly at the nucleus. So, this is not surprising to me.
There is an alternative way to do knock-down of lincRNAs at the nucleus by using a bunch of DNA oligos complementary to your lincRNA. Those oligos will trigger the RNAseH activity and induce the degradation of the lincRNA within the nucleus.
Look to this paper for more info.
http://mbio.asm.org/content/4/1/e00596-12.full
Hope it helps. Best
Paco
I am aware of that, but the problem is that I have a clear cell phenotype which makes me believe that I am depleting something but i am just not able to show it at the RNA level by qrtPCR. I am using dharmacon and ambion sIRNAs.so can not be target off effect.
confusing...
Hi again
You said that you have Cts from 27-30 for your lincRNA?... I suppose that those Cts do not change when compared with the controls transfected without RNAi oligos.
Really confusing... unless you are in fact knocking down something else which causes the phenotype.
yes correct! the Ct do not really change after siRNAs, which makes me wonder that I am depleting something else as you suggested.
This happened with 2 different lncRNAs and different siRNAs (Dharmacon and Ambion) targeting different regions. But beautiful phenotype!
Oh Gosh !... just forgot your lincRNA and study the phenotype... :)
For sure that you already align the sequences of the oligos with the whole genome... Maybe some ectopic effect.
Last crazy suggestion. Do you think that would be possible to test some Jurassic techniques like northern blot to check the expression?. For that you will need a pretty decent levels of lincRNA in a few ug of total RNA.
Hi Lovorka, sometimes RTqPCR gives strange results...as Francisco suggested perhaps Northern blot (NB) is better to confirm KD. Even you could do extraction of Cytoplasmic and Nuclear RNA plus Total RNA and see them by NB...for me it is not really "jurassic" technique ;), because you can really detect full length of RNAs, however by RTqPCR you only detecting a piece of your maybe very long RNA...well, whether if it is not possible to do NB, you can perform more "sexy and modern" technique such as FISH-RNA...Good Luck!
OK.. Jorge.. I mean "Jurassic" because is a the first technique that dinosaurs like me used to learn in their first steps in the molecular biology labs a long, long time ago.
I personally agree totally with you with the suggestion of the FISH... Normally it will give good results for lincRNAs. Great input !.
Dear Francisco, to me it was completely opposite!!...I grew with PCR techniques in molecular biology courses and just few years ago I started with NB...sometimes young people forget this kind of robust technique from old school...maybe dinosaurs are resurrecting!! :) :)
I have decided to go for RNA seq and RNA fish! There are so many isotope a of lncrna that maybe I am not catching the "reAl"one. Whhat is the current sequencing depth suggested for lncrna? 100 million reads?is that enough?thanks
Hi
100 million reads must be more than enough. I am getting good results with 50 million reads, but of course depends on the abundance of your golden lincRNA.
Tons of luck !. Best
Paco
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