Would shRNA be less efficient (as it's processed by cellular machinery and may therefore end up in the cytoplasm rather than nucleus, unlike siRNA which is blasted into the cell and so more likely to end up in the nucleus)?
We're interested in non-coding RNAs acting at the level of transcriptional regulation, so their knockdown has to be efficient.
More info:
Just to make it harder, I'd like to knock these ncRNAs down in primary T cells rather than cell lines.
Thanks again in advance
Hi Catherine, i have cloned shRNA into a lentiviral vector against non coding RNA using the protocol available from addgene. http://www.addgene.org/pgvec1?f=c&cmd=showcol&colid=170&page=2#oligos
I have used it on cell lines on ncRNA that are expressed the cytosol. I have had no luck using this method for nuclear specific ncRNA. So i am not sure if it will be useful for you if your ncRNA is primarily in the nucleus.
Susan
Yes, I used both and I found that in some cases (majority) the maximum KD was at 24-30 hs and the expression was regained in full at 48 hs. Different to what you would expect with protein coding RNAs. I'd recommend to test a few time points to determine the time at which you get the maximum KD. Cheers
We did in vivo
please see
E. González-González, P. P. López-Casas and J. del Mazo “Gene silencing by RNAi in mouse Sertoli cells”. Reproductive Biology and Endocrinology. 6:29 (BioMed Central) doi:10.1186/1477-7827-6-29 (2008). http://www.rbej.com/content/6/1/29
I have already knock-down ncRNA in nucleus with both siRNA and shRNA. Stable transfection of shRNA induce a long-term effect even if is less potent (in term of inhibition) than transient transfection with siRNA. I have used the same sequence in the two methods (si vs shRNA). Transfection of primary non adherent cell enhance the complexity of your project. Read Berteaux et al, 2008 (use of siRNA), sh RNA of the same gene are not published.
PLKO lentiviral shRNA vectors works well. Please see Loewer et al. Nature Genetics
http://www.ncbi.nlm.nih.gov/pubmed/21057500
The knockdown of noncoding RNAs (whether by shRNA or siRNA) may be heavily impacted by (a) its localization in the cell (if its nuclear, whereas the RNAi machinery is cytoplasmic, knockdown will be more difficult) (b) the degree to which si/shRNA recognition sites on the ncRNA are unavailable; either by tertiary structures or being DNA- or protein-bound and (c) whether a single si/shRNA can induce a sufficient level of degradation to fully inhibit the ncRNA (are functional domains remaining intact).
On another note, siRNAs do not get into the nucleus with standard transfection techniques. Even if they did, RISC and other necessary components for RNAi are primarily cytoplasmic.
Hi everyone!
I really feel that this question might be common to all those who are working on ncRNA. Considering Lousie's point I would rather look into the possibilities of trying out LNA or a chemically modified RNA aptamer. Whether nuclear or cytoplasmic RNA we have a higher chance of knockdown.
Please comment if you find the argument reasonable.
Best Regards,
Gaurab
To underline the points Louise mentioned regarding the efficient knockdown of lncRNA it maybe worth to take a look at the following articles as well:
RNA 15: 1797-1804,2009 (http://rnajournal.cshlp.org/content/15/10/1797.long)
Developmental Cell 19, 625–638, 2010 (http://www.ncbi.nlm.nih.gov/pubmed/?term=20951352)
best wishes
Jörg
si/shRNA don't work very well (or at all) for nuclear/chromatin bound lncRNAs. There are new RNA-specific CRISPR now but I would use modified LNAs. Message me for details. a
This blogpost of techniques to disrupt lncRNA function with siPOOLs (RNAi), antisense oligos and CRISPR might help http://blog.sitoolsbiotech.com/2018/02/disrupting-lncrna-function/
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