Hi all, I am trying to clone a big 3'UTR(~3kb) of a gene that is predicted to be a target for my miRNA of interest. When I scan through the 3'UTR sequence I retrieve from Ensemble, I can locate within it (and most importantly, before the predicted miRNA binding site) several polyA signals. Do you think this is a problem?
Are these used in vivo? There are many false calls when scanning through the database, but if there are legitimate polyA sites present and mRNA variants called, they may indeed be real. That said, polyA site seems to vary on cell context (see for example Sandberg et al., Science. 2008 Jun 20;320(5883):1643-7) you can assay this by transfecting your construct and analyzing the RNA (see Licatalosi et al., Nature 2008 from our lab). This is probably physiologically important (i.e. the miRNA site may be regulated in part by either being there or not, via alternative polyadenylation. For the assay, if the sites are used, you can mutate them...
Are these used in vivo? There are many false calls when scanning through the database, but if there are legitimate polyA sites present and mRNA variants called, they may indeed be real. That said, polyA site seems to vary on cell context (see for example Sandberg et al., Science. 2008 Jun 20;320(5883):1643-7) you can assay this by transfecting your construct and analyzing the RNA (see Licatalosi et al., Nature 2008 from our lab). This is probably physiologically important (i.e. the miRNA site may be regulated in part by either being there or not, via alternative polyadenylation. For the assay, if the sites are used, you can mutate them...
hi robert, thanks for your answer. these are to be used for in vitro luciferase assays. i am indeed making a mutated (in the appropriate seed seq) construct for every 3UTR i will use. i will check out the papers you mention, thank you again!
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