I tried to synthesise an artificial miRNA precursor using T7 transcription (I cloned the sequence of my pre-miRNA with a T7 promoter sequence). I ran the RNA on a gel, but I see a smear for the double stranded structure (the one that is hybridised and not denatured), and for the single-stranded structure (denatured at 95°C) I see a clear band (but still a smear in the background). I am planning to inject in insects these pre-amiRNAs as we usually do with dsRNA.
Have you ever tried to synthesise a pre-miRNA (mine is 174-bp long), and if yes, how did you do? How did it look on a gel?
Thanks in advance for your precious help ;-)
Hi,
I use to synthetize RNA using T7 promoter for other things than miRNA and in the gel I have the correct band and lower of it I have the truncated transcripts that form the smear in agarose gel. After injecting the resulting transcript without purification to Xenopus oocytes I always obtained a functional protein. In your case I think the only problem you are facing is the estimation of the right amount of the complete transcript to inject. The smear if it’s lower than your expected band will not cause any problem
I hope this will help
I use T7 normally to generate RNA probes for in situ hybridisation. Important point is to linearise your plasmid before in vitro transcription. You should see a clear band (which might appear on agarose gel a bit smaller then expected) 10-fold the intensity of the plasmid input. If not your enzyme is lazy (try a different brand of T7 polymerase) or you have an RNase contamination.
we did an in vitro pre-miRNA processing assay from synthetic P32-labeled pre-let-7. Check following paper, figure 3 to see how it looks on the gel.
http://www.ncbi.nlm.nih.gov/pubmed/17167072
I can provide more detailed protocol of the procedure if needed.
In a non-denaturing RNA gel, the smears are likely due to inter-molecular binding of arms of the pre-miRNA resulting in aggregates of varying molecular weights.
The intended hairpin istructure (with intra-molecular hybridization of the 5p and 3p arms) is favored to form at lower concentrations and with rapid annealing. Melt the aggregate at low concentration in an annealing buffer (94°C for 4 min) and quickly place the tube in ice. Then analyze using a non-denaturing RNA gel.
Many thanks for your precious help! I'll have a look at all this. I'll follow your advises and will let you know!!
Kay-Dietrich Wagner, thank you for your comment. Actually, I am not using the linearised plasmid as a template, but a fragment obtained after digestion of the plasmid. This fragment contains the T7 promoter and the pre-miRNA sequence. If ever I still have problems, I will definitely just linearise the plasmid as a template.
Can you give me the amount you use in a reaction (I regularly use 1 pmol of DNA in a 20 µL reaction, with the T7 megascript kit from Ambion).
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