pri-miRNA transcript characterization
Posted 08 March 2011 - 01:15 AM
I'm looking for a way to characterize the pri-miRNA of a miRNA. The problem is that we just discover this miRNA and don't know where the pri-miRNA starts.
Anyone knows how to do such a thing ?
Posted 08 March 2011 - 12:55 PM
Posted 09 March 2011 - 03:38 AM
Since it is pretty hard to detect precursor miRNA, if you have cloned and know the mature miRNA sequence, you should blast the genome to find where it is derived, then use the sequence from that region to predict secondary structure of RNA to see if stem-loop structure can be formed by the sequence.
I already did that (prediction with miRDeep ans sRNAloop and experimentaly by custom stemloop RT-PCR )
Now we want to know the pri-miRNA (not the pre-miRNA)
Posted 07 December 2011 - 10:49 PM
as i already mentioned in another post the best way is race or primer extension method.i have tried race it works well for other genes.but about my precursor i have not been able to either clone it or do a race.it seems that when a precursor expresses lowly these methods do not work well or my protocol for these methods is wrong.to prove that this sequence is a real precursor i cloned it and overexpressed it in cell line and i did pcr with a primer overlapped with probable mature form,the pcr product was cloned and i saw that the ends of pcr product exactly is one nucleotide after the predicted mature region indicating that this sequence is processed but it was not acceptable by my tutor.he says that we should clone endogenous precursor
i appreciate any idea about this issue
the topic in this forum if expanded might be helpful for many