transfecting cells with mature miRNA or precursor miRNAs? - (Sep/11/2010 )

hey fellow miRNAists

as a part of my thesis, I have to transfect several eukaryotic cell lines with hs-miRNA 143 and 124. although I am still in the very beginning steps and trying to construct what is needed to proceed to the next level, I actually cant decide which one is more effective ,and of course, cost-effective; mature miRNAs or precursors. so, I though it would be grate to ask you, people with much much more experience.

thank you

mahsa on Sat Sep 11 12:44:44 2010 said:

Hi,
if you want a good number of mature and functional miRNAs in your cells be sure to transfect a construct with the precursor. transfecting ssRNA of mature miRNA will lead to nothing and is less "physiological". you can purchase expression vectors by a number of companies and choose between viral and plasmid based systems. just make sure to use a scrambled sequence expression vector as a negative control
Fiz

Fizban on Mon Sep 13 07:46:09 2010 said:

hey Fiz, thanks for the respopse.
do you have any experience with Ambion Pre-miR™ miRNA Starter Kit?
all the luck
Mahsa

mahsa on Tue Sep 14 13:01:52 2010 said:

sorry but no. i made my overexpressing constructs myself. Ambion is a very good company anyway but nowadays there are a lot of companies selling overexpressing constructs. you just have to decide if u want to use mimicks (ds hairpins to be matured by the cells) or plasmid/viral constructs harbouring the precursor sequence.
you could also try ADDGENE, a no profit company selling constructs made available by research laboratories. they sell what they have for very low prices.
bye
Fiz

Fizban on Wed Sep 15 12:51:48 2010 said:

thanks for the information
good luck
Mahsa

I'm not sure from your description what exactly you mean with "mature" versus "pre-miRNA". If you overexpress them from a plasmid they will always be double stranded to begin with. In this case use pre-miRNAs as they are more readily processed by Drosha and Dicer. Take like 50 basepairs upstream and downstream of the hairpin into your construct. This of course can only be done from a polymerase II promotor.
If you plan on using polymerase III promotors like U6 or H1 for higher expression levels you just have to take the hairpin but make sure your cells are not sensitive to the Exportin-5 block that can occur when having these snRNA levels.
Transfection of mature miRNAs (of course the double stranded ones again) from Ambion will be more effective in terms of knockdown with the same molarity than plasmid transfection with polymerase II promotors. Since there is some loss from transcription, processing, export.

So it really depends how strong your effect has to be or how physiological the setting should be. This depends on the cell sensitivity too. U6/H1 will provide very strong effects but potential toxicity from exportin-5 competition with endogenous miRNA and usually not very good processing efficiencies so you end up with quite some hairpin loop that is not cut into the miRNA or exported. Polymerase II will have good to excellent processing efficiency, a pretty physiological level of expression that seems to not compete with the endogenous miRNA pathways - but the knockdown effects will be lower as less miRNA is produced to begin with.