miRNA overexpression troubleshooting - (Aug/20/2013 )

 Hi guys

I'm dazzled over sth seemed to be able to do that less than one month but now it's more than three months and I cant reach to a conclusion. Any idea or tested protocol is greatly appreciated. I have some repeatable extremely significant data of Luciferase assay due to over expression of miRNA(Ambion pre-miR and 3UTR vector of its target).

But when I transfect cells(N2A) with different concentrations of miR,each time it produces a completely different result! two times a bit downregulating the target but the other two times up regulating the target a bit.

I have checked the expression of protein in the nontransfected cells and it is really well expressed. I even switched to using plasmids expressing pre-miR (having GFP reporter) but the number of GF^positive were too much low(transfecting premiRwith lipofectamin here is the details)

seeding 250000 cells per well 24hrs before transfection using 50,70,100umolar concent. of oligo

transfecting exactly as Invitrogen protocol scales adjusted for a 6well cell plate(cells are 50-60% confluent if not I wait until they reach to this confluency)

changing media between 12-24hrs after transfection.extraction of protein after 24 and 48hrs

(washing cells with cold PBS and all procedures on ice using ripa  as chemical method of lysis)

plz share ur ideas and experiences while working with N2A mouse cells not only  if in the context described above

thanks

Hi gingerx, welcome to bioforum. 

Just to clarify a bit that the so called premiRs sold by Ambion are actually miR mimics. Do you know the miR transfection efficiency with N2A cells by including a fluorescence labeled control mimics? If you have never transfected RNA oligos into the cells you should buy the control also from Ambion to get a sense how well the oligos enter the cells.

>>But when I transfect cells(N2A) with different concentrations of miR,each time it produces a completely different result! two times a bit downregulating the target but the other two times up regulating the target a bit.

By target, do you mean the vector encoded target or endogenous gene? Did you measure endogenous target expression? If you cotransfect miRNA mimics with plasmid, how exactly does the protocol look like? Did you also include controls for transfection efficiency of the plasmid such as renilla luciferase plasmid? 

pcrman on Wed Aug 21 03:15:17 2013 said:

Hi gingerx, welcome to bioforum. 

 

Just to clarify a bit that the so called premiRs sold by Ambion are actually miR mimics. Do you know the miR transfection efficiency with N2A cells by including a fluorescence labeled control mimics? If you have never transfected RNA oligos into the cells you should buy the control also from Ambion to get a sense how well the oligos enter the cells.

 

>>But when I transfect cells(N2A) with different concentrations of miR,each time it produces a completely different result! two times a bit downregulating the target but the other two times up regulating the target a bit.

By target, do you mean the vector encoded target or endogenous gene? Did you measure endogenous target expression? If you cotransfect miRNA mimics with plasmid, how exactly does the protocol look like? Did you also include controls for transfection efficiency of the plasmid such as renilla luciferase plasmid? 

I meant endogenously expressed protein supposed to be target sorry if I was not clear

I had renilla ctrl in my luciferase assay(co transfecting miR plus UTR plasmid). this luciferase assay always works great! Thanks for your suggestion I should've ordered a ctrl from ambion really earlier but I didn't think the whole procedure comes out so tricky!