nonreproducible knockdown efficiency with Plemir-micorRNA from Open Biosystem - (Sep/14/2010 )
I have tried to overexpress the interested microRNAs using the plemir vector from Open Biosystem. I did see very good knockdown (at least 70%) of at least one target for each MicroRNA by western blot. But the problem is that the knockdown at the protein level is not reproducible at all even the same cell line was infected with the microRNA virus. I have been seeing this inconsistent western blot result for at least 2 different micrRNA constructs.
Does any of you has the same experience when you using Plemir from Open Biosystem?
Appreciate your suggestions and advice.
Were the repeats done with same infected cells or cells infected at a different time? Are you sure the infection efficiency is the same and miRNA overexpression is the same?
pcrman on Sun Sep 19 05:56:41 2010 said:
Were the repeats done with same infected cells or cells infected at a different time? Are you sure the infection efficiency is the same and miRNA overexpression is the same?
The same cell lines were infected at a different time by the same virus, which was also made at different time. I am not sure the infection efficiency is the same but it's always at least over 95% since there's RFP within the construct.
Right now I suspect that the virus titer may affect the results since each microrna targets so many genes and the knockdown efficiency may vary each time based on the accessibility of mature microRNA, and even the process efficiency of mature microRNA from the precursor.
It's seems that the only choice is try to increase the virus titer though I don't know it's right or not.
What I don't understand first hand about the Biosystems constructs ... if the miRNA is processed the polyA tail is removed from the mRNA upstream of the miRNA, correct?
Than ... how on earth is there any translation of the tRFP CDS possible if the miRNA is processed? Wouldn't a tRFP expression appear in cases of no miRNA processing? Meaning the more tRFP expression you have the less miRNA processing? If I understand the pLemir schematics correctly only the Puromycin resistance has a positive correlation with the miRNA expression - but not necessarily the miRNA PROCESSING.