Dharmacon anti-miR help - (Jan/28/2011 )
I am trying to knock-down a particular miR in a mouse mammary epithelial cell line using lipofectamine 2000, assaying after 48hrs with little success.
I have used both 50 and 100nM of anti-miR which killed cells both in the negative control and the miR inhibitor wells and caused an increase in miR expression (potentially through feedback loop or just by killing all Tx'd cells).
I have also used 5nm, 10nm and 20nM of anti-miR which had no change in cell viability however also did not decrease the expression of the miRNA.
I am using the Qiagen miScript system to assay miR expression which we have validated in our lab and has given similar results to TaqMan probes.
Does anyone have any experience with Dharmacon anti-miRs and can suggest what I should try next? Any suggestions would be great appreciated,
P.S. the endogenous expression of the miR is relatively normal in these cells (Ct ~ 25-30)
You can try designing siRNA to target the loop sequence of the precursor miRNA. It works well sometimes.
Hi, have you tried looking at the expression level changes in your miRNA target proteins by Western Blot? Sometimes RT-PCR doesn't work for this kind of assays, probably due to dissociation of the probe and miRNA during PCR... I have been working with APL cell lines and trying to knockdown miRNAs using LNA oligos, but no luck, it's been a year we're switching to a lentiviral system.
Pcrman your recommendation sounds interesting, I'd like to try something like that. Can you recommend a paper for it? Thanks!