I am working with glioblastoma U-87 cell lines.
I have pCMV-miR expression vector ~8kb.
I tired both Transfast, Fugene HD, PEI, Jetprime. They had max %30 efficiency.
Transfast best with 1:1 ratio and Fugene HD is best with 1:3 ratio. Jetprime and PEI does not works well.
Unfortunatelly, even with Fugene and Transfast I could not react over %80 efficiency with passage 11 cells.
I use opti-mem during transfection for transfast and incubate them all at 15 minutes.
I have endo-free plasmid with 1.8 purity.
I need to slience a gene with microRNA but this efficiency is not enough.
Would you please suggest me any advice to improve efficiency.?
Have you tried using a U87-specific transfection reagent? If you want to have really high efficiency, especially for glioblastoma cells, then you need to have cell markers and other factors present in the transfection reagent for all cells to get transfected. Check this kit (https://altogen.com/product/u87-transfection-reagent-glioblastoma-cells/) and see if it works for you - it should give at least 80% transfection efficiency for siRNA, while with a complex condenser it should also give high transfection efficiency with plasmids.
Looking at your application.... I suggest to clone your mir sequece inside a lentivirus vector and make high titer virus ... Then you can transduce your cells to give you better efficiency of knockdown.....
Is not possible to silence your gene without using a plasmid vector?
If you can directly transfect siRNA or miRNA, our Viromer BLUE can give you a high efficiency.
https://viromer-transfection.com/wp-content/uploads/2015/09/U87-transfection-viromer.pdf
Mirna mimic may down regulate genes which are not real target of mirnas due to high transfection. The more, I am specially looking for u87 cell lines. My mirna's biogenesis may be blocked in this cell line. So mimic wont give me the real results.
Hi Enes,
Actually I think 80% is very good efficiency in transfection however, I try to help you. I think if you change the media about 1-3 hr before starting your transfection, it helps. Change media with OptiMEM. Then about 3-6 hours after transfection you'd better to change the media with normal complete media.
Also, I think it could be very helpful if you do co-transfection. I'm not sure about your experiment but sometimes if you do it with a protein which enhances the biogenesis of that special smallRNA or whatever, you'll get better transfection.
Hope this helps.
Ah thanks for your answer and please forgive me for my bad english. I tried to say that " I could not reach over %80 efficiency " in other words; I try to reach this efficiency but could not reached yet. To see slience effect of miRNA I need this ratio but I reached max %30.
I performed transfection in also opti-mem. Result was the same
Have you tried using a U87-specific transfection reagent? If you want to have really high efficiency, especially for glioblastoma cells, then you need to have cell markers and other factors present in the transfection reagent for all cells to get transfected. Check this kit (https://altogen.com/product/u87-transfection-reagent-glioblastoma-cells/) and see if it works for you - it should give at least 80% transfection efficiency for siRNA, while with a complex condenser it should also give high transfection efficiency with plasmids.
- Badges
- Science method