How to know siRNA transfection efficiency? - (Feb/16/2006 )
Hi:
I´d like to use a siRNA in chondrocytes, a kind of fibroblastic cell. I´ve read two siRNA secuences that work well. I think that I will use a kit from Invitrogen to transfect my cells but I don´t know what is the best way to know the percentage of cells that have been transfected. Maybe I coud do a western against the protein product of the gene that I have silenced. What about a PCR of the silenced gene? Thank you!
I´d like to use a siRNA in chondrocytes, a kind of fibroblastic cell. I´ve read two siRNA secuences that work well. I think that I will use a kit from Invitrogen to transfect my cells but I don´t know what is the best way to know the percentage of cells that have been transfected. Maybe I coud do a western against the protein product of the gene that I have silenced. What about a PCR of the silenced gene? Thank you!
The easiest way to correlate transfection efficiency is to co-transfect the siRNA with GFP plasmid.
With transient tranfection, it is also possible to see protein level decreased on a western blot, but it really depends on the transfection efficiency and how effective the siRNA itself is in downregulating the protein. So, actually, it is not a good indication of transfection efficiency.
If your siRNA is as large as GFP, its a good idea to use GFP as a control, if it is larger you can use GFP only to control your method, not the efficiency...
The downregulation can be better controlled by Northern Blot then Western Blot! I transfected SH-SY5Y (a Neuroblastoma cell line) and have never seen a effect in Western but in Northern Blot!
Good Luck
Anja
The downregulation can be better controlled by Northern Blot then Western Blot! I transfected SH-SY5Y (a Neuroblastoma cell line) and have never seen a effect in Western but in Northern Blot!
Good Luck
Anja
so siRNA effects can be only limited to Northen blot and cannot be seen on Western?
would that still be enough to prove the si is working?
there is some control-siRNA that is coupled to some fluorophore (I use the one from qiagen)....that is good for evaluating the transfection efficiency.....
the most "straight" way to proove, that siRNA is working would be northernblot or Realtime PCR (or semi-quantitative PCR....it is easy, cheap....and you should expect at least reductionof 50-80%....so it should be easily detectable...I do RT/Northern)
Protein effect is great in addition to RNA-Level.....but I would think it should be done additionally