co transfection plasmid and siRNA - message from gmgabriel68 (Sep/23/2007 )
Hi !
I’m co-transfecting a transcriptional plasmid encoding for a gene report and the siRNA targeting the mRNA of reporter gene, and a second plasmid which is supposed to revert the silencing of the reporter gene.
The amount of plasmid is filled up to 2ug and the transfectant reagent is Lipofectamine 2000 (10 ul) in a 6 well format plate.
Q1: have you done an experiment of co-tranfection plasmid and siRNA?
Q2: Do you track the efficiency of the co delivery transfecting at the same time a plasmid encoding for a second reporter gene (lacZ) and one siRNA havingCY3, in order to check both pDNA-siRNA delivery?
I added this controls and the system does not work well anymore.
Q3: Am I saturating the cationic lipid ?
Adding the plasmid control (50ng) and Cy3 (60 pmoles), a complete silencing of the reporter gene has been observed from 75 to 2.5nM.
Q4: Did anyone try to use less than 2.5 nM to knockdown the expression of a gene encoded from a plasmid ?
best,
gmg68
I’m co-transfecting a transcriptional plasmid encoding for a gene report and the siRNA targeting the mRNA of reporter gene, and a second plasmid which is supposed to revert the silencing of the reporter gene.
The amount of plasmid is filled up to 2ug and the transfectant reagent is Lipofectamine 2000 (10 ul) in a 6 well format plate.
Q1: have you done an experiment of co-tranfection plasmid and siRNA?
Q2: Do you track the efficiency of the co delivery transfecting at the same time a plasmid encoding for a second reporter gene (lacZ) and one siRNA havingCY3, in order to check both pDNA-siRNA delivery?
I added this controls and the system does not work well anymore.
Q3: Am I saturating the cationic lipid ?
Adding the plasmid control (50ng) and Cy3 (60 pmoles), a complete silencing of the reporter gene has been observed from 75 to 2.5nM.
Q4: Did anyone try to use less than 2.5 nM to knockdown the expression of a gene encoded from a plasmid ?
best,
gmg68
I have done slightly differently.
A1: yes, all the time, no problem.
A2: I did with an EGFP cnstruct for quick check gene transfection rate and siRNA delivery rate. SiRNA delivery is always near 100%, while gene transfection rate is cell type dependent. When you do these type of experiments, keep total DNA (+siRNA) dosages and DNA to lipid ratio constant. Sometime a third or forth plasmid is introduced to keep these parameter the same for all experiment groups.
A3: 1:5 ratio seems to be fine.
A4: It depends on what type of siRNA do you use. If a longer siRNA (27 mer) is used, you can get down to 0.5-1 nM concentration.