I have been trying to transfect MIA PACA 2 using lipofectamine 2000. However, the real time PCR results (Ct values) are the same for the control and the experiment.
Lipofectamine 2000 transfection definitely works for PACA 2 cells.
However, failure is possibly due to
1. Either siRNAs were not functional towards the specific gene.
or 2. Transfection technique problem. ( Try overexpression and perform WB to confirm your transfection is successful.)
Repeat the knockdown and do WB to confirm siRNA works. Ideally, we order 4 siRNAs and select the best 2 for further use.
I recommend whatever suggestions given by Ms Kousalya and read the following article which I found in Thermofisher scientific website
https://www.thermofisher.com/in/en/home/brands/product-brand/lipofectamine/lipofectamine-rnaimx.html?gclid=CjwKCAiAgvKQBhBbEiwAaPQw3EK9oUMmFhhimGZkrRDbyr72EqocndJMA3UBR7kUr5ZQVhMcn03YvhoCi0cQAvD_BwE&ef_id=CjwKCAiAgvKQBhBbEiwAaPQw3EK9oUMmFhhimGZkrRDbyr72EqocndJMA3UBR7kUr5ZQVhMcn03YvhoCi0cQAvD_BwE:G:s&s_kwcid=AL!3652!3!552917785801!p!!g!!sirna%20transfection&cid=bid_clb_tfx_r01_co_cp0000_pjt0000_bid00000_0se_gaw_nt_pur_con
I agree. In my opinion Lipofectamin(R)2000 should work.
First, I would propose to determine transfection efficacy with small RNAs (maybe with a Cy5-dye).
2 sources of uncertainty are reasonable: a) inhibitory efficacy of the siRNA (possibly not optimal in this case), b) the experimentator (check and revise the protocoll and performance, routine will help to optimize performance and outcome).
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