siRNA transfection in MLE12 cells - (Jun/23/2008 )
I am having problems transfecting an siRNA into a murine alveolar epithelial type II MLE12 cells using Lipofectamine. I have tried to increase the amount of Lipofectamine and siRNA, independently. Does anyone have any ideas?
Never used this cell line. Do you have a labeled siRNA to control for transfection efficiency?
You may have done the following, but well, here it is..
1. You should call various company tech reps to find out if their company's transfection reagent work with this cell line.
2. Check literature to see what people have used in the past for this cell line.
3. If never done, I would borrow or ask for samples of various transfection reagents from neighbour labs/comanies to test many.
4. Dharmacon RNAi oligos can be tested with 4 diff. reagents they used to sell as a test kit.
5 as pcrman: use labeled RNAi oligos as transfection control.
I don't use MLE12s, but MLE15s and found that I was able to get around 80% knockdown using Dharmafect 1 reagent with siRNA at 100nM. I just made sure that the cells were about 50-70% confluent and well spaced to maximize transfection efficiency. I transfect for 24 hours, then add fresh media for another 24 hours (48 hrs total) and my protein of interest was down.