I was trying to do a knockdown using RNAiMax and siRNA and examine its effects on EGFR pathway in MDA-MB-468 breast cancer cell line (L15 media, Air incubation) on day 1, 2 and 3 after transfection. However, I found the p-EGFR (Y1068) and total EGFR levels in my control (scrambled RNA) significantly decreased on day 2 and almost undetectable on day 3.
I tried to minimize final cell density difference so I seeded decreased cell number for longer time point. The cells are not over confluency over the majority area of the well except some on the well-edges. I used reverse transfection (knockdown and seed at the same time) and didn't change media at day 2/3 because I was concerned about knockdown efficiency.
I'm wondering do anyone has similar experience and how could you maintain the EGFR activation over the period of experiment? Thank you so much!
Did you test that your levels of p-EGFR and total EGFR were indeed non-existent in KD cells in comparison to control cells?
Is it possible that the medium, where cells are cultivated after transfection, contains by any means EGF that could activate receptor mediated degradation of your EGFR receptor? This could explain your results.
Thanks Jaime for your answer.
To clarify, I have three control negative siRNA samples harvested on day1, 2and 3. The pEGFR and total EGFR are still detectable on day2/3, but much weaker compared to day1. I didn't have a control without siRNA or RNAiMax.
You should have a control without transfection and consider changing the medium of culturing after transfection
Thanks Brian and Jamie for your suggestions.
I have tried a transfection with or without negative siRNA and/or RNAiMax. To my great surprise, my negative siRNA transfection sample (compared to RNAiMax only) reduced my pEGFR/total EGFR significantly. I guess this will explain what I have observed earlier.
Thank you again.
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