I've recently been performing shRNA transfections and was hoping to get some advice on why I'm not seeing any knockdown via western blot
I transfected by MDAMB468 breast cancer cell lines with shRNA against PTEN from the pLKO.1 lentiviral vector. I selected for the transfected cells using 1ug/ml puromycin for approximately 6 days. During this time there was very few cell death amongst the cells compared to my control plate (untransfected cells with 1ug/ml puromycin), suggesting that the transfection was successful.
After the 6 days I harvested my cells for protein and RNA extraction. I performed a western yesterday to assess knockdown, but noticed that there was hardly any differences (if any!) compared to my shGFP control. I haven't done a qpcr yet but I plan to this week.
I was wondering if anyone has experienced something similar, especially when working with PTEN? could it be that the cells need to be selected for a longer period (i.e. more than a week) before assessing protein knockdown?