Hi everyone,
I am about to work with gene silencing using plasmid shRNA that I bought from Santa Cruz Biotech. I am planning to do preliminary testing on different concentration of transfection reagent and different incubation time. There are few question I would like to ask:
1. I am thinking of transfect the esophageal cancer cell with selected shRNA of interest gene. May I know if it is ok if I transfect the cell then subculture to multiply the transfected cell? Does this method is stable for plasmid shRNA because some people suggest to use lentivirus shRNA.
2. Since Im going to subculture the transfected cell, does tripsinize the cell for next assay will affected the stability of transfected cell and the transfection efficiency?
3. If we just have a tiny amount of shRNA, how can we make sure there is enough shRNA throughout the entirety of our study?
4. Throughout my reading, qRT PCR is the best way to check the successfull of gene knockdown. Is there any other way to assess the efficiency of silencing?
I am also open and looking forward for any extra tips, do's and dont's and successful technique to conduct shRNA gene silencing. Thanks in advance. Best regards Nur Syafiqah Rahim
1. If you are using the electroporation method, then it is Ok to do subculture, but if you are using chemical reagents like Lipofectamine or any liposome-based product, then kindly perform subculturing after 72 hours. The lentivirus method also goes well with the knockdown experiment, but make sure you calculate the viral load before transfection. I will also suggest if you are using any reagent, kindly stick to the protocol mentioned by the manufacturer.
2. No trypsinization will not affect your gene downregulation if cells are completely transfected with your knockdown vector. So vector selection is also essential. We work with vectors that have puromycin resistance like PLKO, which helps in the selection of those cells which have knockdown of selected genes.
3. If you have a tiny amount of shRNA, then I will suggest you do cloning of that shRNA in PLKO.1TRC vector, which is available in addgene then you transfect that vector inside your cancer cells.
4. Yes, you are right qRT PCR is the best method to check, but I will also suggest you do western blotting of your specific gene. Because we know shRNA works at the RNA level, and we observed around 60-90% knockdown of the particular gene but many times, it has been observed that knockdown is present inside the cells, but the stability of the proteins also gets affected, which delays the proteasomal degradation and enhanced its stability.
Dear Sakat Patel,
Thank you so much for your thorough reply. When you say in response to question 1 that subculturing should be done after 72 hours, do you mean the incubation period for transfections?
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