I am attempting to knockdown a protein in HeLa cells and observe its effect on another protein that I am transfecting into the cells. I am attempting to knockdown (KD) a protein (clathrin) in HeLa cells by transfecting them with siRNA oligos and oligofectamine. I am waiting 48 hours following the siRNA transfection for the KD to take effect and then attempting to transfect the cells again using Lipofectamine 2000 with a plasmid containing cDNA for my protein of interest. My KD seems to be working, as verified by western blots, but I am unable to transfect enough of my protein into the cells as well. This lack of expression has been verified through microscopy and western blots. Is there a better way that I can transfect siRNA oligos and cDNA plasmids?
Transfection using lipofectamine may require endocytosis. Since you are knocking down clathrin, perhaps you are effectively blocking your transfection mechanism. Perhaps try a different transfection method such as electroporation/nucleofection.
http://www.thermofisher.com/uk/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/cationic-lipid-mediated-delivery/how-cationic-lipid-mediated-transfection-works.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2975437/
I normally have no problem transfecting cDNA into cells. My problem is getting BOTH the siRNA and cDNA into the cells and functioning simultaneously.
Transfection using lipofectamine may require endocytosis. Since you are knocking down clathrin, perhaps you are effectively blocking your transfection mechanism. Perhaps try a different transfection method such as electroporation/nucleofection.
http://www.thermofisher.com/uk/en/home/references/gibco-cell-culture-basics/transfection-basics/gene-delivery-technologies/cationic-lipid-mediated-delivery/how-cationic-lipid-mediated-transfection-works.html
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2975437/
Maybe you should try whit another method for the second round. For example lentiviral transduction.
I have no problem transfecting siRNA first and then 24h later cDNA . But I use RNAiMAX to transfect the siRNA and Lipofectamine2000 for the cDNA.
I found that very little RNAiMAX is needed to transfect the siRNA (I use 1ul of RNAiMAX to transfect 20 uMoles of siRNA) maybe you can use less transfection reagent for the first transfection and then the second transfection is gonna work better.
Try changing the second transfection method.
I agree with Alex. Most of the transfection is clathrin-mediated endocytosis based process. Since you knock down clathrin, the cellular machinery is compromised and hence no effect for the second transfection. Hence, you may try co-transfection with Lipofectamine 3000 (this works for siRNA as well pDNA).
In case you need stepwise transfection (first with siRNA then with pDNA to evaluate the effect of siRNA-mediated knockdown with your (protein) cDNA plasmid), then I suggest to use CaCl2 mediated or magnetofection for the cDNA plasmid.
Another possible way is to use a puromycin based siRNA expression lentivector along with Dox ineducable lentivector (for your protein of interest (cDNA plasmid)). The selection of puromycin followed by Dox induction will serve your purpose I guess.
Note: Have you confirmed the cDNA orientation integrity of the protein of interest in your plasmid (by sequencing) and their expression pattern in HeLa?
Have you tried transfecting the cDNA first, followed by siRNA transfection?
I think cDNA transfection and then expression can be challenging depending on the construct, while siRNA transfection is usually more straight forwards.
As transient transfection can difficultly reach 100% cell expression, you may also generate a stable clone of your cDNA transfection if your vector contain ori and appropriate antibiotic selection.
I also think lipo3000 is better than 2000 in the forward plasmid DNA transfection.
Good luck!
- Badges
- Science method