siRNAs transfection in Keratinocytes
Mei Xu
1. Plate HFK cells into 6 well plate, O/N
2. When cells are 50% confluent and look healthy, do transfection as following:
Tube A: mix 45ul serum free medium and 5ul lipofectimin RNAiMax from
Invitrogen (Catalog Number: 13778-075)
Tube B: mix 140ul serum free medium and 10ul siRNAs (20uM)
Sit for 10 mins, and then mix up Tube A and B, let sit for another 20 mins
Add to the cells, mix well
3. 48 to 72 hours later, collect cell lysates for both mRNA and protein detection
Notes:
1) Each gene is different. It is better to check both mRNA and protein level for a
serial time points the first time. My data show that knockdown of Myc protein
level can be detected at 48 hours, and most other genes I have tested can be
detected at 72 hours.
2) Too much siRNAs and RNAiMax reagents show some cytotoxicity, it is better to
check cells the second day after transfection and decide whether changing
medium is needed (sometimes cells look healthy, sometimes they don’t, I don’t
know why). I also found that half of the reaction works for some siRNAs with less
cytotoxicity.
3) It works better to mix up several different siRNAs for one target gene, in
comparison to one siRNA.