siRNA transfection - (Mar/31/2008 )
Hello ,
I have problem with siRNA tranfection into HUVEC and HASMC, I used Lipofectamin 2000 from invitrogen .The conc of my siRNA was 20um and I have used 25ul ,50 ul 2.5 ul with lipofectamine amount 1ul ,2ul ,3ul and 5ul .I could not get any significant reduction in mRNA .Could anyone has idea for these kinds of cells and any thranfection reagent is good for such purpose.
Ela
I have problem with siRNA tranfection into HUVEC and HASMC, I used Lipofectamin 2000 from invitrogen .The conc of my siRNA was 20um and I have used 25ul ,50 ul 2.5 ul with lipofectamine amount 1ul ,2ul ,3ul and 5ul .I could not get any significant reduction in mRNA .Could anyone has idea for these kinds of cells and any thranfection reagent is good for such purpose.
Ela
Lipo2000 should give you 20-30% transfection if you use a reporter gene, like EGFP. No experience with siRNA on these cells. The key is, how does positive control siRNA go? I hope you used one and so you can figure out what went wrong.
Thanks a lot, there is another problem with HUVEC because I could not transfect the cells .I have used to transfect in OptiMUM but the cells started due 5-6 hrs and changed 2 hr post-transfection.When I transfected in complete medium , the aggregation form 6hrs postransfection .I have done one experiement ,I transfected in complete medium and 4 hrs later I changed the medium into copmlete medium again but nothing happen .
Do this way: dilute DNA and lipo2000 separately in serum free OPti-MEM. incubate 20 min, mix both, incubate 10 min, add to cells, then add one volume of 10% FBS-Opti-MEM for 4-12 hrs. Change medium afterwards. check cells in 24 hrs-36 hrs. good luck
Thanks , I want to know after the mixture add to the cells , howlong shall I wait before add one volume 10% serum to Optimum or I add 10% serum Optimum direct after adding a mixture.
Ela