HUVEC siRNA transfection

We have tried a number of transfection reagents for HUVEC and GeneFECTOR works the best for transfecting siRNA oligonucleotides.  And it is cheap.  We have seen some batch-to-batch variation with this reagent that seems to correspond to the reagent concentration being a little higher or lower than it is supposed to be.  If you see toxicity, then there is probably too much reagent and you need to drop the concentration.  This protocol gives 100% transfection – i.e. with a good siRNA you can see 100% silencing by western blotting.

Materials

Opti-MEM (Life Technologies)
GeneFECTOR (Venn Nova)
siRNA stock at 20µM

Transfection

  1. Plate cells the day before transfection.
  2. The next day, wash the cells x3 with OptiMEM and leave in half the normal volume (i.e. 1ml for a 6-well plate).
  3. Set up two tubes for each transfection at room temperature, according to the table below.  Do not vortex.
  4. Add Tube A to Tube B by gentle pipetting.  Leave for 5 min at room temperature.
  5. Add the mixture dropwise to the plate while swirling.
  6. Incubate at 37 °C for 3 h.
  7. Change the medium back into normal HUVEC medium and leave for 48 h.
TUBE A TUBE B
Dish OptiMEM (µl) siRNA (µl) OptiMEM (µl) GeneFECTOR (µl)
12-well
48.5
1.5
47
3
6-well
97
3
94
6
6 cm
242.5
7.5
235
15
10 cm
485
15
470
30

 

 

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