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Transfecting Rh4 and Rh30 - (Jul/11/2008 )

I am trying to transfect some siRNA into human Rhabdomyoasrcoma cell line Rh4 and Rh30

siRNA used: GAPDH siRNA from ambion
reagent used: Lipofectamine 2000
6-well plate, 50% confluence with transfecting
before transfection, remove RPMI1640, wash twice with PBS (to make sure no antibiotics left)
add 1ml of Opti-MEM

Transfection mix: 5ul of Lipo2000 in 125ul Opti-MEM + siRNA in 125ul Opti-MEM (final conc: 25uM -100uM)

After 5hrs of transfection, I removed the siRNA and reconstituted with RPMI1640

One day later, some cells were dead

I planned to extract RNA 2 days post-transfection, but at 48hr, literally all cells were dead...

What should I do??

Have anyone tried to transfect these cells??


My collegue also uses Rh30 cells.
For siRNA transfections he uses successfully X-tremeGENE siRNA Transfection Reagent (Roche) and for trfxn for protein expression FugeneHD (Roche).
Their transfection reagents are muss less cytotoxic than lipofectamine (a real killer agent) with even the same or better trfxn efficiency.
However you have to do the optimization titration (ratio of reagent to siRNA) first but then it should work really nice.
Best success...