Split a plate of confluent HeLa or tet-off cells that has been passed 7 times or less and has not been allowed to become overly confluent. Count cells and add 1.0 x 106 (HeLa) or 1.2 x 106 (tet-off) cells to each well of a 6-well plate. Bring the total volume in each well of the 6-well plate to 2mL
On the following day: transfect cells with siRNAs and DNA plasmids (if desired). You will transfect siRNAs to a conc. of 200nM (2uL/sample from 100uM stock) (you may need to optimize here).
Set up the transfection as follows:
Incubate mixture of 250uL OMEM *2uL of 100uM siRNA stock for 5 min at room temp. (remember control siRNAs)
Incubate mixture of 250uL OMEM and *8-10uL (Xrn1= 8.3, Dcp2= 8.3, TTP= 10) (you may need to optimize) of Lipofectamine 2000 Rgt. at room temp. for 5 min.
Combine the 250uL of OMEM/Lipifectamine mix with the 250uL of 250uL of OMEM/siRNA mix (Do this for each sample). Incubate the samples for 20 min at room temp.
After 20 mins, remove 6-well plates from incubator and aspirate away all media. Add 500uL of antibiotic-free DMEM to each well. Then, add 500uL of siRNA/Lipofectamine mixture to each sample to bring the total volume to 1mL. Place plates back in incubator for ~6hrs.
After 6hrs, aspirate the transfection mix from each well. Wash cells with PBS. Add 2mL of antibiotic free DMEM to each well. Place plates back in incubator.
On the following day, split 100uL of cells to chamber slides for immunofluorescence or split 250-350uL of cells to 12-well plate for pulse chase assay. Also, split one extra well to be used as a protein sample to monitor protein knockdown. Incubate cells O/N in the incubator.
On the following day, conduct experiments as desired, but remember that for many proteins (at least for TTP, Xrn1, and Dcp2) the optimal protein knockdown appears at ~45-55 hrs after the transfection began. For more stable proteins, you may have to conduct a repeat knockdown 48hrs after the original one to achieve efficient knockdown.
With extra cell samples (one control, one knockdown), collect cell lysates and monitor knockdown by western blot.