Eccles:AJ siRNA txn
Forward transfection of siRNA into mammalian cells by using Lipofectamine 2000
This protocol is for use with cells seeded into a 24-well plate with siRNA at 100 nM final concentration in 200 uL/well. Scale volumes according to taste and need, and siRNA stock concentration (the volumes below are for 50 uM siRNA stock).
- Lipofectamine 2000 (Li2K; Invitrogen). Stored inside the fridge door in Hercus 203.
- Opti-MEM (Invitrogen).
- siRNA (no more than 100 nM final concentration, titrated according to specific siRNA and cell line). Stored in the special box in Hercus 203 -20dC.
- sterile 1.5 mL tubes.
Transfection is done according to the manufacturer's instructions (PDF), but with final volume of 200 uL in 24-well plate, rather than 100 uL (I find 100 uL too little to easily cover bottom of 24-well, and the reagent volumes are on the small side to accurately pipette).
For one transfection i.e. one well:
- Dilute appropriate volume of siRNA in Opti-MEM without serum (or other medium without serum) to final volume of 50 uL, and mix gently and well by pipetting.
- 0.4 uL (50 uM) siRNA
- 49.6 uL Opti-MEM
- Mix Li2K gently before use, then, in a separate tube, combine Li2K with Opti-MEM, and mix well by pipetting.
- 1 uL Li2K
- 49 uL OptiMEM
- Allow mixed Li2K/OptiMEM to incubate at RT for 5 min.
- Combine siRNA mix with Li2K mix, mix well by pipetting, and incubate at RT for 20 min (this is for the the siRNA to complex with the transfection reagent).
- Add 100 uL (equal volume) of Opti-MEM, and mix well by pipetting. Final volume is now 200 uL with siRNA at 100 nM.
- remove normal growth media from cells, wash 2 x with 500 uL OptiMEM.
- Add 200 uL transfection mix from (5).
- 4-6 hours post-tranfection, add equal volume (200uL) normal growth media (MEM-alpha for NZM cells) or OptiMEM containing 2x the normal FCS percentage for your particular cell type (to achieve 1x FCS).
- For replicate transfections, make up master mixes.
- Assay for mRNA knockdown by using qPCR 24 hours post-transfection, and for protein 48-72 hours post-tranfection depending on target protein stability.
Three different siRNAs - scrambled negative control, GAPDH positive control, target gene - will be transfected into cells seeded at two different densities in duplicate => 3 siRNAs x 2 duplicates => 6 wells/cell density x 2 cell densities = 12 wells in total => 12 transfections in total (don't forget to factor-in transfection reagent-only controls, if you're doing them - no siRNA tubes, but additional Li2K wells).
Make up the siRNA mix for each respective siRNA in a separate tube - there are 4 tranfections (wells) for each siRNA (n=2 density 1 + n=2 density 2 => 4 wells/siRNA):
|x1 (uL)||x4 (uL)|
|siRNA (50 uM)||0.4||1.6|
The siRNAs are prepared in three sperate tubes, but the Li2K can be made up in one tube containing the appropriate amount of Li2K and Opti-MEM for all the transfections, i.e. 12 (again, don't forget to factor-in tranfection reagent-only controls, if you're doing them - no siRNA tubes, but additional Li2K wells):
|x1 (uL)||x12 (uL)|
From Invitrogen's protocol...
- Plasmid and siRNA co-transfection are possible. Co-transfections have been tested with Lipofectamine 2000 in GripTiteTM cells (293 derived cells) plated at 1.8 x 105 cells/well in a 24-well format (0.5ml medium, no antibiotics). 200 ng of two different reporter plasmids were co-transfected with 10pmol of siRNA following the standard Lipofectamine 2000 protocol, with 2 uL of Lipofectamine 2000 per well. The total volume of the transfection mixes was 100 uL, and it was added to the medium already in the wells.
AJ April 2007.