Eccles:AJ siRNA txn

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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).

Reagents

• 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).

Protocol

For one transfection i.e. one well:

1. 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
2. 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
3. Allow mixed Li2K/OptiMEM to incubate at RT for 5 min.
4. 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).
5. Add 100 uL (equal volume) of Opti-MEM, and mix well by pipetting. Final volume is now 200 uL with siRNA at 100 nM.
6. remove normal growth media from cells, wash 2 x with 500 uL OptiMEM.
7. Add 200 uL transfection mix from (5).
8. 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).
9. For replicate transfections, make up master mixes.
10. Assay for mRNA knockdown by using qPCR 24 hours post-transfection, and for protein 48-72 hours post-tranfection depending on target protein stability.

Example

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):

siRNA and media (one tube/siRNA)

	x1 (uL)	x4 (uL)
siRNA (50 uM)	0.4	1.6
Opti-MEM	49.6	198.4
Total	50	200

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):

Li2K (one tube for total number of wells)

	x1 (uL)	x12 (uL)
Li2K	1	12
Opti-MEM	49	588
Total	50	600

siRNA/plasmid co-tranfection

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.