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RNAi - siRNA Transfection
Transfection of siRNA is a method used to introduce siRNA into host cells. Short interfering RNA molecules (siRNA) are artificial double-stranded RNA molecules that can be used to silence target gene expression via RNA interference (RNAi). Our siRNA products typically consist of three different siRNA molecules that target the same gene, and an antibiotic resistance gene. Host cells are grown in transfection medium, which contains siRNA molecules, and a transfection reagent.
See below for a general protocol and recommendations.
Materials Required
- Tissue culture plate: 6 well, 12-well, 24-well, or 96-well
- Deionized water/DEPC water
- Normal Growth Medium
- siRNA duplex
- Transfection reagent (e.g. Lipofectamine)
- Fetal Bovine Serum (FBS)
- Pipette and pipette tips
- Vials/tubes
- 37°C Incubator
A. Sample and Reagent Preparation
- Sample: Seed 2 x 105 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. Incubate the cells at 37°C in a CO2 incubator, and grow cells to a 60-80% confluency. This typically takes 18-24 hours.
- siRNA duplex solution: Before resuspending, briefly centrifuge the tube to ensure the lyophilized siRNA is at the bottom of the tube. Resuspend the siRNA duplex to an appropriate concentration with DEPC water. For example, resuspend one tube of 5 nmol siRNA in 250 μl of DEPC water to produce 20 μM siRNA duplex solution.
- 2X Normal Growth Medium: Prepare normal growth medium with 2 times the typical serum and antibiotics concentrations.
- Transfection Working Solution: Add the siRNA duplex solution directly to a vial of Transfection Reagent using a pipette according to the below volumes. Mix gently by pipetting the solution up and down and incubate the mixture 15-45 minutes at room temperature. Add Normal Growth Medium to the solution according to the volumes below. For example, for a 24-well plate, and a final siRNA concentration of 50 nM, add 1 μl of lipofectamine to 1.25 μl of 20 μM siRNA duplex solution. Then, add 497.75 μl of Normal Growth Medium to prepare a final medium volume of 500 μl. Note: the optimal siRNA: Transfection medium ratio should be determined experimentally.
|
Plate |
Final volume of medium |
Lipofectamine |
siRNA (20 μM) |
Volume of medium |
Final concentration of siRNA |
|
96-well |
0.25 μl |
0.5 μl |
99.25 μl |
100 nM |
|
|
0.25 μl |
0.25 μl |
99.50 μl |
50 nM |
||
|
0.25 μl |
0.05 μl |
99.70 μl |
10 nM |
||
|
24-well |
500 μl |
1 μl |
2.5 μl |
496.50 μl |
100 nM |
|
1 μl |
1.25 μl |
497.75 μl |
50 nM |
||
|
1 μl |
0.25 μl |
498.75 μl |
10 nM |
||
|
12-well |
1 ml |
2 μl |
5 μl |
993.00 μl |
100 nM |
|
2 μl |
2.5 μl |
995.50 μl |
50 nM |
||
|
2 μl |
0.5 μl |
997.50 μl |
10 nM |
||
|
6-well |
2 ml |
5 μl |
10 μl |
1985.00 μl |
100 nM |
|
5 μl |
5 μl |
1990.00 μl |
50 nM |
||
|
5 μl |
1 μl |
1994.00 μl |
10 nM |
B. Transfection protocol
- Wash the cells once with Normal Growth Medium (6-well plate – 2 ml; 12-well plate – 1 ml; 24-well plate – 500 μl; 96-well plate - 100 μl). Aspirate the medium and proceed immediately to the next step.
- Add the Transfection Working Solution prepared previously dropwise to each well, covering the entire layer. Gently mix by swirling to ensure the entire cell layer is immersed.
- Incubate the cells 5-7 hours at 37°C in a CO2 incubator, or under conditions normally used to culture the cells. Longer transfection times may be desirable depending on the cell line.
- Add half of the volume added in step 1 of 2X Normal Growth Medium to cells (6-well plate – 1 ml; 12-well plate – 0.5 ml; 24-well plate – 250 μl; 96-well plate - 50 μl). If toxicity occurs, remove the transfection mixture and replace with 1X growth medium.
- Incubate the cells for an additional 18-24 hours under conditions normally used to culture the cells.
- Aspirate the medium and replace with fresh 1X normal growth medium.
- Assay the cells using the appropriate protocol 24-72 hours after the addition of fresh medium in the step above.