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Search Thermo Fisher Scientific
Lipofectamine® RNAiMAX Reagent is a proprietary formulation specifically developed for highly efficient delivery of Stealth™ RNAi or short interfering RNA (siRNA) to mammalian cells for RNAi analysis. This reference provides a recommended procedure to transfect Stealth™ RNAi or siRNA into human umbilical vein endothelial cells (HUVEC cells- ATCC, Cat. No. CRL-1730) using Lipofectamine® RNAiMAX (Cat. Nos. 13778-075, 13778-150). Lipofectamine® RNAiMAX has a broad range of activity, enabling achievement of maximal knockdown levels with a minimum of optimization required.
Guidelines for Transfection
Follow these important guidelines when transfecting Stealth™ RNAi or siRNA into HUVEC cells using Lipofectamine® RNAiMAX:
Have the following reagents on hand before beginning:
Note: Use low-passage cells; make sure that cells are healthy and greater than 90% viable before transfection.
Use this procedure to forward transfect Stealth™ RNAi or siRNA into HUVEC cells in a 24 well format (for other formats, see Recommended Reagent Amounts and Volumes). In forward transfections, cells are plated in the wells, and the transfection mix is generally prepared and added the next day. All amounts and volumes are given on a per well basis.
To qualitatively assess transfection efficiency, we recommend using a K1F11 Stealth™ Select RNAi (available through www.lifetechnologies.com/rnaiexpress; for human cells, oligo HSS105842 is a good choice). Adherent cells in which K1F11/Eg5 is knocked down exhibit a "rounded-up" phenotype after 24 hours due to mitotic arrest (Weil, D. et al. Biotechniques 2002, 33: 1244-1248); slow growing cells may take up to 72 hours. Alternatively, growth inhibition can be assayed after 48-72 hours. Note: The BLOCK-iT™ Fluorescent Oligo (Cat. No 2013) is optimized for use with Lipofectamine® 2000, and is not recommended for Lipofectamine® RNAiMAX.
Due to the broad range of maximal activity exhibited by Lipofectamine® RNAiMAX, a range of cell densities and volumes of Lipofectamine® RNAiMAX can be used for transfection. For transfecting HUVEC cells in 24-well format, 0.5-1.25 µl Lipofectamine® RNAiMAX and 30,000 – 40,000 cells per well is suitable. For extended time course experiments (72 hours), consider using the lower cell number; for short-term experiments (24 hours), consider the higher cell number.
The final concentration of RNAi duplex can be varied between 1-50 nM. A concentration of 10 nM RNAi duplex is suitable to knockdown many target genes. However, the optimal concentration of RNAi duplex will vary depending on the efficacy of the duplex, and should be determined empirically.
To transfect HUVEC cells in different tissue culture formats, vary the amounts of Stealth™ RNAi or siRNA, Lipofectamine® RNAiMAX, cells, and medium used in proportion to the relative surface area, as shown below.
Note: 20 µM Stealth™ RNAi or siRNA = 20 pmol/µl.
Culture vessel | Cells plated per well | Dilution medium | RNAi duplex amount | Final RNAi duplex conc. | Lipofectamine® RNAiMAX2 | |||||
Start point | Acceptable Range | Reverse transf. (µl) | Forward transf. (µl) | Start point (pmol) | Acc. Range (pmol) | Start point (nM) | Acc. Range (nM) | Start point (µl) | Acc. Range (µl) | |
96-well
|
7,500
|
5,000-10,000
|
20
|
2 x 10
|
1.2
|
0.12-6
|
10
|
1-50
|
0.2
|
0.1-0.25
|
48-well
|
15,000
|
10,000-20,000
|
40
|
2 x 20
|
2.4
|
0.24-12
|
10
|
1-50
|
0.4
|
0.25-0.5
|
24-well
|
35,000
|
30,000-40,000
|
100
|
2 x 50
|
6
|
0.6-30
|
10
|
1-50
|
1
|
0.5-1.25
|
6-well
|
100,000
|
90,000-120,000
|
500
|
2 x 250
|
30
|
3-150
|
10
|
1-50
|
5
|
2.5-6.25
|
¹ Surface areas may vary depending on the manufacturer.
² If the volume of Lipofectamine® RNAiMAX is too small to dispense accurately, and you cannot pool dilutions, predilute Lipofectamine® RNAiMAX 10-fold in Opti-MEM® I Reduced Serum Medium, and dispense a 10-fold higher amount (should be at least 1.0 µl per well). Discard any unused diluted Lipofectamine® RNAiMAX.