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Transfecting Stealthâ„¢ RNAi or siRNA into HUVEC Cells Using Lipofectamineâ„¢ RNAiMAX
Sponsored,vendor-submitted protocol    Sponsored by Invitrogen Corporation, part of Life Technologies    Published in November 2009 (p.61) DOI: 10.2144/000113031

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 synopsis provides a recommended procedure to transfect Stealth™ RNAi or siRNA into human umbilical vein endothelial cells (HUVECs) using Lipofectamine™ RNAiMAX. Lipofectamine™ RNAiMAX has a broad range of activity, enabling achievement of maximal knockdown levels with minimal non-specific effect using the smallest amount of siRNA concentration.

Materials

HUVEC cells (Cat# CRL-1730, ATCC) maintained in Ham's Kaighn's Modification F12 (F12K) (Cat# 21127-022, Invitrogen) supplemented with 2 mM L-Glutamine (Cat# 25030-081, Invitrogen), 0.1 mg/ml Heparin sodium salt from porcine intestinal mucosa (Sigma, St. Louis, MO, USA), 0.05 mg/ml Endothelial Cell Growth Supplement (ECGS) (BD, Franklin Lakes, NJ, USA), 10% Fetal Bovine Serum (FBS) (Cat# 16000-044, Invitrogen). This medium should be prepared fresh every 2 weeks.

Note: Use low-passage cells; make sure that cells are healthy and greater than 90% viable before transfection.

StealthRNAi (or siRNA) of interest LipofectamineRNAiMAX Reagent (Cat# 13778-075 or 13778-150, Invitrogen)

Opti-MEM® I Reduced Serum Medium (Cat# 31985-062, Invitrogen) Methods

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.

  1. One day before transfection, plate 35,000 cells in 500 µl of growth medium without antibiotics. The cell density should be 30–50% confluent at the time of transfection.

  2. For each well to be transfected, prepare RNAi duplex-Lipofectamine™ RNAiMAX complexes as follows:

    1. Dilute 6 pmol RNAi duplex in 50 µl Opti-MEM® I Reduced Serum Medium. Mix gently.

    2. Mix Lipofectamine™ RNAiMAX gently before use, then dilute 1 µl in 50 µl Opti-MEM® I Reduced Serum Medium. Mix gently.

    3. Combine the diluted RNAi duplex with the diluted Lipofectamine™ RNAiMAX. Mix gently and incubate for 10–20 min at room temperature.

  3. Add the RNAi duplex-Lipofectamine™ RNAiMAX complexes to each well containing cells. This gives a final volume of 600 µl and a final RNA concentration of 10 nM. Mix gently by rocking the plate back and forth.

  4. Incubate the cells 24–48 h at 37°C in a CO2 incubator until you are ready to assay for gene knockdown. Medium may be changed after 4–6 h, but this is not required.

Important Guidelines for Transfection

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. Recommended Reagent Amounts and Volumes

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.


Figure 1. StealthRNAi was transfected into HUVEC cells using LipofectamineRNAiMAX Reagent. (Click to enlarge)



Table 1. (Click to enlarge)


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