Stealth™/siRNA Transfections Oligofectamine™

Introduction

Oligofectamine™ Reagent is a proprietary formulation for the transfection of oligonucleotides 1 and short interfering RNA (siRNA) 2,3 into eukaryotic cells.  The reagent formulation has been changed to enhance its stability at cold temperatures (+4°C) while continuing to provide the highest specific activity and lowest non-specific effects on cell growth.
 
Performance may be enhanced in some assays.

Guidelines for Transfection
 

  1. Use the procedure at www.lifetechnologies.com/rnai; click on Protocols.
  2. Use an initial oligonucleotide concentration of 200 nM for transfection. Optimal oligonucleotide concentrations may range from 50-250 nM; optimize as necessary. Always include a control oligonucleotide in the experiment to assess non-specific effects.
  3. Prepare complexes using the amount of oligonucleotide and Oligofectamine™ recommended on below Optimization may be necessary. Note: We recommend using Opti-MEM® I Reduced Serum Medium (Cat. No. 31985-062) to dilute Oligofectamine™ and oligonucleotide before complexing.
  4. Transfect cells at 30-50% confluence. Optimize as necessary. Maintain the same seeding conditions between experiments. Use cells within 20 passages of optimization.
  5. Do not add antibiotics to media during transfection as this causes cell death. 
  6. For optimal results, perform transfection in medium without serum. Transfection in the presence of serum may be tested, if desired. Test any serum-free medium for compatibility with Oligofectamine™.  

Protocol

Use the following procedure to transfect adherent mammalian cells in a 96-well format. For other formats, see Scaling Up Transfections. All amounts and volumes are given on a per well basis.
 
  1. One day before transfection, plate cells in 100 µl of growth medium without antibiotics so that cells will be 30-50% confluent at the time of transfection.

  2. For each transfection sample, prepare complexes as follows:

    • Dilute 1 µl of a 20 µM stock oligonucleotide in 16 µl of Opti-MEM® I Reduced Serum Medium (or other medium) without serum. Mix gently.
    • Mix Oligofectamine™ gently before use, then dilute 0.4-0.8 µl in Opti-MEM® I Medium (or other medium) without serum to a final volume of 3 µl. Mix gently and incubate for 5-10 minutes at room temperature.
    • Combine the diluted oligonucleotide with diluted Oligofectamine™ (total volume = 20 µl). Mix gently and incubate for 15-20 minutes at room temperature (solution may appear cloudy).

  3. While complexes are forming, remove the growth medium from the cells and wash once with medium without serum. Add 80 µl of medium without serum to each well containing cells.

  4. Mix the 20 µl of complexes (Step 2c) gently, and add to the cells.

  5. Incubate cells at 37 °C in a CO2 incubator for 4 hours.

  6. Add 50 µl of growth medium containing 3X the normal concentration of serum without removing the transfection mixture.

  7. Assay for gene activity at 24-72 hours post-transfection or as appropriate for your cell type and target.

Optimizing Transfection

To obtain the highest transfection efficiency and low non-specific effects, optimize transfection conditions by varying cell density as well as oligonucleotide and Oligofectamine™ concentrations

Scaling Up Transfections

To transfect cells in different tissue culture formats, vary the amounts of Oligofectamine™, oligonucleotide, cells, and medium used in proportion to the relative surface area, as shown in the table below. See top of page for additional recommendations about amounts of oligonucleotide to transfect.

Culture vessel
Relative surf. area vs. 96-well
Oligo (µl of 20 µM stock) & dilution vol. (µl)
Oligofectamine™ (µl) & final dilution vol. (µl)
Plating medium vol.
Total vol. per well
Added vol. medium with 3X serum
 96-well
     1
1 µl in 16 µl
0.4-0.8 µl to 3 µl
    80 µl
100 µl
    50 µl
 24-well
     5
2.5 µl in 40 µl
1-2 µl to 7.5 µl
  200 µl
250 µl
  125 µl
 12-well
   10
5 µl in 85 µl
1-3 µl to 10 µl
  400 µl
500 µl
  250 µl
 6-well
   25
10 µl in 175 µl
2-4 µl to 15 µl
  800 µl
1 ml
  500 µl

References

1.   Li, Y., et al. (2002) J. Biol. Chem. 277, 11352.
 
2.   Elbashir, S.M., et al. (2001) Nature 411, 494.
 
3.  Harborth, J., et al. (2001) J. Cell Sci. 114, 4557.
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