What are the critical parameters to when optimizing transfection conditions?

The following parameters may be assessed, in an effort to maximize transfection efficiencies:

The amount of siRNA/shRNA being delivered

The optimal amount will be dependent upon the cell line, and target gene, under study. For most experiments, maximal potency, with minimal off-target effects, is achieved between 1nM to 100nM siRNA or shRNA plasmid.

The amount of transfection reagent

This is dependent upon the transfection reagent being used and should be optimized carefully. For QIAGEN’s transfection reagents you will find helpful starting conditions for optimization based on real experimental data on our Transfect Protocol database (http://www.qiagen.com/transfectionprotocols/default.aspx).

Length of transfection complex formation incubation period

Many chemical transfection reagents have a “sweet spot”, at which time a transfection complex of optimal diameter is formed. This is typically between 5 to 30 minutes, depending upon the nature of the reagent. Refer to the reagent manufacturer’s recommendations.

Cell line

Optimal transfection conditions are extremely cell line-dependent. The amount of siRNA/shRNA and transfection reagent, as well as the amount of time that the transfection complex should be left on the cells, will vary from one cell line to another. Helpful information is always available at the Transfect Protocol Database (http://www.qiagen.com/transfectionprotocols/default.aspx).

Cell density

This will be cell line-dependent. For most adherent cell lines, cultures that are 60-80% confluent at the time of transfection are typically optimal. For suspension cultures, densities between 0.5-1.0 X 106 cells/ml are typically optimal.

Cell passage number

Transfection efficiency declines the longer the cells are kept in culture. It is recommended that cell cultures that have been in culture beyond 10 passages NOT be used for transfection. Always take care to make sure that the cell cultures to be transfected are actively dividing, and are at least 90% viable, prior to transfecting.

Traditional vs. reverse transfection protocol

In some instances, plating cells onto wells or plates containing transfection complexes may result in increased transfection efficiency, compared to the traditional approach of adding transfection complexes to an established culture. An additional benefit to such reverse transfection protocols is that seeding and transfecting cells on the same day shortens the experimental timeline by a full day.

Electroporator settings

When utilizing electroporation to deliver siRNA/shRNA to cells that are difficult to transfect via conventional chemical methods, the voltage, pulse length, and pulse number are three critical factors which will require optimization. For additional information, refer to your instrument’s user’s manual.

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