Critical parameters for successful transfection

General guidelines for successful transfection

Successful transfection is influenced by many factors. The health and viability of the cell line, quality of the nucleic acid used, transfection reagent, duration of transfection, and the presence or absence of serum can all play a part.

Cells should be grown in an appropriate medium, supplemented with serum or growth factors, as required for the viability of that cell line. Do not use cells and media that you know are contaminated (e.g., with yeast or mycoplasma). If there is any doubt, reseed cells from a frozen, uncontaminated stock. Always ensure the medium is fresh if any components are unstable since missing key components may harm cell growth. Keep the incubation conditions constant at 37°C, CO2 at the correct level (usually 5–10%) and 100% relative humidity.

Every cell line has optimal culture conditions. Refer to the American Type Culture Collection [ATCC] web site.

Some transfection protocols require serum-free conditions for optimal performance, since serum can interfere with many commercially available transfection reagents. This should be checked for your protocol.
As a guide, cells should be transfected at 40–80% confluency. Too few cells will cause the culture to grow poorly without cell-to-cell contact. Too many cells results in contact inhibition, making cells resistant to uptake of foreign nucleic acid. Actively dividing cells will yield best results.
Keep the number of passages low (<50). In addition, the number of passages for cells used in a set of experiments should be consistent. 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. Cell characteristics can change over time with immortalized cell lines, and cells may not respond to the same transfection conditions after repeated passages, resulting in poor expression.

The optimal amount of nucleic acid varies widely, depending on the type of nucleic acid, number of cells, size of culture dish/plate, and the cell line used.

Increasing the quantity of transfected nucleic acid significantly may not yield better results. In fact, if initial transfection results are satisfactory, a reduced nucleic acid quantity should be tested (keeping the optimal reagent: nucleic acid ratio constant).

In some cases, a range of nucleic acid concentration may be suitable for transfection; although efficiency will decrease, sometimes markedly, outside of this range.

Too little nucleic acid may result in the experimental response not being present. Conversely, too much nucleic acid can prove toxic to cells.

This is dependent upon the transfection reagent being used and should be optimized carefully.

Many chemical transfection reagents have an ideal time window, in which a transfection complex of optimal diameter is formed. This is typically between 5 and 30 minutes, depending upon the nature of the reagent. Refer to the reagent manufacturer’s recommendations.

In general, transfection reagents need to be in contact with cells for a period of time before additional medium is added or the medium is replaced (to help minimize toxic effects of the reagent). The optimal transfection time depends on the cell line, transfection reagent, and nucleic acid used.

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.

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