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Tips from the Bench: Transfection Tip

Compare Transfection Reagents

Compare Transfection Reagents Side-by-Side Using the Same Batch of Cells

Transfection results can vary day-to-day. In order to evaluate transfection reagents under optimal conditions, test each reagent side-by-side on the same day with the same batch of cells. Using historical comparisons (transfections performed on different days) to compare transfection reagents can lead to incorrect conclusions.

As an example, see the data presented in Figure 1. If a researcher compared transfection results generated using Reagent A on Day 1 to those obtained using Reagent B on Day 2 (Figure 1A), they would conclude that Reagent A was the better transfection reagent for these cells, which is incorrect.

When tested side-by-side, Reagent B outperformed Reagent A on both days (Figure 1B). Most importantly, the performance patterns were consistent from day-to-day although the overall luciferase activity varied.

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Transfection Results Vary Day-to-Day

Transfection Results Vary Day-to-Day. Cells were transfected in duplicate with a luciferase expression vector (1.5 µg/well) using Reagents A and B. Twenty-four hours post-transfection, the cells were harvested and assayed for luciferase activity. Identical sets of transfection were performed on two separate days (independent experiments). (A) Reagent A data on Day 1 compared to Reagent B data on Day 2. (B) Complete transfection results from Days 1 and 2.


Obtain the Best Performance by Following Reagent-specific Protocols

Each transfection reagent is supplied with a manufacturer's recommended protocol. The amount of reagent used per microgram of plasmid DNA often varies between transfection products. Figure 2 shows transfection results with varying amounts of three different reagents based on the manufacturer's suggested ranges. With each reagent there are optimal levels for highest expression.

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Reagent Titration to Optimize Transfection Performance

Reagent Titration to Optimize Transfection Performance. Cells were transfected with a luciferase expression vector (1.5 µg/well) using three different transfection reagents according to the manufacturer's recommendations. Each reagent was titrated based on its respective protocol to identify the optimal transfection conditions.

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