Setting up successful transfection experiments for the first time can be a daunting task, especially when working with stubborn cells that are refractory to transfection. Variables such as cell line, passage number, confluency, DNA quality and even transfection reagent formulation all contribute to the likelihood of success (or failure) of a given transfection experiment.
Below, we highlight our top “Tips from the Bench” for optimizing plasmid DNA delivery.
Maintain a similar passage number between experiments to ensure reproducibility. A low passage number can make cells more sensitive to transfection whereas a high passage number can render cells refractory to transfection.
Use healthy, actively dividing cells to maximize transfection efficiency. Mirus Bio recommends plating cells the night before a transfection experiment at a density that will promote cell division to obtain 75-90% confluency for transfection the following day.
µl TransIT®-LT1 Reagent / µg DNA
Cell confluency can affect transfection efficiency. In this example, a plasmid expressing luciferase was transfected into cells that were either 75% (red) or 100% covering the plate confluent (grey). The non-dividing, over-confluent cells showed lower luciferase expression and activity. The optimal number of cells to transfect will depend on both the cell type and application of your transfection experiment.
Contaminants, such as protein, carbohydrate and lipids, may affect transfection efficiency and gene expression levels. Ensure that the plasmid preparation exhibits an A260/A280 ratio of > 1.8. Traces of contaminating endotoxin can be removed using Mirus Bio’s MiraCLEAN® Endotoxin Removal Kit.
Identifying the TransIT® reagent volume to DNA mass (µl to µg) to use in your transfection is an easy first step in optimizing gene delivery. We colloquially refer to this as the “reagent:DNA ratio.”
The best reagent:DNA ratio to use varies between reagents as well as by cell type, so when working with a new experimental system (e.g. new transfection reagent, new cell line, etc.) we suggest performing a small-scale pilot transfection to determine the optimal reagent:DNA ratio that achieves the highest transfection efficiency with minimal toxicity. For example, vary the concentration of TransIT®-LT1 Transfection Reagent from 2-8 µl per 1 µg DNA to find the optimal ratio.
You can download this handy, step-by-step Optimization Protocol (PDF) for DNA transfection in a 12-well plate to find what reagent:DNA ratio to use going forward. Once determined, this optimal ratio can be used in smaller as well as larger scale formats.
The Transfection Experts at Mirus Bio have already determined optimal reagent:DNA ratios for many popular cell lines and TransIT® reagents. You can find this information in our Reagent Agent® database.
After mixing the DNA and transfection reagent, incubate to form complexes for 15-30 minutes at room temperature, before adding the mix to your cells. Transfection efficiency may decrease if the complex formation exceeds an hour.
Depending on the gene being expressed and the experimental design, post-transfection incubation time can have a dramatic effect on experimental outcome. Protein expression is typically detectable as early as 4 hours post-transfection and can persist for many days. In general, maximal protein expression occurs 48 hours post-transfection. The time point for optimal gene expression can be determined by varying post-transfection incubation times from 4 to 72 hours.