Transfection Efficiency

Methods abound for quantifying RNA and protein post-transfection at both the population as well as single cell level. qPCR and western blotting are commonly used to determine the relative expression of RNA and protein, respectively, between transfected cells and a negative control condition. It is essential to account for potential differences in cell number or sample processing between conditions; thus, normalization to a reference gene or spike-in controls should be practiced to ensure accurate comparisons of gene expression.

In the siRNA-mediated knockdown example below, mRNA expression of MAPK3, Lamin A/C and ABC A1 was first normalized to GAPDH expression to account for differences in cell number and sample processing. For this experiment, GAPDH is a reference gene because its expression is not expected to be affected by the transfected siRNA, and its expression level is a “reference” for the baseline amount of total RNA in a given sample. MAPK3, Lamin A/C and ABC A1 are target genes because the transfected siRNA was designed to “target” their transcripts for degradation. After normalization, transfection efficiency was then assessed by comparing gene expression between the ‘target gene siRNA’ condition and negative controls (‘untreated’ and ‘control siRNA’). In knockdown experiments, high transfection efficiency is reflected by lower gene expression. Thus, knockdown in the primary hepatocytes shown below was deemed efficient because only the target gene expression was decreased and only by the target gene siRNA transfection. 

Efficient Knockdown of Endogenous Genes in Primary Hepatocytes Using TransIT-TKO® Reagent. Primary mouse hepatocytes were transfected with the indicated siRNAs or a non-targeting control siRNA using the TransIT-TKO® Reagent. Twenty-four hours post-transfection, the amount of each mRNA was measured relative to GAPDH mRNA levels using qRT-PCR and then scaled to the expression level of the specific target mRNA in the cells alone (untreated) controls.

In addition to relative gene expression levels, researchers may assess transfection efficiency by the percentage of cells that exhibit gene expression within a single cell population. Flow cytometry and other methods that parse the distribution of gene expression in a pool of cells can be used to calculate transfection efficiency.

Transfection Efficiency = Number of Expressing Cells ÷ Total Number of Cells.

For knockdown or knockout experiments, the calculation would instead be: 1 – (Number of Expressing Cells ÷ Total Number of Cells).

What constitutes as ‘expressing’ will differ by the assay method, gene under study and experimental objectives. In general, it is ideal if there is a clear distinction between the population of expressing and non-expressing cells.

However, what to do if the success of a transfection experiment is not easily detected by gene expression? For example, CRISPR genome editing can be subtle, introducing base pair mutations with no detectable effect at the protein level. In these cases, many clever sequence-based approaches have been developed to detect successful genome editing, such as the T7 endonuclease 1 mismatch assay and GUIDE-seq.^1,2 Label IT® technology from Mirus Bio can also be used to assess transfection efficiency independently from gene expression-based methods. Label IT® works by non-specifically attaching a fluorophore or tag to nucleic acids before they are transfected. Nucleic acid uptake can then be tracked by quantifying Label IT® signal.

Tracking of Plasmid Localization and Expression. COS-7 cells transfected with Label IT® Tracker™ Cy®5 (blue) labeled pEYFP-nuc and TransIT®-LT1 Reagent in serum containing media. Images were acquired at 3, 8, and 20 hours post-transfection. The blue signal indicates the cellular localization of the labeled plasmid, the green signal indicates cellular autofluorescence, and the yellow signal is the expression of the nuclear yellow fluorescent protein (YFP) reporter.

Though uptake of the transfected nucleic acid is required for all transfection applications, it may not be sufficient. A plasmid or mRNA may be internalized by cells but not efficiently transcribed or translated, respectively. Similarly, an siRNA or CRISPR/Cas complex may successfully enter cells but be unable to effectively bind its target to exert its function. Which metrics and strategies are used to measure transfection efficiency will ultimately depend on experimental objectives.

For example, in virus production experiments, a bountiful yield of infectious viral particles is usually the best indicator of success–not necessarily expression of each individual viral component. In the words of Mirus Field Application Scientist, Leisha Kopp, “A picture is not worth a (thousand words) functional titer assay.” In the lentivirus production experiment highlighted below, TransIT-VirusGEN® Transfection Reagent was used to transfect the pLKO.1-puro-CMV-TurboGFP™ transfer plasmid with packaging plasmid mixes from two different manufacturers.

From the fluorescent microscopy pictures of the transfected cells, one would conclude that transfection efficiency was nearly 100% (brightfield images not shown) regardless of the packaging mix used. The transfection efficiency, when considering transgene expression and plasmid delivery, is indeed very high. However, assaying the lentivirus for ability to transduce target cells, i.e. functional titering, revealed a large difference in yield that was dependent on the packaging mix used for transfection. This example underscores the importance of selecting a relevant measure of transfection efficiency for your unique experimental goals.

Common reporter assays to measure transfection efficiency

Reporter assays are those that are coupled to easily observable signal like fluorescence, luminescence and absorbance. Reporter assays for transfection thus include reporter genes or RNA whose expression is easily detected post-transfection. In the table below, we highlight four common reporters and their unique features.