Our products speak for themselves in quality and performance. REQUEST AN EVALUATION

The TransMission: Transfection 101

Transfection Efficiency

Last Updated: January 26, 2023

Share: Twitter or LinkedIn

Explore Related Info & Links:

"How successful was your transfection experiment?"

Transfection efficiency is a measure used to answer this question. Because success can be defined in multiple ways--number of cells expressing a gene, % knockdown, plasmid uptake, etc.--how to calculate transfection efficiency will depend on the type of transfection experiment. Researchers frequently use easily tractable reporter assays for determining and optimizing transfection efficiency for their experimental system.

In this TransMission Transfection 101 we will cover:

  • The many meanings of transfection efficiency
  • Common reporter assays to measure transfection efficiency
  • How to improve transfection efficiency

The many meanings of transfection efficiency

Table 1 below summarizes how transfection efficiency is described for various types of transfection experiments.

Table 1. Measures of Transfection Efficiency

Transfection Application Measure of Efficiency
Gene expression RNA and/or protein level of transfected gene
Transcript expression
Protein level of transfected mRNA
Gene knockdown
Target RNA and/or protein level
Genome editing
Quantity of edited genomic sequence or edited RNA and/or protein level
Nucleic acid uptake
Intracellular level of transfected nucleic acid
Virus production Viral titer


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. 

    Copy Link Enlarge Image
    Efficient Knockdown of Endogenous Genes in Primary Hepatocytes Using TransIT-TKO Reagent

    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.

    Copy Link Enlarge Image
    Tracking of Plasmid Localization and Expression

    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.

    importance of functional titer


    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 Table 2, we highlight four common reporters and their unique features.

    Reporter Description
    Fluorescent protein, e.g. GFP
    Originally isolated from jellyfish,3 they are a class of proteins with stable, intense fluorescent properties.
    Fluorescence can be quantitatively or qualitatively measured without cell lysis or addition of other materials.
    An enzyme that acts on a luciferin substrate, resulting in luminescence.
    Allows for a quantitative readout of photon emission. Highly sensitive with a broad dynamic range, ideal for determining relative transfection performance between samples. Most luciferase assays require cell lysis.
    An enzyme that catalyzes hydrolysis of certain sugars, e.g. X-gal and ONPG. Hydrolysis products can be easily detected.
    Many different sugar substrates are available, which allows colorimetric, fluorescent or chemiluminescent detection of ß-galactosidase with high sensitivity. Most ß-galactosidase assays require cell lysis.
    Secreted alkaline phosphatase (SEAP)
    An enzyme secreted from cells that catalyzes hydrolysis of various phosphate substrates, e.g. PNPP.
    Cells transfected with SEAP expression constructs do not need to be lysed to assay for activity. Since lysis is not required, culture media can be sampled at multiple times throughout an experiment. SEAP assays can be colorimetric or fluorescent in nature.


    The reporter gene can be used on its own to establish optimal transfection conditions before starting experiments with your actual genes of interest. Reporters can also be coupled in cis or trans to the gene of interest. Cell lines which constitutively express a reporter are useful for assaying knockdown or CRISPR gene editing.

    How to improve transfection efficiency

    Now that we’ve covered common reporters and ways to measure transfection efficiency, you may be wondering how to maximize transfection efficiency. Below is a list of transfection variables to examine for improved transfection efficiency. Much of the same information is also available in this Transfection Troubleshooting Infographic.

    Cell Health

    Cell physiology and gene expression are intertwined. Thus, not surprisingly, your transfection results may be linked to cellular health. Have you tried these troubleshooting tips related to cell health?

    • Optimize the density or confluency of cells at transfection.
    • Ensure cells appear and are growing as expected before beginning a transfection experiment.
    • Thaw and culture a new aliquot of cells if needed.
    • Check that the cell culture medium does not contain transfection-incompatible additives like polyanions such as dextran sulfate or heparin, which can inhibit transfection. If necessary, the transfection medium can be replaced with the transfection-incompatible medium 4 hr post-transfection. NOTE: A media change is not typically needed or recommended for transfection with TransIT® reagents.

    Transfection Complex Formation Conditions

    TransIT® reagents come together with nucleic acids to form a complex with a net positive charge. Successful complex formation relies on:

    • Using a serum-free complex formation medium, e.g. serum-free Opti-MEM® or PBS
    • Correct volume of reagent and mass of nucleic acid (more is not always better)
    • Optimal ratio between reagent volume and nucleic acid mass. This ratio (µl:µg) can be determined by testing a range from 1:1 to 5:1. See: mirusbio.com/optimize
    • Mixing well after each addition of nucleic acid and reagent, and not disturbing the complex afterward
    • Correct incubation time specified in the protocol before adding to cells

    Assay Time and Method

    Have you considered that your transfection is working, but how you are assessing transfection is the true culprit? Choosing an optimal time to harvest and assess your transfected cells can be beneficial. For example, peak protein expression for transfected mRNA is typically earlier than for transfected plasmids. Furthermore, factors like mRNA half-life, protein half-life and translation rate can depend on the sequence of your construct. For gene silencing experiments, knockdown may be observed at the transcript level (e.g. with PCR-based methods) before it is observed at the protein level (e.g. with western blotting). Use proper controls with all assay methods to have greater confidence in the validity of the results of your transfection.

    Plasmid or Nucleic Acid Sequence and Quality

    Larger plasmids are typically more difficult to transfect than smaller plasmids--though large plasmids are known to be successfully delivered with TransIT® reagents. For any given mass of plasmid, there will be less molecules of a larger plasmid than a smaller plasmid. You can increase the "dose" of the plasmid by delivering more transfection complexes, e.g. if normally you transfect with 3 µl of TransIT® reagent and 1 µg of plasmid, try doubling the dose with 6 µl of TransIT® and 2 µg of plasmid.

    Check also that the nucleic acid you are transfecting is of the correct sequence and free of contaminants. For example, endotoxin is a common contaminant of plasmid preparations from E. coli that is known to negatively impact transfection. mRNA is prone to degradation through freeze-thaw and exposure to nucleases. Sequencing your construct and checking its integrity with gel electrophoresis should thus be included to ensure maximum efficiency. Designing a more biologically active sequence may also be necessary, e.g. test multiple gRNA or RNAi sequences.

    Gene Delivery Method

    You may need to try a different transfection reagent or delivery method. Use Mirus Bio’s Reagent Agent® (mirusbio.com/ra) to find proven solutions for your cell type and nucleic acid of interest. Consider other techniques like delivery with viral vectors or electroporation, which may be more effective for some cell types that are simply not well suited for transfection with chemical reagents.

    At Mirus Bio, our R&D team has developed transfection reagents with improved nucleic acid uptake/release and protein expression, knockdown and virus production capabilities. Our dedicated Technical Support and Field Applications staff are also available for free consultations to tackle your unique transfection experiments and requirements. Please contact techsupport@mirusbio.com with questions or to schedule a consultation.

    Happy Gears

    Did this post help you understand transfection efficiency? Let us know in the Comments section below! We’d love to hear from you. 


    1. Sentmanat, M., et al., Scientific Reports (2018).
      DOI: 10.1038/s41598-018-19441-8
    2. C. Nobles, Methods Mol. Biol (2021).
      DOI: 10.1007/978-1-0716-0822-7_6
    3. Shimomura, O., et al., J. Cell. Comp. Physiol. (1962).
      DOI: 10.1002/jcp.1030590302


    No Comments

    The TransMission

    Feedback or questions?
    We'd love to hear from you. Email techsupport@mirusbio.com or call us at 888.530.0801.