Hello Jiaxuan Yang

There are three main factors that will help to increase the transfection efficiency.

1. Transfecting healthy cells that are still actively dividing. Healthy Cells are happy and will have high transfection-rate. Cell viability is important.

2. Optimizing the nucleic acid (both high-quality and proper amount) is a part of the transfection efficiency equation. You must ensure that the RNA you’re introducing into the cell is as close to homogenous as possible. Otherwise, there would be too many variables to account for when analyzing the results. Prepare a purified form of nucleic acid and then prepare different concentrations to test. For instance, by preparing concentrations incrementally from 25-250 ng of nucleic acid to be tested against your cell population, you can optimize the transfection efficiency per cell type.

3. Correct the cell population per individual well. When too many cells are present within a single well, you may experience a low transfection rate due to contact inhibition. Contact inhibition is what prevents cells from proliferating without control. When the entire well is filled with the cell population, it may inhibit some of the regulatory functions of cells, proliferation being one of them, and gene uptake being another. Attempt to have around 60-80% confluency for optimal transfection efficiency. Please note that cell transfection efficiency doesn’t increase with the amount of nucleic acid concentration in a one-to-one fashion. With too much, you may observe cell toxicity.

For example, if you are using a 24-well plate format, you may use a ratio of 500 ng mRNA : 1ul transfection reagent in a total volume of 50ul complexed mRNA/lipid per well. This will be a good starting point.

FYI: mRNA tends to transfect more efficiently than DNA as more cells in a population typically will take up mRNA than DNA. mRNA can yield better results than DNA in some contexts, for instance, when transfecting primary or terminally differentiated cells. With primary cells, DNA transfection efficiency can be 30% or lower. But with mRNA, you can get about 80% transfection efficiency.

Also note that primary human macrophages are well known to be extremely hard to transfect, primarily due to their recognition of foreign nucleic acids, and their initiation of immune responses to exogenous siRNA molecules.

Please refer to the paper attached below.

Article Transfection of hard-to-transfect primary human macrophages ...

Hope the above suggestions may help improve the mRNA transfection efficiency.

Good Luck!

To improve mRNA transfection efficiency in macrophages, consider optimizing the amount of mRNA and transfection reagent (e.g., MessengerMAX). Ensure cells are seeded at an appropriate density, typically 70-90% confluency, to maximize uptake.

Use high-quality, intact mRNA and enhance transfection with additional reagents or lipid-based carriers to improve delivery.

Incubate the cells with the transfection mix for 4-6 hours, followed by medium replacement to reduce toxicity.

If lipid-based reagents are not efficient, try electroporation. It’s important to monitor cell viability at higher reagent concentrations to balance transfection efficiency with cell health.

To improve mRNA transfection efficiency in macrophages using Thermo Fisher MessengerMAX, optimize the mRNA-to-reagent ratio (e.g., 1:2 or 1:3), use high-quality capped and polyadenylated mRNA, and ensure macrophages are healthy and at 70–90% confluency. Perform transfection in serum-free medium like Opti-MEM, and return to complete medium 4–6 hours later. Minimize immune activation by using modified mRNA (e.g., pseudouridine) or pre-treating cells with inhibitors like Ruxolitinib. Prolong incubation of complexes (up to 6 hours) while monitoring for cytotoxicity. If efficiency remains low, consider alternative methods such as electroporation or testing different transfection reagents.