Transfection techniques are pivotal advances in molecular biology that allow scientists to reliably introduce DNA, RNA, ribonucleoproteins (RNPs), and other molecules into cells. Several delivery methods are available, including chemical and physical approaches like electroporation, as well as viral-based transduction. Transfection requires optimization based on the molecule and target cell type to achieve the highest efficiency and viability.
Among several crucial optimization steps, researchers must validate protocols for accurate transfection efficiency. Due to the diversity of molecules that scientists can transfect, transfection efficiency assessment methods are equally varied, ranging from genetic analyses to protein expression assays.
Methods that enable scientists to accurately assess the number of DNA sequence copies integrated into a host genome after transfection provide valuable insights into efficiency. Researchers have accomplished this via droplet digital PCR (ddPCR), which provides a more accurate copy number measurement compared to conventional PCR.1 PCR-based techniques cannot, however, confirm the presence of protein following transfection.
Western blotting is a reliable method to confirm the expression of a specific protein as well as the expected protein size. For instance, researchers demonstrated the successful expression of human β-cardiac myosin heavy chain S1L via western blotting, monitoring transfection efficiency across a range of DNA construct concentrations.2
If scientists require quantitative protein expression data, flow cytometry can be used to measure both the relative expression of a protein and the proportion of cells that express that protein. For example, researchers have done this by measuring the expression of a transfected fluorescent protein such as green fluorescent protein (GFP) or using target-specific antibodies.3
Scientists often use a combination of methods to verify and validate their methods. No matter the construct or cell type, workflows can be tailored to optimize transfection success.
Learn more about assessing transfection efficiency.
