Transfection is a laboratory technique that introduces foreign genetic material, such as DNA or RNA, into eukaryotic cells, specifically animal cells, without using viral infection. This process allows scientists to alter the genetic content of host cells, enabling the study of gene function, protein expression, and cellular behavior. Transfection is widely applied in biological research and biotechnology, contributing to areas like gene therapy, vaccine development, and the production of recombinant proteins.
Understanding Transfection Efficiency
Transfection efficiency measures the success rate of introducing foreign genetic material into cells and achieving its expression. It quantifies how many cells in a population have successfully taken up the nucleic acid and are expressing the desired gene or protein. This metric is assessed using methods like reporter genes, such as Green Fluorescent Protein (GFP), which allows visual counting of fluorescent cells, or through functional assays measuring gene activity. Higher efficiency leads to more reliable experimental results, as a larger proportion of cells participate in the study. Optimizing this efficiency is a common goal in molecular biology experiments.
Cellular Considerations for Optimal Transfection
The characteristics and condition of target cells influence transfection success. Cell type is a major factor; primary cells, isolated directly from tissue, are often more challenging to transfect than immortalized cell lines. Cell health and viability are also important; cells should be actively dividing and exhibit over 90% viability before transfection. Additionally, cells must be free from contamination, such as bacteria, yeast, mold, or mycoplasma, which can compromise cell health and transfection.
The passage number of cells, or how many times they have been subcultured, should be kept low, ideally under 50 passages, as repeated passages can alter cell characteristics and reduce efficiency. The cell’s growth phase, or confluency, is another factor; for adherent cells, 40-80% confluency is recommended at transfection. Too few cells can lead to poor growth, while too many can cause contact inhibition, reducing nucleic acid uptake. Maintaining consistent incubation conditions, including temperature (37°C), CO2 levels (5-10%), and 100% relative humidity, also supports cell health and transfection success.
Nucleic Acid Quality and Quantity
The quality of the genetic material, whether DNA or RNA, influences transfection efficiency. High purity is essential; the nucleic acid should be free from contaminants like proteins, RNA, salts, organic solvents (e.g., phenol), or endotoxins. Contaminants can interfere with transfection or be toxic to cells, reducing uptake or expression. Purity is assessed by measuring absorbance ratios with a spectrophotometer: an A260/A280 ratio of around 1.8 for DNA indicates good purity, and a ratio between 2.0 and 2.2 for A260/A230 suggests minimal contamination.
The integrity of the nucleic acid, meaning it is intact and not degraded, is important for successful gene expression. Gel electrophoresis can confirm its integrity and topology for plasmid DNA. Supercoiled plasmid DNA is preferred for transfection due to its higher efficiency compared to nicked or linear forms. The appropriate concentration of nucleic acid is also important; too little may not yield a detectable response, while excessive amounts can be toxic. The optimal amount varies by cell type and experimental setup and is determined through titration. For sensitive applications like primary cell transfection or in vivo studies, endotoxin levels in plasmid DNA should be below 0.1 EU/µg; for standard cell lines, 0.1 to 10 EU/µg is acceptable.
Optimizing Transfection Methods and Reagents
The selection and optimization of the transfection method and reagents influence efficiency. Chemical methods, such as lipid-based or polymer-based reagents, form complexes with nucleic acids to facilitate their entry into cells. The optimal ratio of nucleic acid to transfection reagent is an important parameter that must be titrated for each cell line and nucleic acid combination. An imbalance can reduce efficiency or increase cell toxicity.
Incubation times during complex formation and exposure to cells require optimization. Complex formation, where the nucleic acid and reagent combine, occurs within 5 to 30 minutes, depending on the reagent. Prolonged incubation of complexes before adding them to cells can lead to larger, less effective complexes and lower efficiency. The presence or absence of serum in the culture medium during transfection varies by protocol. Some reagents work best in serum-free conditions during complex formation to avoid nuclease interference, while many protocols recommend adding complexes to cells in complete, serum-containing medium for cell health and efficiency. Post-transfection, culture media changes depend on reagent toxicity and experimental goals.
Troubleshooting Low Transfection Efficiency
When facing low transfection efficiency, a systematic troubleshooting approach is beneficial, involving revisiting the conditions of the cells, nucleic acid, and protocol. Compromised cell health is a common issue, stemming from contamination (e.g., bacteria, mycoplasma) or using cells past their optimal passage number. Ensuring cells are actively dividing and at the recommended confluency (40-80% for adherent cells) prior to transfection can improve uptake. If the nucleic acid is degraded or impure, it will hinder expression; verifying its integrity and purity can resolve this.
Problems with the transfection reagent or protocol contribute to low efficiency. This includes using a suboptimal ratio of nucleic acid to reagent, incorrect incubation times for complex formation (which can be detrimental), or the presence of antibiotics or serum during complex formation that interferes with the reagent’s action. Some reagents may be sensitive to freezing or improper storage, reducing activity. If basic adjustments do not yield improvements, considering alternative transfection reagents or methods, such as electroporation for difficult-to-transfect cells, may be necessary.