Transfection is one of the most widely used techniques in cell-based experiments. It involves introducing exogenous nucleic acids into cells using various methods to alter cellular properties, thereby enabling research into gene function and protein expression. Based on different mechanisms, transfection methods can be generally categorized into three types: physical methods (e.g., electroporation, gene gun, microinjection), chemical methods (e.g., calcium phosphate, cationic liposomes, cationic polymers), and biological methods (e.g., retroviruses, adenoviruses).
As the most commonly used transfection reagent in laboratories, what precautions should be taken when using liposome-based transfection reagents?
Figure1: Mechanism of Liposome-Mediated DNA Transfection
Reagent Section
Q1. What is the working principle of liposome-based transfection reagents?
Liposomes carry a positive charge and bind nucleic acids via electrostatic interactions, forming nucleic acid–liposome complexes. These complexes are adsorbed onto the cell membrane and enter the cell through membrane fusion or endocytosis. Eventually, the DNA is released from the endosomes into the cytoplasm or nucleus, where it can be transcribed and expressed.
Q2. Do liposome-based transfection reagents require freezing for storage?
No, they should not be frozen. Liposome-based transfection reagents must be stored at 4°C. Avoid prolonged or repeated exposure to air, as oxidation of the liposomes can negatively affect transfection efficiency.
Operation Section
Q3. Can serum be present when preparing transfection complexes?
No. It is recommended to use serum-free medium (commonly Opti-MEM) when preparing transfection complexes. Negatively charged proteins in serum can interfere with the binding between liposomes and nucleic acids, affecting complex formation.
Q4. Can antibiotics be added during transfection?
No antibiotics should be added at any stage of the transfection process. Transfection increases cell membrane permeability, which can amplify the cytotoxic effects of antibiotics, leading to cell death.
Q5. Does the transfection process need to be terminated?
No. Liposome complexes remain stable for up to 6 hours. If the medium was not changed before transfection, it is recommended to replace it with fresh culture medium after 4–6 hours to ensure sufficient nutrients for cell growth. However, if the medium was refreshed prior to transfection, no further medium change is necessary.
Experimental Optimization Tips
Figure2: Workflow of Liposome-Based Transfection Using Hieff Trans Reagent
Q6. What are the key considerations for liposome-based transfection?
Cell plating density: For DNA transfection, 90%–95% confluency; for RNA transfection, 30%–50%.
High-purity DNA improves transfection efficiency.
Use serum-free medium to dilute both DNA and transfection reagent.
Avoid antibiotics in the culture medium during transfection.
Store reagents at 4°C, and avoid repeated, prolonged exposure to air.
Optimize DNA and reagent ratios when first using a reagent. A typical DNA to transfection reagent ratio is 1:2–1:3.
Q7. Can multiple plasmids be co-transfected? Can DNA and RNA be co-transfected?
Yes, multiple plasmids can be co-transfected, but a pilot experiment is recommended. The total DNA amount is usually kept constant, while the ratio of each plasmid should be adjusted based on size, structure, and desired expression. Co-transfection of DNA and siRNA is also possible, but siRNA transfection efficiency may be lower in such cases.
Q8. Are there any requirements for cell condition during transfection?
Use healthy cells with viability over 90%. Passage the cells 24 hours before transfection to allow recovery. Avoid over-passaged cells, and ensure the absence of contaminants such as mycoplasma, fungi, or bacteria. Always use fresh growth medium.
Q9. Does plasmid size affect liposome-based transfection efficiency?
Yes. As plasmid size increases, transfection efficiency tends to decrease. For optimal results, plasmid size should ideally be under 10 kb, as larger plasmids may significantly reduce transfection efficiency.
Product Overview
|
Product Name |
Specification |
Cat No. |
|
0.5 mL/1 mL/5 mL |
40802ES02/03/08 |
|
|
0.5 mL/1 mL/5 mL |
40808ES02/03/08 |
|
|
500 μL/5× 500 μL |
40804ES76/86 |
|
|
0.5 mL/1 mL |
40806ES02/03 |
|
|
25 μL/100 μL/1 mL |
40810ES25/01/03 |
|
|
1.5 mL/10 mL/100 mL |
40820ES04/10/60 |
|
|
Hieff Trans™ Suspension Cell-Free Liposomal Transfection Reagent |
100 μL/0.5 mL/1.0 mL/5×1 mL |
40805ES01/02/03/08 |