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Published on: December 8, 2023
Transfection protocol for antisense oligonucleotides affects uniformity of transfection in cell culture and
Chantal A Reed1, Eigen R Peralta, Lisa M Wenrich
1Isis Pharmaceuticals, Carlsbad, CA 92008, USA. chantal_reed@isisph.com
Abstract:
In an effort to optimize the transfection of cell lines with antisense oligonucleotides, we examined cellular accumulation of a labeled oligonucleotide by flow cytometry. We were surprised to observe that a routinely used transfection protocol, a fixed lipid/oligonucleotide ratio, resulted in variable transfection efficiency depending on the concentration of oligonucleotide used. A significant population of cells, especially at lower doses of oligonucleotide and cationic lipid, were untransfected. We investigated lipid/oligonucleotide ratios, different lipid preparations, and different cell types and found that these variables did not alter the percentage of cells transfected at these lower doses of oligonucleotide. However, when lipid-oligonucleotide complexes were formed at the high dose and then diluted into a solution of lipid or a complex of lipid and unlabeled, negative control oligonucleotide, a constant percentage of cells was transfected. Under these conditions, mRNA target reduction dose-response curves were also shifted to lower doses. We hypothesize that poor transfection observed at a low concentration of lipid-oligonucleotide complex when diluted in medium is due to loss of active complexes, either by adsorption to the substrate or by changes in physical characteristics of complexes. By maintaining a constant lipid concentration, more consistent transfection was achieved.
Insights
Optimizing cell transfection requires careful consideration of lipid-oligonucleotide complexes. Maintaining a constant lipid concentration ensures consistent transfection efficiency, especially at lower doses, by preventing complex loss.
Area of Science:
- Molecular Biology
- Cell Biology
- Biotechnology
Background:
- Antisense oligonucleotides (ASOs) are crucial for gene silencing but require efficient delivery into cells.
- Standard transfection protocols using a fixed lipid/oligonucleotide ratio can yield variable transfection efficiencies.
- Low concentrations of oligonucleotide and cationic lipid often result in a significant population of untransfected cells.
Purpose of the Study:
- To optimize the transfection of cell lines using antisense oligonucleotides.
- To understand the factors influencing variable transfection efficiency at low oligonucleotide concentrations.
- To develop a more consistent and effective method for delivering oligonucleotides into cells.
Main Methods:
- Flow cytometry was used to quantify cellular accumulation of labeled oligonucleotides.
- Various lipid/oligonucleotide ratios, lipid preparations, and cell types were investigated.
- Lipid-oligonucleotide complexes were formed at high concentrations and then diluted to assess transfection consistency.
Main Results:
- A fixed lipid/oligonucleotide ratio showed variable transfection efficiency dependent on oligonucleotide concentration.
- Diluting pre-formed lipid-oligonucleotide complexes in lipid or control oligonucleotide solution resulted in constant transfection percentages.
- This dilution method shifted mRNA target reduction dose-response curves to lower doses, indicating improved efficacy.
Conclusions:
- The loss of active lipid-oligonucleotide complexes, possibly due to adsorption or altered physical characteristics, explains poor transfection at low doses.
- Maintaining a constant lipid concentration during complex formation and dilution is key to achieving consistent transfection.
- This optimized approach enhances antisense oligonucleotide delivery and gene silencing efficacy in cell lines.
