Quantitative measurement removes the guesswork from siRNA transfection optimization
What challenges are associated with nucleic acid transfection?
The best way to move DNA and RNA nucleic acids into cells, known as transfection, varies from one type of cell to another. Enough nucleic acid must be used to achieve a biological effect, but the researcher must also take care not to use too much and waste precious reagent resources. Also, lipid-based and electroporation transfection methods may not be well tolerated by some cells and can even cause cell death. For these reasons, BEFORE you do your experiments, transfection conditions must be optimized for each unique combination of cell type, transfection method and nucleic acid reagent used.
How to find the best transfection conditions
The best conditions for transfection can be found by varying the concentration of each reagent – usually a nucleic acid control and lipid transfection reagents. The lowest amount of lipid transfection reagent that still delivers biologically effective levels of nucleic acids can be found using a concentration range. Similarly, the best concentration of nucleic acid must also be determined empirically. The concentration of these two reagents should be independently varied to determine the optimum concentration of each one. See Figure 1 in the associated application note for an example.
What is the best way to analyze transfection results?
There is no one right answer to this question. Analysis will depend on what is being transfected, and how it can be monitored. A DNA control plasmid commonly has a fluorescent reporter, and time will be needed for fluorescent protein expression levels to rise before results should be read. RNA oligos may be directly labeled allowing analysis after a shorter incubation period. Other non-fluorescent methods including phenotypic changes or selection by antibiotic resistance are also possible.
The application note - Determining transfection conditions using siGLO Transfection Indicators and the Cytell Cell Imaging System - provides you with a real-world example of siRNA transfection and analysis. It not only shows how to set up transfection optimization, but also how to use the Cytell Cell Imaging system to determine which conditions are optimal!
You may also be interested in these related resources:
- A general protocol for use of DharmaFECT transfection reagents to deliver siRNA (or microRNA) into cultured mammalian cells.
- This protocol outlines reverse transfection of siRNA.
- A comprehensive portfolio of competitively priced, proven transfection reagents for a wide range of applications.
- Moving nucleic acids into cells
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