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TransMessenger Transfection Reagent Handbook

ng a particular cell type/RNA combination.

Cell density at transfection
Table 1 lists the recommended number of adherent cells to seed per culture well the day prior to transfection of single-stranded RNA. The optimal confluency for transfection of adherent cells with single-stranded RNA is 8090%, while the optimal confluency for transfection of adherent cells with siRNA is 5080%. The optimal confluency should be determined for every new cell type to be transfected and kept constant in future experiments. This is achieved by counting cells prior to seeding and by keeping the time period between seeding and transfection (minimum 24 hours) constant. This will ensure that the cell density is not
too high and that the cells are in optimal physiological condition on the day of transfection.

Table 1. Recommended number of cells to seed the day before transfection of single-stranded RNA * Be sure that cells are seeded a minimum of 24 hours before transfection of RNA. Actual cell number used depends on cell type and size. Sufficient cells should be seeded such that the culture is 8090% confluent on the day of transfection. The volume of medium used is not critical. Use a volume suitable for the cell culture format. Amount of RNA
The amount of RNA used is a critical factor for efficient transfection. Toxic effects may arise if too much RNA is used, while conversely, expression levels may be too low if insufficient RNA is used. Therefore, the amount of RNA transfected should be optimized for every new RNA and/or new cell type used. The recommended amount of RNA for transfection using 6-well plates i
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