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TransMessenger Transfection Reagent Handbook

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 4. Pipetting scheme for optimization of siRNA transfection in 24-well plates

Guidelines for Transfection of siRNA Duplexes Using TransMessenger Transfection Reagent

The procedure below is based on the TransMessenger Reagent standard procedure and RNAi studies using targets described by Elbashir et al. (2). This procedure is provided as a starting point for optimization of siRNA transfection in mammalian cells using TransMessenger Transfection Reagent. For specific cell type and targets, optimal conditions could be different from those described here. Please be sure to read the background information and the protocol notes on pages 9 and 10 of this handbook before starting. 1. The day before transfection, seed 50,000 to 100,000 cells (depending on cell type and the time point of analysis) per well of a 24-well plate in 0.5 ml appropriate growth medium containing serum and antibiotics.
Make sure that cells are in good condition and are seeded 24 h before transfection. Cells should be 5080% confluent on the day of transfection. 2. Incubate cells under their normal growth conditions (typicall
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