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Griffin:siRNA transfection: Difference between revisions
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→Verify transfection efficiency
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The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. | The efficiency of transfection may depend on the cell type, but also on the passage number and the confluency of the cells. The time and the manner of formation of siRNA-liposome complexes (e.g. inversion versus vortexing) are also critical. | ||
Verify the transfection efficiency with the use of a fluorescent dye-labeled siRNA (FITC, CY5, etc.). A flow cytometer can be used to measure cell transfection efficiency. The transfection efficiency must be measured 5-7 hours after cells are first exposed to the transfection reagent. This is important as the siRNA reagent complex with the siRNA will maximal enter the cell during this time frame. After 5-7 hours, the cells could begin to degrade or exotise the control siRNA-FITC conjugate and this will. When transfecting adherent cells, 5-7 hours after transfection, wash the slide then fix and mount the cells prior to visualization under a fluorescent scope. | Verify the transfection efficiency with the use of a fluorescent dye-labeled siRNA; [http://datasheets.scbt.com/sc-36869.pdf Control siRNA (FITC Conjugate)-A: sc-36869] (FITC, CY5, etc.). A flow cytometer can be used to measure cell transfection efficiency. The transfection efficiency must be measured 5-7 hours after cells are first exposed to the transfection reagent. This is important as the siRNA reagent complex with the siRNA will maximal enter the cell during this time frame. After 5-7 hours, the cells could begin to degrade or exotise the control siRNA-FITC conjugate and this will. When transfecting adherent cells, 5-7 hours after transfection, wash the slide then fix and mount the cells prior to visualization under a fluorescent scope. | ||
'''1)''' In a six well tissue culture plate, seed 2 x 105 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. | '''1)''' In a six well tissue culture plate, seed 2 x 105 cells per well in 2 ml antibiotic-free normal growth medium supplemented with FBS. | ||
