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Simple optimization experiment
This tool assists users by providing a ready-to-transfect plate to test 24
different transfection conditions. Transfection efficiency is typically scored
at 24 hours after tranfection by fluorescence microscope observation and 72
hours after tranfection for cell death observation, mRNA level and protein
level. Therefore, the number of cells initially plated should reach 70%-100%
confluence after 72 hours. Different transfection reagents recommend different
amounts of reagent for the same amount of siRNA. Please consult the manufacture
for the suggested range of volumes and then optimize an experiment based on
those ranges.
Optimization test design
Users should design a series of conditions based on the cell lines and
transfection reagents intend to use in their final experiments.
The following figure provides an example arrangement for an optimization
assay. In this example, users can test four different transfection reagent
volumes (Different rows) and three different cell densities (Different Columns).
Example optimization arrangement (the yellow one uses no cells for cell
viability background detection)
About Reverse Transfection:
Delivery of siRNAs into cultured cells requires the use of a transfection
reagent. Traditionally, the first step in a transfection is to plate the cells
24 hours before transfection. Reverse transfection is a timesaving, effective
alternative method in which cells are transfected as they are plated. Compared
to traditional plating protocols, equivalent or improved transfection efficiency
is seen for many cell types tested. In addition, the reverse transfection
process is an entire day shorter than traditional transfection. Taken together,
SABiosciences recommends reverse siRNA transfections.
Simple optimization procedures
- 24 hours after transfection, check the fluorescence signal in TEC1 after
washing the cell with phenol-red free cell culture media.
- 72 hours after transfection, check the cells in TEC2 wells to see the cell
death. Inreased cell death correlates with higher transfection and knockdown
efficiency.
Optional: 72 hours after transfection, collect cell lysates for mRNA
knockdown confirmation. Purify the total RNA from the corresponding wells of the
TEC1 and Positive Control. Synthesize cDNA from the same amount of isolated RNA.
Use real-time PCR to identify the transfection conditions with the greatest
GAPDH knockdown (>80%) by comparing the Positive Control Wells to the TEC1
wells.
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