I am trying to knock down a gene of interest using siRNA in SH-SY5Y cells. But the amount of siRNA I use is mostly based on the well surface area etc. I use 50pmol/well in a 6-well plate.
So is it fair to use the same amount of siRNA in cells that are undifferentiated as well as ones that will be treated with siRNA "post-differentiation"? Even though I am starting with the same number of cells, the cells that are undifferentiated are rapidly dividing and are about 60-70% confluent on the day of transfection (24 hours after plating) and even more by the time I process them for extractions (48 hours post siRNA treatment). So the number of cells is way more here and they are more confluent.
On the other hand, the differentiated cells, are not dividing, only extending their processes so the number of cells in this case is essentially the same as the starting number. They are more spread apart and not in direct contact with each other like the undifferentiated ones. So how do we account for that difference while calculating how much siRNA should be added to each well?
Also, what about the effect of confluence on final results? Is it fair to compare the data from gene expression between the differentiated and undifferentiated cells?
Under experimental conditions that you shared, it is desirable to have a large excess of siRNA for an extended period of time. Assuming that cells upon differentiation do not loose the ability to take up siRNA and do not secrete a nuclease, which can degrade the siRNA, it is acceptable to use equal excess amounts of siRNA for your experiment in which the aim is to achieve appreciable (>70%) gene expression knockdown in both cell types.
Under experimental conditions that you shared, it is desirable to have a large excess of siRNA for an extended period of time. Assuming that cells upon differentiation do not loose the ability to take up siRNA and do not secrete a nuclease, which can degrade the siRNA, it is acceptable to use equal excess amounts of siRNA for your experiment in which the aim is to achieve appreciable (>70%) gene expression knockdown in both cell types.
You can Optimize how much siRNA should be added to each well.
Yes, compare the data from gene expression level and not by confluency between the differentiated and undifferentiated cells.
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