Thursday, May 3, 2012

Testing 1, 2, 3

Recently we have received a number of questions along the lines of, "How much dsRNA should I use and how many cells?"

If you are doing a screen with our libraries, we offer two different concentrations of dsRNA (one recommend for the bathing method, the other recommend for transfection). As a result, you can change cell number but you're stuck with our dsRNA concentrations. Thus, you should optimize things with those concentrations in mind, and for 384-well format plates.

If, however, you are designing a smaller-scale study or otherwise have control over dsRNA concentrations, you can play around with both dsRNA and cell concentrations, in various plate formats, in order to find an optimal set-up for your assay. Above is an example layout for testing what concentration of dsRNA and cells to use for an assay. In this case, we were designing a test for a 96-well format experiment using the bathing method of dsRNA delivery. We chose dsRNA directed against thread (th) as a positive control for RNAi and did a read-out of cell viability to assess knockdown. Including several negative controls (LacZ and EGFP in addition to empty wells) allowed us to make sure dsRNAs did not reach non-specific toxic levels and get a sense of the overall variability of the assay. In our test, wells in columns 1 and 2 got the smallest number of cells and less dsRNA, and wells in columns 11 and 12 got the largest number of cells and more dsRNA.

For other plate formats, other dsRNA delivery methods, different cell types, etc. you can use the same general strategy to design a test and carefully determine an optimal set of conditions for your assay. Doing this type of testing at the beginning can save time and improve results later in the process.

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