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EtOH washes using a spray bottle to completely fill each well, followed by wiping down excess EtOH on the electrode strips (bottom side) and allowing the transfection plates to completely dry in the culture hood.
This spreadsheet automatically identifies wells with low Renilla luciferase signal, highlights wells that exhibit repression compared to the negative control, and provides measures of repression across the entire plate (Figure 2).
While this measure may not be used directly to indicate a miRNA target, it is useful for identifying outliers, problematic wells, or patterns in the data that are not solely attributed to direct miRNA regulation.
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Mix cells in culture plate with pipette positioned vertically in the center of the well, as cells will tend to aggregate on the sides of the well unless mixed properly.
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NOTE: Multiple plates can be read and data stored on the same excel file, however issues may be encountered with multiple plate reads where the luminometer program will crash.
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NOTE: If multiple plates are being measured sequentially on one luminometer, create buffer master mixes with everything except ATP and substrates, adding these reagents followed by pH adjustment immediately before use with each plate.
However, the optimization of transfection efficiency and cell survival with each cell line will have to be optimized using multiple buffer conditions, pulse codes, and number of cells.
Furthermore, these predictions do not take into account mechanisms of mRNA processing that exclude miRNA target sites, such as alternative polyadenylation 25, RNA editing 26, RNA methylation 27, and cooperative binding.
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LIFE screen allows the detection of subtle effects of miRNA targeting on a large number of genes, and the identification of multiple components of gene networks that are regulated by a given miRNA 24.
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While these algorithms are useful to identify candidate miRNA targets for subsequent experimental validation, these high error rates limit the efficacy of bioinformatic approaches for systematic miRNA target detection.
