Note that poorly conserved miRNAs are generally lowly expressed, and are therefore less likely to exert a function. For regulation to occur, both the miRNA and its target mRNA must be expressed in the same cell and at the same time, and importantly, the miRNA must be expressed at a level sufficient to mediate consequential repression. This level can vary somewhat, depending on the size of the cell and the identity of the miRNA, but as a rough guideline, the miRNA typically must exceed 1000 molecules per cell for repression to be detectable.

The consideration of mRNA expression levels is particularly important when performing global analyses that measure the extent to which predicted targets change after perturbing a miRNA. A common mistake is to include all predicted targets in such an analysis, without filtering out those that are too lowly expressed to be accurately quantified. Another common mistake is to use all predictions when comparing the performance of two approaches that yield different numbers of predictions. Instead, the same number of predictions should be compared, first filtering out all that are too lowly expressed to be accurately quantified and then considering only the top-ranked predictions of the less stringent list of predictions.