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Why use a miRNA sensor?

The two most common methods for quantifying the miRNA profile of a cell line are by miRNA microarray and quantitative polymerase chain reaction (qPCR). However, these methods specifically measure the amount of miRNA relative to a control (a known abundant miRNA such as miRNA-7 or the healthy cell miRNA level). It has been indicated that the physical amount of miRNA present doesn't always have a strong correlation to the repression of the associated gene(Mullokandov et. al Nature 2012). By using a miRNA sensor, we can directly measure the activity of the miRNA, which is one of the aspects of miRNA being utilized in our circuit.

Mullokandov et. al, referring to the figure to the left, remarked in their 2012 Nature article: "We detected the expression of more than 310 miRNAs (Fig. 2a). Our library included sensors for 165 of these miRNAs (188 when considering families), but we detected the suppression of only 67 sensors (Fig. 2b). Thus, 59% of the expressed miRNAs that we sampled did not have suppressive activity."(1)

However, this does create a contradiction in our methods. The sources we used to chose miRNA candidates did report their results using amounts of miRNA rather than miRNA activity. Because miRNA sensors are not yet widely used to characterize the miRNA profile of a cell, it was nearly impossible to find information on miRNA activity in endometriotic versus healthy endometrium. Therefore, our team had to settle with choosing miRNA candidates based on the relative amount of miRNA present in endometrial cells.

Using miRNA activity rather than abundance will allow more control over the output of our circuit.

Using a miRNA Sensor

The sensor, courtesy of Jeremy Gam, consists of a red fluorescent protein, mKate, which is constitutively controlled by the human elongation factor-1 alpha (hEF1a) promoter. After mKate, you will notice here are four yellow blocks. These represent four miRNA target site domain repeats. After the target sites is a blue fluorescent protein (BFP) also controlled by the hef1a promoter. When there is miRNA present, it will bind to the miRNA target site on the mKate mRNA and repress the expression of mKate. The miRNA, however, will have no effect on the expression of the BFP, which is used as a positive control (transfection marker).

The more miRNA activity present, the less mKate (red fluorescent protein) will be produced.