Revision as of 17:54, 16 October 2016

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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."

Although the sources we used to chose miRNA candidates did report their results using amounts of miRNA rather than miRNA activity, using miRNA sensors to characterize the miRNA profile of a cell is not widely used as of yet. Because this phenomenon of miRNA amount not always corresponding to miRNA activity has not been explained, it was decided that choosing candidates based on the fold difference reported between eutopic endometriotic and healthy endometrium was the best method in selecting miRNA-ts to characterize.

Utilizing 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 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 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.