Team:MIT/Experiments/miRNA/more background
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.
How a miRNA sensor works
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 guide RISC to the target site on the mKate mRNA, degrade the mRNA, and repress the expression of mKate. There will be 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.
How miRNA sensors are used
The general use of miRNA sensors is to detect the relative miRNA activity of a few selected miRNAs in a desired cell line under varying conditions. This information could be used to characterize a cell line. With the addition of varying amounts of small interfering RNA (siRNA), which acts similarly to miRNA, data about a miRNA-ts sensitivity can also be measured. In this example, five different miRNA sensors are inserted into a cell line for the purpose of getting a small scope miRNA profile. Flow cytometry is used to measure the relative fluorescence emitted from the proteins produced by the miRNA sensor and a scatter plot like the one above can be produced. On the x-axis is the transfection marker,blue fluorescent protein in relative fluorescent units. On the y-axis is the inverse of the miRNA activity, red fluorescence also in relative fluorescence units. It is important to look at the relationship between the red and blue fluorescence because plasmids are usually taken up by a cell in a 1:1 ratio. This means that a transfected cell that has red fluorescent protein knocked down by miRNA will show a slope less than one and indicates higher miRNA activity. Low miRNA activity will be indicated by a trend with a slope of 1. In this data set, it can be interpreted that only miRNA #1, represented by blue dots, has a low miRNA activity because the relationship between red and blue fluorescence has a slope of 1.
The scatter plot data can be represented further by a bar graph. Our miRNA reporter, red fluorescence, is normalized using the transfection marker, blue fluorescence, per a cell. This allows us to simplify our figure and use the x-axis to now label the different miRNA sensors used. The new y-axis is the red to blue fluorescence ratio representing the relative miRNA activity in the cells. This allows viewers to quickly identify which miRNAs have the highest miRNA activity in a desired cell line.
