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General Experimental Set-Up and Data Analysis

Tissue Culture Conditions

Media: Dulbecco's modified eagle medium, Ham's F12 nutrient mixture, 1% penicillin streptomycin, 10% fetal bovine serum
Incubator: 37°C and 5% CO2
tHESC were split when confluent, typically every six days.
Experiments took place in 24 well plates, seeded with 200,000 cells/well, with a total volume of 500uL/well.

Read miRNA sensors about here

Transient Transfection

Electroporation

Our team carried out electroporation using the Neon Transfection System. First the cells are washed, resuspended in buffers, and and DNA is added. Next, the cells undergo electroporation. Electroporation is the process of subjecting cells to high-voltage electric shocks in order to break holes through the membrane and allow the uptake of DNA.(1) After the transfection, the cells are returned to normal culture conditions in order to heal and replicate.

Flow Cytometry Analysis

Program Used:CytoFlo by Brian Teague

Flow cytometry occurs 48 hours after transfection, and if applicable, 24 hours after induction of a small molecule (such as the hormone estrogen). After the data is collected, anaylsis of the flow cytometry data commences.

1) After inputting each unique sample with their respective variables, we observe the total cell population using forward scatter height vs. width. The upper right of the graph represents pieces that are most likely cell aggregates and the lower left represents debris and dead cells. The middle oval is the desired cell population we wish the analyze.

2)Only looking at the experimental samples, we look at a graph with the transfection marker vs. cell count. The threshold of transfection is set in the dip between the untransfected cell population and the hump of known transfected cell. From now on, we will only be analyzing the population above this threshold, the transfected cells.

3)The next step is when the cell population is divided into specific bins. This distinguishes sections of the cell population by different number of copies of the transfection marker taken up. To avoid the strange behavior seen at low and high transfection marker copy number, we might decide to only analyze the middle bin(s).

4)After deciding the bins to look at, we apply the variables we specified in the very beginning. For example, we can look at the varying red fluorescence output due to different amounts of siRNA induction, as seen in our first experiment.

Sensitivity of miRNA Target Sites

Purpose

We want to understand the sensitivity of our miRNA target site constructs.

Set Up

The miRNA target site 451a was specifically chosen to study in tHESC because a preliminary experiment indicated tHESC had very little miRNA 451a activity(Figure 1). siRNA was designed and ordered to be complementary to the miRNA target site using Integrated DNA Technologies. Tert-immortalized human endometrial stromal cells were co-transfected with our 451a miRNA sensors and varying concentrations of siRNA (0nM, 1nM, 10nM, 50nM, and 100nM). 48 hours later, flow cytometry was performed and red fluorescence from the mKate protein was measured to indicate the amount of repression from the siRNA and therefore, the sensitivity of the miRNA target site (Figure 3). As described above, flow cytometry analysis was carried out in those four steps listed. Before experimental variables were applied, it was decided to only analyze bins 14-19 due to their uniform behavior (Figure 2).

Results

Figure 1

Because the resulting trend of endogenous miRNA-451a in tHESC had a slope ~ 1, it is concluded that the miRNA activity of 451a is very minimal and therefore could be treated with siRNA without fear of influence from endogenous miRNAs.

Figure 2

Before finalizing the analysis, it was decided that bins 14-19 were most representative of the data.

Figure 3

When increasing siRNA-451a 0 to 1 nM, there is a 10 fold repression of red fluorescence. Saturation occurs at approximately 10 nM. More experiments would need to be done to understand the responsiveness between 0 to 1 nM.

miRNA Profile of tHESC

Purpose

To confirm that tHESC under varying conditions (1nm estrodial and no estrodial) would have distinct miRNA profiles.

Set Up

Tert-immortalized human endometrial stromal cells were transfected with our eight candidate miRNA sensors. Twenty-four hours after transfection, half of the samples were induced with 1nM of estrodial . 48 hours later, flow cytometry was performed and red fluorescence from the mKate protein was measured to indicate the amount of repression from endogenous miRNA (Figure 5).Before the finalized analysis, as seen in figure 5, the different miRNA activity detected by the different miRNA sensors was evident by looking at the different cell populations (Figure 4).

Results

Figure 4

Cells containing either the 142-5p or 21-5p miRNA sensor belonged to two distinct cell populations.

Figure 5

Red Relative Fluorescence was normalized using the relative fluorescence of the transfection marker, BFP. The fold difference between estrodial and no estrodial conditional miRNA activity amongst the eight miRNA candidates ranged from 0 to 0.5 fold.