Difference between revisions of "Team:MIT/Experiments/miRNA/more experiments"
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<h2 style="font-family: Verdana;"><a href=https://2016.igem.org/Team:MIT/Experiments/miRNA/more_background>Read miRNA sensors about here</a></h2> | <h2 style="font-family: Verdana;"><a href=https://2016.igem.org/Team:MIT/Experiments/miRNA/more_background>Read miRNA sensors about here</a></h2> | ||
<img src = "https://static.igem.org/mediawiki/2016/c/cd/T--MIT--microRNAsensor.PNG" style = 'padding: 5px'; width: 400px; height = 400px; float: right; border:5px;'> | <img src = "https://static.igem.org/mediawiki/2016/c/cd/T--MIT--microRNAsensor.PNG" style = 'padding: 5px'; width: 400px; height = 400px; float: right; border:5px;'> | ||
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<h3 style="color:#000000;text-decoration:underline"> Electroporation </h3> | <h3 style="color:#000000;text-decoration:underline"> Electroporation </h3> | ||
<img src = "https://static.igem.org/mediawiki/2016/b/b2/T--MIT--electroporation2.PNG" style = 'padding: 5px'; width: 600px; height = 250px; float: center; border:5px;'> | <img src = "https://static.igem.org/mediawiki/2016/b/b2/T--MIT--electroporation2.PNG" style = 'padding: 5px'; width: 600px; height = 250px; float: center; border:5px;'> | ||
| − | <p></p> | + | <p style="font-family: Verdana;">Our team carried out electroporation using the <a href=https://www.thermofisher.com/us/en/home/life-science/cell-culture/transfection/transfection---selection-misc/neon-transfection-system.html>Neon Transfection System</a>. 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.<a href=https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2975437/>(1)</a> After this process, the cells are returned to normal culture conditions in order to heal and replicate.</p> |
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Revision as of 03:15, 19 October 2016
General Experimental Set-Up and Data Analysis
miRNA Sensor
Read miRNA sensors about here
Transient Transfection
Electroporation
Our team carried out electroporation using the Neon Transfection System. 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 this process, the cells are returned to normal culture conditions in order to heal and replicate.
Flow Cytometry Analysis
Program Used:CytoFlo by Brian Teague
Sensitivity of miRNA Target Sites
Purpose
We want to understand the sensitivity of our miRNA target site constructs.
Set Up
We chose one miRNA target site, 451a, and designed and ordered siRNA complementary to the target site. We co-transfected tert-immortalized human endometrial stromal cells with our miRNA sensors and varying concentrations of siRNA then measured the red fluorescence output to gauge repression.
Results
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
Set Up
Results
