Difference between revisions of "Team:MIT/Experiments/miRNA"

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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center> How can we detect endometriosis using miRNA? </h1>   
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<h2 style="color:#FF9733; text-decoration:underline> What is miRNA?</h2>
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<h2 style="color:#FF9733; text-decoration:underline"> What is miRNA?</h2>
 
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For those who are not familiar with miRNA, miRNA are small units of RNA <b>found in all mammalian cells</b> and play a regular role in <b>gene regulation</b>. miRNA is single stranded and only about 20 nucleotides long. With the help of other proteins(which form what is called the 'RISC complex'), miRNA is guided to a complementary part on the mRNA (miRNA target site) where miRNA binds and cleaves messenger RNA. This inhibits gene expression post transcriptionally.  
 
For those who are not familiar with miRNA, miRNA are small units of RNA <b>found in all mammalian cells</b> and play a regular role in <b>gene regulation</b>. miRNA is single stranded and only about 20 nucleotides long. With the help of other proteins(which form what is called the 'RISC complex'), miRNA is guided to a complementary part on the mRNA (miRNA target site) where miRNA binds and cleaves messenger RNA. This inhibits gene expression post transcriptionally.  
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<img src = "https://static.igem.org/mediawiki/2016/7/70/T--MIT--miRNA_profile.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
 
<img src = "https://static.igem.org/mediawiki/2016/7/70/T--MIT--miRNA_profile.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
 
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<h2 style="color:#FF9733; text-decoration:underline> Why is miRNA a good biomarker?</h2>
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<h2 style="color:#FF9733; text-decoration:underline"> Why is miRNA a good biomarker?</h2>
 
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There are hundreds of different miRNAs that can make up a specific cell's miRNA profile. The profile and activity of miRNA varies between different <b>cell types </b> and different <b>cells states</b>.This makes it is possible to tell the difference between cells from the uterus and liver, and the difference between healthy and diseased cells based on miRNA profiles.
 
There are hundreds of different miRNAs that can make up a specific cell's miRNA profile. The profile and activity of miRNA varies between different <b>cell types </b> and different <b>cells states</b>.This makes it is possible to tell the difference between cells from the uterus and liver, and the difference between healthy and diseased cells based on miRNA profiles.
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<h2 style="color:#FF9733; text-decoration:underline> What is the miRNA profile of Endometriosis?</h2>
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<h2 style="color:#FF9733; text-decoration:underline"> What is the miRNA profile of Endometriosis?</h2>
 
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Cells from the uterus (referred to as 'eutopic endometiotic cells') of women with endometriosis have been found to have dysregulated miRNA in comparison to cells from the uterus of women who do not have endometriosis (referred to as 'eutopic endometrial cells')(<i>Burney et. al 2009, Joshi NR et. al 2015, Qun Pan et. al 2007</i>). In order to study the uses of these miRNAs in our circuit, eight candidates were chosen based on fold difference in miRNA level between the the endometrial biopsy from women with and without endometriosis. The eight candidates are shown in the table below:
 
Cells from the uterus (referred to as 'eutopic endometiotic cells') of women with endometriosis have been found to have dysregulated miRNA in comparison to cells from the uterus of women who do not have endometriosis (referred to as 'eutopic endometrial cells')(<i>Burney et. al 2009, Joshi NR et. al 2015, Qun Pan et. al 2007</i>). In order to study the uses of these miRNAs in our circuit, eight candidates were chosen based on fold difference in miRNA level between the the endometrial biopsy from women with and without endometriosis. The eight candidates are shown in the table below:
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center> How can our circuit sense miRNA activity? </h1>   
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> How can our circuit sense miRNA activity? </h1>   
  
 
<img src = "https://static.igem.org/mediawiki/2016/c/c2/T--MIT--miRNA_ts_setup.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
 
<img src = "https://static.igem.org/mediawiki/2016/c/c2/T--MIT--miRNA_ts_setup.PNG" style = 'padding: 5px 10px 5x 10px; width: 400px; height = 400px; float: right; border:10px;'>
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center> Are the target sites sensitive to miRNA levels? </h1>  
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> Are the target sites sensitive to miRNA levels? </h1>  
  
 
<img src = "https://static.igem.org/mediawiki/2016/9/9c/T--MIT--tHESC_siRNA.PNG" style = 'padding: 5px'; width: 200px; height = 350px; float: right; border:5px;'>
 
<img src = "https://static.igem.org/mediawiki/2016/9/9c/T--MIT--tHESC_siRNA.PNG" style = 'padding: 5px'; width: 200px; height = 350px; float: right; border:5px;'>
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center> Probing tHESC for miRNA Levels </h1>
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<h1 style="color:#000000; background-color:#FF9733; -moz-border-radius: 15px; -webkit-border-radius: 15px; padding:15px; text-align: center"> Probing tHESC for miRNA Levels </h1>
  
 
<p> <img src = "https://static.igem.org/mediawiki/2016/3/3b/T--MIT--tHESC_profile.PNG" style = "padding:10px; width: 250px; height = 250px; float: right; border:10px;">  
 
<p> <img src = "https://static.igem.org/mediawiki/2016/3/3b/T--MIT--tHESC_profile.PNG" style = "padding:10px; width: 250px; height = 250px; float: right; border:10px;">  

Revision as of 17:17, 16 October 2016

A page

How can we detect endometriosis using miRNA?




















What is miRNA?


For those who are not familiar with miRNA, miRNA are small units of RNA found in all mammalian cells and play a regular role in gene regulation. miRNA is single stranded and only about 20 nucleotides long. With the help of other proteins(which form what is called the 'RISC complex'), miRNA is guided to a complementary part on the mRNA (miRNA target site) where miRNA binds and cleaves messenger RNA. This inhibits gene expression post transcriptionally.



Why is miRNA a good biomarker?


There are hundreds of different miRNAs that can make up a specific cell's miRNA profile. The profile and activity of miRNA varies between different cell types and different cells states.This makes it is possible to tell the difference between cells from the uterus and liver, and the difference between healthy and diseased cells based on miRNA profiles.


Found in all mammalian cells


Unique to cell type


Unique to cell states


What is the miRNA profile of Endometriosis?

Cells from the uterus (referred to as 'eutopic endometiotic cells') of women with endometriosis have been found to have dysregulated miRNA in comparison to cells from the uterus of women who do not have endometriosis (referred to as 'eutopic endometrial cells')(Burney et. al 2009, Joshi NR et. al 2015, Qun Pan et. al 2007). In order to study the uses of these miRNAs in our circuit, eight candidates were chosen based on fold difference in miRNA level between the the endometrial biopsy from women with and without endometriosis. The eight candidates are shown in the table below:

Figure Burney et. al 2009 One of the graphs observed in order to choose miRNA candidates to characterize. The three miRNAs, miR-34c-5p, miR-9, miR-34-b are shown to be downregulated and have a significant fold difference (p*=0.05 p**<0.05) compared to their respective levels in healthy eutopic endometrial cells. These measurements were from an endometrial biopsy and taken using quantitative polymerase chain reaction (qPCR), similar to the other sources we also studied.










How can our circuit sense miRNA activity?

Our circuit utilizes the natural function of miRNA to regulate gene expression. Depending on the miRNA activity in a cell, different levels of our desired gene will be expressed. This allows our circuit to produce a differential output depending on whether the cell has dysregulated miRNA. This is achieved by attaching 4 tandem sites complementary to the affected miRNA following a gene of interest. This is called the miRNA target site (miRNA-ts).

Read more about our decision to use miRNA sensors




Are the target sites sensitive to miRNA levels?

These 4 tandem sites for miRNA binding were tested by coding for them distal to the gene for red fluorescent protein. We were able to see a ten fold repression upon increasing the concentration of siRNA from 0 to 1 nM. Saturation appeared at about 10 nM.

Read more about our experiment testing miRNA target site sensitivity

Probing tHESC for miRNA Levels


Unlike more common cell lines like MCF7 and HEK293, tHESC is not a highly characterized cell line. This meant that the levels of our eight miRNA candidates were completely unknown in tHESC. We set out to characterize our miRNA target sites (miRNA-ts) in tHESC using a miRNA sensor.

What is tHESC?

Read more about our experiment to characterize miRNA targets sites in tHESC