Team:Heidelberg/Project/miMeasure
From 2010.igem.org
(→=Flow cytometry measurements) |
(→Confocal microscopy measurements) |
||
Line 29: | Line 29: | ||
====Confocal microscopy measurements==== | ====Confocal microscopy measurements==== | ||
- | We used microscopy analysis to determine the EGFP expression in relation to EBFP2. EBFP2 serves as a normalization for transfection efficiency. Nine miMeasure constructs with different binding sites were designed. | + | We used microscopy analysis to determine the EGFP expression in relation to EBFP2. EBFP2 serves as a normalization for transfection efficiency. Nine miMeasure constructs with different binding sites were designed. Those were cloned downstream of EGFP behind the miMeasure construct, whereas the EBFP2 expression stays unaffected. The GFP/BFP-ratio stand for the level of GFP-expression normalized to one copy per cell. We modified binding sites for the synthetic miRNA by introducing random basepairs surrounding the seed region as described above, thereby changing the knockdown efficiency. In figure 1 we plotted the knockdown percentage of our constructs. The miMeasure construct and negative control were [https://2010.igem.org/Team:Heidelberg/Notebook/Methods#Transfection co-transfected] with either shRNA miRsAg expressed from a CMV promoter on pcDNA5 or an inert synthetic RNA as control in 1:4 ratio, respectively. |
[[Image:M12-M22_HeLa_daten.jpg|thumb|500px|center|'''GFP/BFP ratio normalized by the negative control''' The data are generated by confocal microscopy of Hela cells, which were transfected with different miMeasure constructs M12-M22 including the negative control (miMeasure construct without binding site). The negative control equals to 1.]] | [[Image:M12-M22_HeLa_daten.jpg|thumb|500px|center|'''GFP/BFP ratio normalized by the negative control''' The data are generated by confocal microscopy of Hela cells, which were transfected with different miMeasure constructs M12-M22 including the negative control (miMeasure construct without binding site). The negative control equals to 1.]] | ||
Line 61: | Line 61: | ||
- | Comparing the GFP/BFP-ratio between the constructs, we can see a significant difference of GFP expression | + | Comparing the GFP/BFP-ratio between the constructs, we can see a significant difference of GFP expression between the negative control and the construct containing the perfect binding site. Since the control is not downregulated due to lack of binding sites, we set it as 100% expression on this chart. It can be seen that the perfect binding sites causes the lowest GFP expression, approximately 50%, while other binding sites range in between 55% and 100% of expression. |
- | + | ||
<!--discussion?The seed region is altered in M22. Since the seed region is considered the most important site for knock-down efficiency, its change diminishes the knock-down capability of the binding site completely. So the GFP expression in this case is as high as the negative control, where no binding site was inserted into the miMeasure plasmid. --> | <!--discussion?The seed region is altered in M22. Since the seed region is considered the most important site for knock-down efficiency, its change diminishes the knock-down capability of the binding site completely. So the GFP expression in this case is as high as the negative control, where no binding site was inserted into the miMeasure plasmid. --> | ||
Line 83: | Line 82: | ||
</div> | </div> | ||
</html> | </html> | ||
- | |||
====Flow cytometry measurements==== | ====Flow cytometry measurements==== |
Revision as of 14:52, 27 October 2010
|
|
||||||||||||||||||||||||||||||||||||||||||