"
Team:Heidelberg/Notebook/miMeasure
From 2010.igem.org
miMeasureAnalysis of binding sites against synthetic miRNA miRsAg5000 Hela cells were seeded on day one in each well of the 96 well plate. Transfection of the constructs (M12-M22) with four different conditions were carried out on day two. The ratio of transfection is 1 (M construct) : 5 (stuffer/ miRsAg/ pcDNA5/ shRNA3) with a total amount of 50ng DNA. Condition a: cotransfection with stuffer (salmon sperm DNA) Condition b: cotransfection with synthetic RNA miRsAg Condition c: cotransfection with empty pcDNA5 Condition d: cotransfection with synthetic shRNA3 A control consisting of the empty miMeasure plasmid (without binding site) was also cotransfected with the same conditions a, b, c and d. The cells were used for measurements on day three.
 Table1
miraPCR: analysis of endogenous miRNAHela and Huh7 cells were transfected with the constructs miM-r12, miM-r22, miM-1.3-7 and miM-3.1-8 (see table2). These constructs contain binding sites against miR-122. Since Huh7 cells express miR-122, the constructs will be affected in the Huh7 cells without cotransfecting any miRNAs, whereas miR-122 were cotransfected for the Hela cells. The transfection is done with lipofectamin. 70ng of total DNA is used and for Hela cells the miRNA to miMeasure construct ratio was 2:1. The cells were imaged one day after trasnfection with the Leica confocal microspe. The result shows the ratio between GFP and BFP normalized to the negative control, where miMeasure constructs without binding sites are transfected. The miMeasure construct with one perfect binding site is also transfected.
Table2
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
