Revision as of 00:01, 27 October 2010

miMeasure

Seeding and transfection of cells for microscopy and flow cytometry

5000 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.

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 (M12-22) including the negative control (miMeasure construct without binding site). The negative control equals to 1.

Table1

**table 1**: used binding sites and their features
sequence	binding site feature	Name/number
ctcagtttactagtgccatttgttc	perfect binding site against miRsAg	M12
ctcagtttactagacgcatttgttc	miMeasure with randomised nucleotides 10-12	M13
ctcagtttactagtaacatttgttc	miMeasure with randomised nucleotides 10-12	M14
ctcagtttactagacggatttgttc	miMeasure with randomised nucleotides 9-12	M15
ctcagtttactagatgtatttgttc	miMeasure with randomised nucleotides 9-12	M16
ctcagtttactagtggcatttgttc	miMeasure with mutated nucleotide 10	M17
ctcagtttactagtgacatttgttc	miMeasure with mutated nucleotide 10	M18
ctcagtttactagtaccatttgttc	miMeasure with mutated nucleotide 9	M20
ctcagttatgtagtgccatttgttc	miMeasure with mutated nucleotide 9	M22

miraPCR: analysis of endogenous miRNA

Hela 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

**table 2**: raPCR designed binding sites and their features
binding site feature	Name/number
miMeasure with 3 aligned perfect binding sites	miM-1.3-7
miMeasure with two imperfect binding sites	miM-3.1-8
miMeasure with randomised nucleotides 9-12	miM-r12
miMeasure with randomised nucleotides 9-22	miM-r22

July
M	T	W	T	F	S	S
			1	2	3	4
5	6	7	8	9	10	11
12	13	14	15	16	17	18
19	20	21	22	23	24	25
26	27	28	29	30	31

August
M	T	W	T	F	S	S
						1
2	3	4	5	6	7	8
9	10	11	12	13	14	15
16	17	18	19	20	21	22
23	24	25	26	27	28	29
30	31

September
M	T	W	T	F	S	S
		1	2	3	4	5
6	7	8	9	10	11	12
13	14	15	16	17	18	19
20	21	22	23	24	25	26
27	28	29	30	-

October
M	T	W	T	F	S	S
				1	2	3
4	5	6	7	8	9	10
11	12	13	14	15	16	17
18	19	20	21	22	23	24
25	26	27	28	29	30	31

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 [[Image:M12-M22_HeLa_daten.jpg|thumb|600px|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 (M12-22) including the negative control (miMeasure construct without binding site). The negative control equals to 1.]]
+<center>
 ====Table1====
-<center>
 {| class="wikitable sortable" border="0" style="text-align: left"
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Team:Heidelberg/Notebook/miMeasure

From 2010.igem.org