Team:Heidelberg/Notebook/miMeasure

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Revision as of 23:31, 26 October 2010

Contents

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
sequencebinding site featureName/number
ctcagtttactagtgccatttgttcperfect binding site against miRsAgM12
ctcagtttactagacgcatttgttcmiMeasure with randomised nucleotides 10-12M13
ctcagtttactagtaacatttgttcmiMeasure with randomised nucleotides 10-12M14
ctcagtttactagacggatttgttcmiMeasure with randomised nucleotides 9-12M15
ctcagtttactagatgtatttgttcmiMeasure with randomised nucleotides 9-12M16
ctcagtttactagtggcatttgttcmiMeasure with mutated nucleotide 10M17
ctcagtttactagtgacatttgttcmiMeasure with mutated nucleotide 10M18
ctcagtttactagtaccatttgttcmiMeasure with mutated nucleotide 9M20
ctcagttatgtagtgccatttgttcmiMeasure with mutated nucleotide 9M22

Table2

table 2: raPCR designed binding sites and their features
binding site featureName/number
miMeasure with 3 aligned perfect binding sitesmiM-1.3-7
miMeasure with two imperfect binding sitesmiM-3.1-8
miMeasure with randomised nucleotides 9-12miM-r12
miMeasure with randomised nucleotides 9-22miM-r22
July
MTWTFSS
1234
567891011
12131415161718
19202122232425
262728293031
August
MTWTFSS
1
2345678
9101112131415
16171819202122
23242526272829
3031
September
MTWTFSS
12345
6789101112
13141516171819
20212223242526
27282930 -
October
MTWTFSS
123
45678910
11121314151617
18192021222324
25262728293031
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