Team:Heidelberg/Notebook/miMeasure

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[[Image:M12-M22_HeLa_daten.jpg|thumb|400px|center|'''HeLa cells two days after transfection with miMeasure''' (A) fluorescence signal GFP channel, 8bit; (B) fluorescence signal BFP channel, 8bit; (C) merge of channels A and B, RGB (D) cells after segmentation and automated cell counting and annotation]]
[[Image:M12-M22_HeLa_daten.jpg|thumb|400px|center|'''HeLa cells two days after transfection with miMeasure''' (A) fluorescence signal GFP channel, 8bit; (B) fluorescence signal BFP channel, 8bit; (C) merge of channels A and B, RGB (D) cells after segmentation and automated cell counting and annotation]]
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<center>
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{| class="wikitable sortable" border="0" style="text-align: left"
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|-bgcolor=#cccccc
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|+ align="top, left"|'''table 3''': Used NEB restriction enzymes.
 +
|sequence||binding site feature||Name/number||
 +
|-
 +
|AATTCctcagt ttactag tgc catttgttcCTGCA ||perfect binding site against miRsAg||M12
 +
|-
 +
|AATTCctcagt ttactag  acg  catttgttcCTGCA||miMeasure with randomised nucleotides 10-12||M13
 +
|-
 +
|AATTCctcagt  ttactag  taa  catttgttcCTGCA ||miMeasure with randomised nucleotides 10-12||M14
 +
|-
 +
|AATTCctcagt  ttactag  acgg  atttgttcCTGCA||miMeasure with randomised nucleotides 9-12||M15
 +
|-
 +
|AATTCctcagt  ttactag  atgt  atttgttcCTGCA||miMeasure with randomised nucleotides 9-12||M16
 +
|-
 +
|AATTCctcagt ttactagtg  g  catttgttcCTGCA ||miMeasure with mutated nucleotide 10||M17
 +
|-
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|AATTCctcagt ttactagtg  a  catttgttcCTGCA||miMeasure with mutated nucleotide 10||M18
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|-
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|AATTCctcagt ttactagt  a  ccatttgttcCTGCA||miMeasure with mutated nucleotide 9||M20
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|-
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|AATTCctcagt t  atg  tagtgccatttgttcCTGCA||miMeasure with mutated nucleotide 9||M22
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|-
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|-||miMeasure without any binding site||NC (negative control)
 +
|-
 +

Revision as of 21:55, 26 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.


HeLa cells two days after transfection with miMeasure (A) fluorescence signal GFP channel, 8bit; (B) fluorescence signal BFP channel, 8bit; (C) merge of channels A and B, RGB (D) cells after segmentation and automated cell counting and annotation


table 3: Used NEB restriction enzymes.
sequencebinding site featureName/number
AATTCctcagt ttactag tgc catttgttcCTGCA perfect binding site against miRsAgM12
AATTCctcagt ttactag acg catttgttcCTGCAmiMeasure with randomised nucleotides 10-12M13
AATTCctcagt ttactag taa catttgttcCTGCA miMeasure with randomised nucleotides 10-12M14
AATTCctcagt ttactag acgg atttgttcCTGCAmiMeasure with randomised nucleotides 9-12M15
AATTCctcagt ttactag atgt atttgttcCTGCAmiMeasure with randomised nucleotides 9-12M16
AATTCctcagt ttactagtg g catttgttcCTGCA miMeasure with mutated nucleotide 10M17
AATTCctcagt ttactagtg a catttgttcCTGCAmiMeasure with mutated nucleotide 10M18
AATTCctcagt ttactagt a ccatttgttcCTGCAmiMeasure with mutated nucleotide 9M20
AATTCctcagt t atg tagtgccatttgttcCTGCAmiMeasure with mutated nucleotide 9M22
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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