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
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