Team:Heidelberg/Notebook/MiMeasure/October

21/10/2010

seeding HeLa and Huh7 in 96-well plates

22/10/2010

Transfection

23/10/2010

seeding HeLa and Huh7 in 96-well plates

24/10/2010

HeLa cells were transfected with miMeasure constructs with GFP expression regulated by different imperfect binding sites for miRsAg. As controls we used miMeasure construct with perfect binding site and no binding site. Each miMeasure was cotransfected with pcDNA plasmid expressing synthetic miRsAg and pcDNA5 expressing synthetic shRNA3.

Transfection of HeLa and Huh7 cells with miMeasure with binding sites for miR122.

Huh7 were transfected with 50ng of miMeasure. HeLa cells were used as a control they were transfected with miMeasure constructs and miR122. Control without miR122 expression plasmid was made.

10/26/2010

microscopy measurements

different binding sites for miRsAg

different binding sites for miR122, HeLa cotransfected with miR122 expression plasmid

• Flow cytometry measurements
  • HeLa and HuH7 cells were seeded two days. One day before the cells were transfected and 100µl of cells are used for the flow cytometry, whereby 10000 cells are measured for each sample.

GFP/BFP ratio of single cells according to flow cytometry analysis

10/27/2010

HuH7 cells seeded and trasnfected one day before are imaged under the confocal microscope.

• constructs:
  • perfect binding sites against miR-122
  • 3 perfect binding sites aligned via miRaPCR
  • 2 imperfect binding sites aligned via miRaPCR

HuH7 cells transfected with different binding sites are imaged with the confocal microscope; 63x; green = EGFP, blue = EBFP2

HeLa cells are also imaged under the confocal microscope and compared to the HuH7 cells in terms of EGFP/EGFP ratio normalized to the negative control.

comparison of HuH7 and HeLa cells": cells were transfected with the different constructs.