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Cy3 labeled control Pre-miR from Ambion binding to cell membrane.

Cy3 Pre-miR Ambion electroporation

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#1 rapane

rapane

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Posted 28 November 2011 - 08:58 AM

We are working with "pre-miRs" from Ambion and using flow cytometry to evaluate delivery eficiency. FAM labeled controls work fine when transfecting with lipofectamine. When using electroporation (e.g. for Jurkat cells or cord blood CD34+ Hematopoietic Stem Cells) we have to use the Cy3 labeled control (because FAM signal in the cytosol becomes undetectable by flow cytometry). The problem is that over 80% of the cells in the control tube without the shock (but with the Cy3 labeled control) becomes labeled. I found in another old topic, that the control pre-miR can bind to the cell membrane, resulting in a false positive (if you dont use the control to identify such problem). Is there anybody having the same trouble? How did you solved it ? We are using a high concentration (50 to 100nM) of the pre-miR (as this is the recomended). When using a FITC labeled siRNA, we can evaluate the eficiency without any problem. Could i use this to check my eficiency, during padronizations ? Thank you all in advance. Rodrigo.





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