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Transfection of human platelets with short interfering RNA.

By Wei Hong, Altaf A Kondkar, Srikanth Nagalla, Wolfgang Bergmeier, Ying Jin, Jay Herman and Paul Bray

Abstract

Platelets contain mRNAs and are capable of translating mRNA into protein, and it has been previously demonstrated that platelets increase their levels of integrin β3 overtime while in blood bank storage conditions. We are unaware of prior attempts to introduce nucleic acids into platelets. Considering the potential clinical and research utility of manipulating platelet gene expression, we tested whether small interfering RNAs (siRNAs) could be transfected into normal human platelets. Multiple conditions were tested, including lipofectamine versus electroporation, different amounts of siRNA, the effect of different buffers and the presence of plasma during transfection, and the time for optimal siRNA incorporation after transfection. Using flow cytometry to assess transfection efficiency, we found that optimal transfection was obtained using lipofectamine, washed platelets, and 400 pmoles siRNA. Cell sorting of transfected platelets suggested that the incorporated siRNA was able to knockdown the level of a targeted mRNA. This is the first ever demonstration that nucleic acids can be introduced directly into platelets, and offers proof of concept for manipulating gene expression in platelets by nonviral methods. Future technical improvements may permit improving the quality and/or lifespan of stored human platelets

Topics: Blood Platelets, Cell Separation, Electroporation, Fluorescent Dyes, Gene Expression Regulation, Genetic Techniques, Humans, RNA, Messenger, RNA, Small Interfering, Time Factors, Transfection, RNA, Messenger, RNA, Small Interfering, Hematology
Publisher: Jefferson Digital Commons
Year: 2011
OAI identifier: oai:jdc.jefferson.edu:cardeza_foundation-1024

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