| Chemical transfection of dye-conjugated microRNA precursors for microRNA functional analysis of M2 macrophages. | |
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MedLine Citation:
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PMID: 22213010 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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BACKGROUND: MicroRNAs (miRNAs) are short non-coding ribonucleic acids known to affect gene expression at the translational level and there is mounting evidence that miRNAs play a role in the function of tumor-associated macrophages (TAMs). To aid the functional analyses of miRNAs in an in-vitro model of TAMs known as M2 macrophages, a transfection method to introduce artificial miRNA constructs or miRNA molecules into primary human monocytes is needed. Unlike differentiated macrophages or dendritic cells, undifferentiated primary human monocytes have been known to show resistance to lentiviral transduction. To circumvent this challenge, other techniques such as electroporation and chemical transfection have been used in other applications to deliver small gene constructs into human monocytes. To date, no studies have compared these two methods objectively to evaluate their suitability in the miRNA functional analysis of M2 macrophages. RESULTS: Of the methods tested, the electroporation of miRNA-construct containing plasmids and the chemical transfection of miRNA precursor molecules are the most efficient approaches. The use of a silencer siRNA labeling kit (Ambion) to conjugate Cy 3 fluorescence dyes to the precursor molecules allowed the isolation of successfully transfected cells with fluorescence-activated cell sorting. The chemical transfection of these dye-conjugated miRNA precursors yield an efficiency of 37.5 ± 0.6% and a cell viability of 74 ± 1%. RNA purified from the isolated cells demonstrated good quality, and was fit for subsequent mRNA expression qPCR analysis. While electroporation of plasmids containing miRNA constructs yield transfection efficiencies comparable to chemical transfection of miRNA precursors, these electroporated primary monocytes seemed to have lost their potential for differentiation. CONCLUSIONS: Among the most common methods of transfection, the chemical transfection of dye-conjugated miRNA precursors was determined to be the best suited approach for the functional analysis of M2 macrophages. J. Cell. Biochem. © 2011 Wiley Periodicals, Inc. |
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Authors:
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Yee Seng Ng; Hernan Roca; David Fuller; Sudha Sud; Kenneth J Pienta |
Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-12-28 |
Journal Detail:
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Title: Journal of cellular biochemistry Volume: - ISSN: 1097-4644 ISO Abbreviation: - Publication Date: 2011 Dec |
Date Detail:
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Created Date: 2012-1-3 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8205768 Medline TA: J Cell Biochem Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Copyright Information:
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Copyright © 2011 Wiley Periodicals, Inc. |
Affiliation:
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Department of Internal Medicine, University of Michigan, Ann Arbor, Michigan. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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