Abstract
Chemically synthetic siRNA and miRNA have become powerful tools to study gene function in the past decade. Fluorescent dyes covalently attached to the 5′ or 3′ ends of synthetic small RNAs are widely used for fluorescently imaging and detection of these RNAs. However, the reliability of fluorescent tags as small RNA markers in different conditions has not attracted enough attention. We used Cy3-labelled small RNAs to explore the reliability of fluorescent tags as small RNA markers in cell cultures involving serum. A strong Cy3-fluorescence signal was observed in the cytoplasm of the cells transfected with Cy3-miR24 in the culture medium containing fetal bovine serum (FBS), but qRT-PCR results showed that little miR24 were detected in these cells. Further study demonstrated that small RNAs were degraded in the presence of FBS, suggesting that it was Cy3-RNA fragments, rather than the original Cy3-miR24, diffused into cells. These phenomena disappeared when FBS was replaced by boiled-FBS, further supporting that the Cy3-fluorescence we observed in cells in the presence of FBS could not represent the presence of intact small RNAs. These findings addressed that fluorescent tags are not reliable for small RNA transfection in the presence of serum in culture.
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Acknowledgements
We thank Dr Xue-Mei Hao and Xiao-Chen Li for professional technical support, and Rong-Chang Li and Yun-Bo Guo for their help. This study was supported by the 973 Major State Basic Research Program of China (2011CB809101) and the National Natural Science Foundation of China (30730013).
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Corresponding editor: B JAGADEESHWAR RAO
MS received 16 January 2013; accepted 22 April 2013
Corresponding editor: B Jagadeeshwar Rao
[Han J, Wang Q-W and Wang S-Q 2013 Fluorescent tag is not a reliable marker for small RNA transfection in the presence of serum. J. Biosci. 38 471–478] DOI 10.1007/s12038-013-9336-5
Supplementary materials pertaining to this article are available on the Journal of Biosciences Website at http://www.ias.ac.in/jbiosci/sep2013/supp/Han.pdf
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Han, J., Wang, QW. & Wang, SQ. Fluorescent tag is not a reliable marker for small RNA transfection in the presence of serum. J Biosci 38, 471–478 (2013). https://doi.org/10.1007/s12038-013-9336-5
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DOI: https://doi.org/10.1007/s12038-013-9336-5