title: The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction.
abstract: BACKGROUND
  MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. The fluorescent labeling and lipofection are two common methods for changing the levels and locating the position of cellular miRNAs. Despite many studies about the mechanism of DNA/RNA lipofection, little is known about the characteristics, mechanisms and specificity of lipofection of fluorescent-labeled miRNAs.
  
  
  RESULTS
  Therefore, miRNAs labeled with different fluorescent dyes were transfected into adherent and suspension cells using lipofection reagent. Then, the non-specific binding and its mechanism were investigated by flow cytometer and laser confocal microscopy. The results showed that miRNAs labeled with Cy5 (cyanine fluorescent dye) could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent. The binding of miRNAs labeled with FAM (carboxyl fluorescein) to K562 cells was obvious, but it was not significant in Hela cells. After lipofectamine reagent was added, most of the fluorescently labeled miRNAs binding to the surface of Hela cells were transfected into intra-cell because of the high transfection efficiency, however, most of them were still binding to the surface of K562 cells. Moreover, the high-salt buffer which could destroy the electrostatic interactions did not affect the above-mentioned non-specific binding, but the organic solvent which could destroy the hydrophobic interactions eliminated it.
  
  
  CONCLUSIONS
  These results implied that the fluorescent-labeled miRNAs could non-specifically bind to the cell surface by hydrophobic interaction. It would lead to significant errors in the estimation of transfection efficiency only according to the cellular fluorescence intensity. Therefore, other methods to evaluate the transfection efficiency and more appropriate fluorescent dyes should be used according to the cell types for the accuracy of results.
authors.affiliation: Division of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China.
authors.forename: Ting
authors.initials: T
authors.lastname: Lu
authors.affiliation: The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health; The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, China.
authors.forename: Zongwei
authors.initials: Z
authors.lastname: Lin
authors.affiliation: Health Division of Guard Bureau, General Staff Department of Chinese PLA, Beijing, China.
authors.forename: Jianwei
authors.initials: J
authors.lastname: Ren
authors.affiliation: Traditional Chinese Medicine Department, Jinan Firefighting Hospital, Jinan, China.
authors.forename: Peng
authors.initials: P
authors.lastname: Yao
authors.affiliation: The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health; The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, China.
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Wang
authors.affiliation: Division of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Cardiovascular Remodeling and Function Research, Chinese Ministry of Education and Chinese Ministry of Health; The State and Shandong Province Joint Key Laboratory of Translational Cardiovascular Medicine, Qilu Hospital of Shandong University, Jinan, China.
authors.forename: Qunye
authors.initials: Q
authors.lastname: Zhang
mesh_terms: D002232:Carbocyanines / Q000737:chemistry / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D005434:Flow Cytometry; D036681:Fluorescence Recovery After Photobleaching; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D020014:K562 Cells; D008081:Liposomes / Q000737:chemistry; D035683:MicroRNAs / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D055672:Static Electricity; D014162:Transfection / Q000379:methods
chemical_list: D002232:Carbocyanines; D005456:Fluorescent Dyes; D008081:Liposomes; D035683:MicroRNAs; C085321:cyanine dye 5