Abstract
BackgroundMicroRNAs 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.Methods and ResultsTherefore, 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.ConclusionsThese 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.
Highlights
MicroRNAs are small noncoding RNA gene products about 22 nt long and regulate the expression of target genes by complementarily binding to their 3 'untranslated region (3'UTR) [1]
The results showed that miRNAs labeled with Cy5 could firmly bind to the surface of adherent cells (Hela) and suspended cells (K562) even without lipofection reagent
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. These results implied that the fluorescent-labeled miRNAs could non- bind to the cell surface by hydrophobic interaction
Summary
MicroRNAs (miRNAs) are small noncoding RNA gene products about 22 nt long and regulate the expression of target genes by complementarily binding to their 3 'untranslated region (3'UTR) [1]. MicroRNAs play important roles in almost all biological processes and the pathogenesis of various diseases including cancer, cardiovascular and endocrine diseases. Methods and technologies of miRNA research were studied in depth in order to better reveal the physiological and pathological significance of miRNAs. Currently, commonly used technologies in miRNA research included the detection of miRNA expression using gene chip, high throughput sequencing and quantitative PCR, discovery and verification of miRNA target genes using a dual luciferase reporter vector, high throughput sequencing of crosslinking immunoprecipitation (HITS-CLIP), et al, wherein the transfection and tracing of miRNA mimic or inhibitor were the key technologies for studying miRNA function [5]. MicroRNAs are small noncoding RNAs about 22 nt long that play key roles in almost all biological processes and diseases. 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
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