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The Non-Specific Binding of Fluorescent-Labeled MiRNAs on Cell Surface by Hydrophobic Interaction

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      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.

      Methods and 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.

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      Most cited references 29

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      Circulating microRNAs as stable blood-based markers for cancer detection.

      Improved approaches for the detection of common epithelial malignancies are urgently needed to reduce the worldwide morbidity and mortality caused by cancer. MicroRNAs (miRNAs) are small ( approximately 22 nt) regulatory RNAs that are frequently dysregulated in cancer and have shown promise as tissue-based markers for cancer classification and prognostication. We show here that miRNAs are present in human plasma in a remarkably stable form that is protected from endogenous RNase activity. miRNAs originating from human prostate cancer xenografts enter the circulation, are readily measured in plasma, and can robustly distinguish xenografted mice from controls. This concept extends to cancer in humans, where serum levels of miR-141 (a miRNA expressed in prostate cancer) can distinguish patients with prostate cancer from healthy controls. Our results establish the measurement of tumor-derived miRNAs in serum or plasma as an important approach for the blood-based detection of human cancer.
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        Characterization of microRNAs in serum: a novel class of biomarkers for diagnosis of cancer and other diseases.

         Xi Chen,  Yi Ba,  Lijia Ma (2008)
        Dysregulated expression of microRNAs (miRNAs) in various tissues has been associated with a variety of diseases, including cancers. Here we demonstrate that miRNAs are present in the serum and plasma of humans and other animals such as mice, rats, bovine fetuses, calves, and horses. The levels of miRNAs in serum are stable, reproducible, and consistent among individuals of the same species. Employing Solexa, we sequenced all serum miRNAs of healthy Chinese subjects and found over 100 and 91 serum miRNAs in male and female subjects, respectively. We also identified specific expression patterns of serum miRNAs for lung cancer, colorectal cancer, and diabetes, providing evidence that serum miRNAs contain fingerprints for various diseases. Two non-small cell lung cancer-specific serum miRNAs obtained by Solexa were further validated in an independent trial of 75 healthy donors and 152 cancer patients, using quantitative reverse transcription polymerase chain reaction assays. Through these analyses, we conclude that serum miRNAs can serve as potential biomarkers for the detection of various cancers and other diseases.
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          Mammalian microRNAs predominantly act to decrease target mRNA levels

          Summary MicroRNAs (miRNAs) are endogenous ∼22-nucleotide RNAs that play important gene-regulatory roles by pairing to the mRNAs of protein-coding genes to direct their repression. Repression of these regulatory targets leads to decreased translational efficiency and/or decreased mRNA levels, but the relative contributions of these two outcomes have been largely unknown, particularly for endogenous targets expressed at low-to-moderate levels. Here, we use ribosome profiling to measure the overall effects on protein production and compare these to simultaneously measured effects on mRNA levels. For both ectopic and endogenous miRNA regulatory interactions, lowered mRNA levels account for most (≥84%) of the decreased protein production. These results show that changes in mRNA levels closely reflect the impact of miRNAs on gene expression and indicate that destabilization of target mRNAs is the predominant reason for reduced protein output.
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            Author and article information

            Affiliations
            [1 ]Division of Endocrinology and Metabolism, Shandong Provincial Hospital affiliated to Shandong University, Jinan, China
            [2 ]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
            [3 ]Health Division of Guard Bureau, General Staff Department of Chinese PLA, Beijing, China
            [4 ]Traditional Chinese Medicine Department, Jinan Firefighting Hospital, Jinan, China
            University of Torino, ITALY
            Author notes

            Competing Interests: The authors have declared that no competing interests exist.

            Conceived and designed the experiments: QYZ ZW. Performed the experiments: TL ZWL. Analyzed the data: TL XWW. Contributed reagents/materials/analysis tools: JWR PY. Wrote the paper: TL QYZ.

            Contributors
            Role: Editor
            Journal
            PLoS One
            PLoS ONE
            plos
            plosone
            PLoS ONE
            Public Library of Science (San Francisco, CA USA )
            1932-6203
            1 March 2016
            2016
            : 11
            : 3
            26930565
            4773022
            10.1371/journal.pone.0149751
            PONE-D-15-40873
            (Editor)
            © 2016 Lu et al

            This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

            Counts
            Figures: 5, Tables: 0, Pages: 17
            Product
            Funding
            This work was supported by 81370270, 91439111 (ZQY), 81570712, 81370890 (WZ), the National Natural Science Foundation of China, www.nsfc.gov.cn; JQ201519 (ZQY), ZR2010HL012, Shandong Provincial Natural Science Foundation; and 2012YD18079, Science and Technology Development Program of Shandong Province, www.sdstc.gov.cn. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
            Categories
            Research Article
            Biology and life sciences
            Genetics
            Gene expression
            Gene regulation
            MicroRNAs
            Biology and life sciences
            Biochemistry
            Nucleic acids
            RNA
            Non-coding RNA
            MicroRNAs
            Research and analysis methods
            Biological cultures
            Cell lines
            HeLa cells
            Research and analysis methods
            Biological cultures
            Cell cultures
            Cultured tumor cells
            HeLa cells
            Biology and Life Sciences
            Cell Biology
            Cell Physiology
            Cell Binding
            Biology and Life Sciences
            Molecular Biology
            Molecular Biology Techniques
            Transfection
            Research and Analysis Methods
            Molecular Biology Techniques
            Transfection
            Physical Sciences
            Materials Science
            Materials by Attribute
            Pigments
            Dyes
            Fluorescent Dyes
            Research and Analysis Methods
            Microscopy
            Light Microscopy
            Fluorescence Microscopy
            Research and Analysis Methods
            Microscopy
            Light Microscopy
            Fluorescence Recovery after Photobleaching
            Engineering and Technology
            Equipment
            Optical Equipment
            Lasers
            Custom metadata
            All relevant data are within the paper and its supporting Information files.

            Uncategorized

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