Innovative microRNA purification based on surface properties modulation.
The increasing interest in circulating microRNAs (miRNAs) as potential non-invasive cancer biomarkers has prompted the rapid development of several extraction techniques. However, current methods lack standardization and are costly and labor intensive. In light of this, we developed a microRNA solid-phase extraction strategy based on charge and roughness modulation on substrate surfaces. PECVD treated silicon oxide (PECVD-SO) and thermally grown silicon oxide (TG-SO) surfaces were functionalized with positively charged 3-aminopropyltriethoxysilanes (APTES) and neutral poly(ethylene glycol) silanes (PEG-s) mixed in different proportions to modulate the density of net positive charges and the roughness of the substrate. Characterization of the surfaces was performed by atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and s-SDTB (sulfosuccinimidyl-4-o-(4,4-dimethoxytrityl) butyrate) assay in order to investigate the surface morphology and chemical composition, respectively. Adsorption and elution efficiency were assessed by fluorescence microscopy by means of synthetic fluorescently labeled microRNAs. We identified PECVD-SO functionalized with 0.1% APTES and 0.9% 21-24 units long PEG-s as a promising surface able to selectively bind microRNAs and release them in the presence of a basic buffer (pH=9) compatible with downstream analyses. MicroRNA integrity was assessed by reverse transcription and real-time PCR and confirmed by electrophoresis (Agilent 2100 Bioanalyzer), while binding competition from circulating DNA and proteins was excluded by fluorescence analyses and real-time PCR. On the contrary, total RNA slightly decreased miRNA adsorption. In conclusion, we showed an innovative and easy solid-state purification method for circulating miRNAs based on charge interaction, which could pave the path to future diagnostic and prognostic assays feasible as a routine test.
Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo, Trento, Italy.
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Santini
Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo, Trento, Italy; CNR - Consiglio Nazionale delle Ricerche, Istituto di Biofisica, via alla Cascata 56/C, I-38123 Povo, Trento, Italy. Electronic address: cpotrich@fbk.eu.
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Potrich
Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo, Trento, Italy; CNR - Consiglio Nazionale delle Ricerche, Istituto di Biofisica, via alla Cascata 56/C, I-38123 Povo, Trento, Italy.
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Lunelli
Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo, Trento, Italy.
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Pasquardini
Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo, Trento, Italy.
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Vaghi
Fondazione Bruno Kessler, Laboratory of Biomolecular Sequence and Structure Analysis for Health, via Sommarive 18, I-38123 Povo, Trento, Italy.
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Pederzolli
D035683:MicroRNAs / Q000302:isolation & purification*; D058626:Plasma Gases / Q000737:chemistry; D012822:Silicon Dioxide / Q000737:chemistry; D013499:Surface Properties
D035683:MicroRNAs; D058626:Plasma Gases; D012822:Silicon Dioxide
Charge modulation; MicroRNA purification; MicroRNA real-time PCR; Surface silanization