Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

Peró-Gascón, Roger; Sanz‐Nebot, Victoria; Berezovski, Maxim V.; Benavente, Fernando

doi:10.1021/acs.analchem.8b00405

Cited by 28 publications

(38 citation statements)

References 57 publications

(110 reference statements)

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“…Peripheral blood (250 µl) was lysed with 750 µl TRIzol LS isolation reagent (Thermo Fisher Scientific, Inc.). Total RNA was extracted using the phenol chloroform method (33). The purity of RNA was determined by A260/A280 using UV-spectrophotometry (Nanodrop ND2000; Thermo Fisher Scientific, Inc.).…”

Section: Methodsmentioning

confidence: 99%

MicroRNA‑494‑3p protects rat cardiomyocytes against septic shock via PTEN

Kong

2018

Exp Ther Med

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The aim of the present study was to investigate the role of microRNA (miR)-494-3p in myocardial injury in patients with septic shock and the underlying mechanism. A total of 22 patients with sepsis and 17 patients with septic shock were included in the present study. In addition, 20 healthy subjects were recruited as the control group. Peripheral blood was collected from all subjects and a rat cardiomyocyte model of myocardial injury was constructed. Reverse transcription-quantitative polymerase chain reaction was used to measure miR-494-3p expression, while cell counting kit-8 assays were performed to assess cell proliferation. Flow cytometry was performed to investigate cell cycle distribution and apoptosis. Lactate dehydrogenase (LDH) assays were performed to measure LDH levels. ELISA was also performed to measure LDH, tumor necrosis factor (TNF)-α and interleukin (IL)-6 levels in cell culture supernatants. Western blotting was employed to detect phosphatase and tensin homolog (PTEN) protein expression and dual luciferase reporter assays were performed to identify the interaction between miR-494-3p and PTEN mRNA. Reduced miR-494-3p expression was correlated with myocardial damage in patients with septic shock. Sera from patients with septic shock downregulated miR-494-3p expression in rat cardiomyocytes. miR-494-3p overexpression inhibited rat cardiomyocyte injury induced by treatment with sera from patients with septic shock. Furthermore, miR-494-3p overexpression reduced the synthesis and release of TNF-α and IL-6 from rat cardiomyocytes. PTEN knockdown alleviated rat cardiomyocyte injury following treatment with serum from patients with septic shock. PTEN was demonstrated to induce the release of TNF-α and IL-6 from rat cardiomyocytes treated with septic shock serum, while miR-494-3p was demonstrated to bind to the 3′-untranslated seed region of PTEN mRNA to regulate its expression. The results of the present study suggest that miR-494-3p is downregulated in the peripheral blood of patients with septic shock and is negatively correlated with myocardial injury. The present study also indicates that miR-494-3p regulates PTEN expression, inhibits sepsis-induced myocardial injury and protects the function of cardiomyocytes. The protective effect and mechanism of action of miR-494-3p indicate that it has potential for use in the clinical diagnosis and therapy of myocardial damage.

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Section: Methodsmentioning

confidence: 99%

MicroRNA‑494‑3p protects rat cardiomyocytes against septic shock via PTEN

Kong

2018

Exp Ther Med

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“…MicroRNAs (miRNA) play an important role in a variety of cellular processes and can be associated with several diseases. Pero-Gascon et al developed a SPE-CE-MS method for the analysis of miRNAs in serum after offline sample preparation [95]. The potential of this method was demonstrated by detecting specific microRNAs only in patients suffering from B-cell chronic lymphocytic leukemia.…”

Section: Biomarkers: Peptides Metabolites and Nucleic Acidsmentioning

confidence: 99%

“…Pero‐Gascon et al. developed a SPE‐CE‐MS method for the analysis of miRNAs in serum after offline sample preparation . The potential of this method was demonstrated by detecting specific microRNAs only in patients suffering from B‐cell chronic lymphocytic leukemia.…”

Section: Analytical Applicationsmentioning

confidence: 99%

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

et al. 2018

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Capillary electrophoresis (CE) offers fast and high‐resolution separation of charged analytes from small injection volumes. Coupled to mass spectrometry (MS), it represents a powerful analytical technique providing (exact) mass information and enables molecular characterization based on fragmentation. Although hyphenation of CE and MS is not straightforward, much emphasis has been placed on enabling efficient ionization and user‐friendly coupling. Though several interfaces are now commercially available, research on more efficient and robust interfacing with nano‐electrospray ionization (ESI), matrix‐assisted laser desorption/ionization (MALDI) and inductively coupled plasma mass spectrometry (ICP) continues with considerable results. At the same time, CE‐MS has been used in many fields, predominantly for the analysis of proteins, peptides and metabolites. This review belongs to a series of regularly published articles, summarizing 248 articles covering the time between June 2016 and May 2018. Latest developments on hyphenation of CE with MS as well as instrumental developments such as two‐dimensional separation systems with MS detection are mentioned. Furthermore, applications of various CE‐modes including capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), capillary gel electrophoresis (CGE) and capillary isoelectric focusing (CIEF) coupled to MS in biological, pharmaceutical and environmental research are summarized.

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“…As for a variety of sample pre-enrichment techniques, increase of sample amount, volume or duration inevitably leads to continuous increase in the signal before breakthrough [28][29][30] . On a similar principle, we assumed that in the sandwich-like format, the signal at test zone should vary constantly with the increase of sampling volume until the saturation of capture probes with all sampling methods of LFAs.…”

Section: Validation In Mir-210 Lfasmentioning

confidence: 99%

Improvement in Detection Limit for Lateral Flow Assay of Biomacromolecules by Test-Zone Pre-enrichment

Zhang

Liu

Wang

et al. 2020

Sci Rep

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Lateral flow assay (LFA) is one of the most prevalent commercially available techniques for point-of-care tests due to its simplicity, celerity, low cost and robust operation. However, conventional colorimetric LFAs have inferior limits of detection (LODs) compared to sophisticated laboratory-based assays. Here, we report a simple strategy of test-zone pre-enrichment to improve the LoD of LfA by loading samples before the conjugate pad assembly. The developed method enables visual LODs of miR-210 mimic and human chorionic gonadotropin protein, to be improved by 10-100 fold compared with a conventional LfA setup without introducing any additional instrument and reagent except for phosphate running buffer, while no obvious difference occurred for Aflatoxin B1 (AFB1). It takes about 6-8 min to enrich every 50 μL of sample diluted with phosphate running buffer, therefore we can get visual results within 20 min. We identified a parameter by modeling the entire process, the concentration of probe-analyte conjugate at test zone when signaling unit being loaded, to be important for the improvement of visual limit of detection. In addition, the test-zone pre-enrichment did not impair the selectivity when miR-210 mimic was adopted as target. Integrated with other optimization, amplification and modification of LfAs, the developed test-zone pre-enrichment method can be applied to further improve LoD of LfAs. Lateral flow assay (LFA), a paper-based in-situ detection platform, has been widely used in diverse fields such as biomedicine, environmental health, quality control and food safety due to its rapid, inexpensive, portable measurements 1. Basing on affinity interactions such as hybridization of oligonucleotide or aptamer-target, antibody-antigen or biotin-streptavidin, the signal of the test zone on the LFA test strip changes with the concentration of a particular analyte of interest when a liquid sample is applied 2,3. There are two basic formats of LFA: sandwich format and competitive format. In addition, the competitive format could be designed in the following two ways: fixing the capture probe of target at test zone (competitive format I) or fixing the competitive analogue at test zone (competitive format II). Generally, there are two strategies to load signaling unit in the conventional LFA. The first one is the direct sampling method with drying signaling unit on the conjugate pad fixed between the nitrocellulose (NC) membrane and the sample pad before sampling. This strategy is the most widely used and easily handled one and the results usually show significant concentration dependence. However, the limit of detection (LOD) is usually poor, which is the main drawback of this conventional LFA. The other strategy is the pre-mixing/pre-incubation sampling method with mixing signaling unit with sample solution to load together onto the sample pad 4. Signaling unit is greatly diluted by sample solution in this strategy, resulting in a prolonged interaction between signaling unit and capture molecules at test zone and ...

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Analysis of Circulating microRNAs and Their Post-Transcriptional Modifications in Cancer Serum by On-Line Solid-Phase Extraction–Capillary Electrophoresis–Mass Spectrometry

Cited by 28 publications

References 57 publications

MicroRNA‑494‑3p protects rat cardiomyocytes against septic shock via PTEN

MicroRNA‑494‑3p protects rat cardiomyocytes against septic shock via PTEN

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

Improvement in Detection Limit for Lateral Flow Assay of Biomacromolecules by Test-Zone Pre-enrichment

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