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1.
Fa Yi Xue Za Zhi ; 35(4): 387-392, 2019 Aug.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-31532143

RESUMO

Abstract: Objective Quantitative analysis and comparison of the expression of ribonucleic acid (RNA) from frozen organs and formaldehyde-fixed and paraffin-embedded (FFPE) tissues. Methods Frozen specimens of human brain, myocardium and liver tissues as well as FFPE samples at different postmortem intervals were collected and mass concentration of RNA was extracted and detected. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) technology was used to analyze the amplification efficiency and relative expression of each RNA marker. Results The mass concentration and integrity of RNA extracted from FFPE samples were relatively low compared with frozen specimens. The amplification efficiency of RNA markers was related with RNA species and the length of amplification products. Among them, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and ß-actin (ACTB) with relatively long amplification products failed to achieve optimal amplification efficiency, whereas 5S ribosomal RNA (5S rRNA) achieved ideal amplification efficiency and showed quite stable expression across various tissues, therefore it was chosen as internal reference marker. The expression quantity of GAPDH and ACTB in frozen specimens with longer postmortem intervals and in FFPE samples with relatively long amplification products was decreased. The expressions of tissue-specific microRNAs (miRNAs), GAPDH and ACTB with relatively short amplification products had consistency in the same tissues and FFPE samples. Conclusion Through standardizing the RT-qPCR experiment, selecting the appropriate RNA marker and designing primers of appropriate product length, RNA expression levels of FFPE samples can be accurately quantified.


Assuntos
Formaldeído , MicroRNAs/análise , Inclusão em Parafina , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real/normas , Primers do DNA , Perfilação da Expressão Gênica , Humanos , Miocárdio
2.
Chem Commun (Camb) ; 55(69): 10288-10291, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31396601

RESUMO

A simple nanopore modification and sensing strategy was developed for the detection of miRNAs. This preparation and sensing approach provides a quick, simple and facile tool for the detection of specific biomolecules with high sensitivity and selectivity, and may find a wide range of applications in bio-analysis.


Assuntos
Ouro/química , Ácidos Nucleicos Imobilizados/química , MicroRNAs/análise , Nanoporos/ultraestrutura , Técnicas Biossensoriais/instrumentação , Desenho de Equipamento , Hibridização de Ácido Nucleico
3.
Chem Commun (Camb) ; 55(71): 10603-10606, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31424058

RESUMO

A truly ratiometric homogeneous electrochemical biosensor has been developed for sensitive miRNA detection based on the unique diffusion/intercalation properties of electroactive dyes without the need for electrode modification or materials preparation.


Assuntos
Técnicas Biossensoriais/métodos , Corantes/química , Técnicas Eletroquímicas/métodos , Substâncias Intercalantes/química , MicroRNAs/análise , DNA/química , Eletrodos , Exodesoxirribonucleases/química , Compostos Ferrosos/química , Limite de Detecção , Metalocenos/química , Azul de Metileno/química , Oxirredução
4.
Chem Commun (Camb) ; 55(71): 10615-10618, 2019 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-31428753

RESUMO

A programmable molecular beacon (MB) with a good discrimination capability for mature and precursor microRNAs was loaded onto the surface of Mo2C quantum dots (QDs) for accurate detection of intracellular mature microRNAs.


Assuntos
Corantes Fluorescentes/química , MicroRNAs/análise , Molibdênio/química , Pontos Quânticos/química , Animais , Técnicas Biossensoriais/métodos , Linhagem Celular Tumoral , Humanos , Limite de Detecção , Camundongos , Imagem Óptica/métodos , Precursores de RNA/análise , Propriedades de Superfície
5.
Chem Commun (Camb) ; 55(70): 10380-10383, 2019 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-31397448

RESUMO

A strategy for the photoelectrochemical detection of miRNA with ultra-low background noise was developed using tungsten diselenide-cysteine-dopamine (WSe2/Cys/DA) as a nanoprobe coupled with mismatched catalytic hairpin assembly target recycling. A superior detection limit of 3.3 aM toward miRNA-221 was achieved.


Assuntos
Cisteína/química , Dopamina/química , Técnicas Eletroquímicas/métodos , MicroRNAs/análise , Sondas Moleculares/química , Nanoestruturas , Processos Fotoquímicos , Selênio/química , Tungstênio/química , Técnicas Biossensoriais , Catálise , Humanos , Limite de Detecção , MicroRNAs/sangue , Estudo de Prova de Conceito
6.
Chem Commun (Camb) ; 55(67): 9959-9962, 2019 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-31364996

RESUMO

On the basis of BSA stabilized tetraphenylethylene nanocrystals (BSA-TPE NCs) as aggregation-induced enhanced electrochemiluminescence (ECL) emitters with high ECL efficiency and good biocompatibility, as well as molecular recognition between ß-CD and ferrocene, an ultrasensitive and versatile ECL biosensing platform was constructed to achieve microRNA detection in cancer cells.


Assuntos
Materiais Biocompatíveis/química , Corantes Fluorescentes/química , MicroRNAs/análise , Nanopartículas/química , Soroalbumina Bovina/química , Estilbenos/química , Animais , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Bovinos , Linhagem Celular Tumoral , Técnicas Eletroquímicas , Compostos Ferrosos/química , Humanos , Limite de Detecção , Medições Luminescentes , Metalocenos/química , Sensibilidade e Especificidade , beta-Ciclodextrinas/química
7.
Chem Commun (Camb) ; 55(62): 9104-9107, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31298232

RESUMO

We have developed a photoluminescent membrane for microRNA detection, consisting of chemically modified mesoporous silica nanoparticles (CaF2:Yb/Ho@MSNs) attached, via single stranded DNA probes, to flexible polyurethane fibres coated with graphene oxide (GO). By detecting the release of the luminescent nanoparticles resulting from complementary co-hybridization between target miRNA sequences and the DNA probe, accurate measurements of the miRNA concentration at high sensitivity levels can be obtained. The constructs therefore offer a route to rapid detection and the potential for early cancer diagnosis.


Assuntos
Técnicas Biossensoriais , Grafite/química , MicroRNAs/análise , Nanopartículas/química , Dióxido de Silício/química , Tamanho da Partícula , Porosidade , Propriedades de Superfície
11.
Anal Chim Acta ; 1078: 176-181, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31358217

RESUMO

Intracellular microRNA (miRNA) analysis in single cell is highly informative and offers valuable insights to its physiological and pathological state, but it must confront the pivotal challenge of gene probe delivery and conditional release. Herein, we report an assembled DNA mini-hexahedron (DMH) that can selectively package and protect miRNA probe, target-cell-specific delivery and release it based on the target sequence recognition for intracellular miRNA detection. In brief, the DMH is self-assembled from six single-stranded oligonucleotide strands through rational design, one of which containing AS1411 sequence for specific uptake. Two fluorescent dye labeled recognition strands are inserted into two DMH edges with quencher groups through partially complementary hybridization. We find that this DMH possesses great biocompatibility, good trans-membrane ability and are able to protect the gene cargo against enzymatic degradation and protein binding. Fluorescence restoration caused by the target-mediated competitive chain replacement reaction allows to simultaneous detection of two cancer-related intracellular miRNAs with little false-positive signal, providing a powerful tool to discriminate healthy normal cell and cancerous cell. Thus, the construct opens a new avenue to circumvent the challenges in gene delivery, specific delivery and intrinsic interferences resistance.


Assuntos
DNA de Cadeia Simples/química , MicroRNAs/análise , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Linhagem Celular Tumoral , DNA de Cadeia Simples/genética , Portadores de Fármacos/química , Fluoresceínas/química , Fluorescência , Corantes Fluorescentes/química , Humanos , MicroRNAs/genética , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética
12.
Analyst ; 144(16): 4917-4924, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31313769

RESUMO

MicroRNAs (miRNAs) are attractive candidates for biomarkers for early cancer diagnosis, and play vital roles in physiological and pathological processes. In this work, we developed a colorimetric and fluorescent dual-mode sensor for miRNA detection based on the optical properties of gold nanoparticles (AuNPs) and the duplex-specific nuclease (DSN)-assisted signal amplification technique. In brief, FAM labelled hairpin probes (HPs) were immobilized on AuNPs, and fluorescence was efficiently quenched by the vicinity of the fluorophores to the AuNPs surface. In the presence of target miRNAs, the HPs could specifically hybridize with miRNAs and the DNA strand in the DNA/RNA heteroduplexes could be subsequently hydrolyzed by DSN. As a result, numbers of fluorophores were released into the solution, resulting in obvious fluorescence signal recovery. Meanwhile, the target miRNAs were able to participate in other hybridization reactions. With the DSN-assisted signal amplification technique, lots of gold nanoparticles were produced with short-chain DNA on their surface, which could aggregate in salt solution and result in a colorimetric detection. The proposed dual-mode strategy offers a sensitive, accurate and selective detection method for miRNAs. One reason is that the stem of the HPs was elaborately designed to avoid hydrolyzation by DSN under optimal conditions, which ensures a relatively low background and high sensitivity. The other is that the dual-mode strategy is more beneficial for enhancing the accuracy and reproducibility of the measurements. Moreover, the unique selective-cutting ability and single-base mismatch differentiation capability of the DSN also give rise to a satisfactory selectivity. This demonstrated that the developed method could quantitatively detect miR-21 down to 50 pM with a linear calibration range from 50 pM to 1 nM, and the analytical assay of target miRNAs in cell lysate samples revealed its great potential for application in biomedical research and clinical diagnostics.


Assuntos
Corantes/química , Endonucleases/química , Ouro/química , Nanopartículas Metálicas/química , MicroRNAs/análise , Técnicas Biossensoriais/métodos , Linhagem Celular , Colorimetria , DNA/química , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico/métodos , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrometria de Fluorescência
13.
Medicine (Baltimore) ; 98(30): e16538, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31348272

RESUMO

BACKGROUND: Atrial fibrillation (AF) is recognized as the most prevalent arrhythmia, and its subsequently serious complications of heart failure and thromboembolism always raise the social attention. To date, the molecular pathogenesis of AF has largely remained unclear. Publications of contemporary studies have evaluated individual miRNAs expression signatures for AF, and findings of different studies are inconsistent and not all miRNAs reported are actually important in the pathogenesis of AF. METHODS: Medline, Embase, and Cochrane Library databases will be comprehensively searched (up to April 30, 2019) for studies identifying miRNA expression profiling in subjects with and without AF. Log10 odds ratios (logORs) and associated 95% confidence interval (95%CI) will be calculated using random-effects models. Subgroup analysis will be performed according to miRNA detecting methods, species, sample types, and ethnicities. Sensitivity analysis will be conducted to detect the robustness of the findings. The methodological quality of studies will be independently assessed using criteria adopted from the Quality Assessment of Diagnostic Accuracy Studies (QUADAS-2). Furthermore, bioinformatics analysis will be performed to identify the potential target genes in AF and the corresponding pathways of dysregulated miRNAs. Two reviewers will independently screen potential studies and extract data in a structured eligibility items, with any disagreements being resolved by consensus. RESULTS: The present systematic review will identify potential biomarkers by pooling all differentially expressed miRNAs in AF studies, as well as to predict miRNA-target interactions and to identify the potential biometric functions using bioinformatics analysis. CONCLUSIONS: This systematic review and bioinformatics analysis will identify several miRNAs as potential biomarkers for AF, and explore the biological pathways regulated by the eligible miRNAs. PROSPERO REGISTRATION NUMBER: CRD42019127594.


Assuntos
Fibrilação Atrial/genética , MicroRNAs/análise , Biomarcadores/análise , Biologia Computacional/métodos , Feminino , Humanos , Masculino , Projetos de Pesquisa , Revisão Sistemática como Assunto
14.
Biochemistry (Mosc) ; 84(4): 380-389, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31228929

RESUMO

MicroRNAs (miRNAs), a family of ∼22-nucleotide non-coding single-stranded RNA molecules, are considered as key post-transcriptional regulators of gene expression that regulate various biological processes in living organism. Many miRNAs have been identified in animals; however, few have been reported in Hynobiidae species. The present study is aimed to identify a full repertoire of miRNAs in Batrachuperus yenyuanensis (Yenyuan stream salamander), which would significantly increase our knowledge of miRNAs in amphibians. A small RNA library was constructed from B. yenyuanensis and sequenced using deep sequencing. As a result, 1,717,751 clean reads were obtained, representing 356 known and 80 novel miRNAs. Additionally, expression levels of eight randomly selected miRNAs in B. yenyuanensis were confirmed using the stem-loop quantitative real-time reverse transcription PCR. In addition, 13,972 targets were predicted for these identified miRNAs, although the physiological functions of many of these targets remain unknown. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis suggested that the predicted targets are involved in a variety of physiological regulatory functions in B. yenyuanensis. These results provide useful information for further research on the miRNAs involved in the growth and development of B. yenyuanensis, as well as adaptation of this species to its high-altitude habitats.


Assuntos
MicroRNAs/metabolismo , Urodelos/genética , Animais , Sequência de Bases , Biblioteca Gênica , Ontologia Genética , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , MicroRNAs/análise , MicroRNAs/química , Reação em Cadeia da Polimerase , Análise de Sequência de RNA , Testículo/metabolismo , Urodelos/metabolismo
15.
Biochemistry (Mosc) ; 84(3): 272-282, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31221065

RESUMO

Differential diagnosis of arrhythmogenic cardiomyopathy (ACM) during the disease latent phase is a challenging clinical problem that requires identification of early ACM biomarkers. Because extracellular nucleic acids are stable, specific, and can be easily detected, they can be used as reliable biomarkers of various diseases. In this study, we analyzed the levels of extracellular microRNAs and mitochondrial DNA in the conditioned medium collected from cardiomyocytes differentiated from induced pluripotent stem cells of ACM patients and healthy donor. Several microRNAs were expressed differently by the affected and healthy cardiomyocytes; therefore, they could be considered as potential ACM biomarkers.


Assuntos
Displasia Arritmogênica Ventricular Direita/diagnóstico , DNA Mitocondrial/análise , MicroRNAs/análise , Biomarcadores/análise , Células Cultivadas , DNA Mitocondrial/genética , Humanos , MicroRNAs/genética
16.
Anal Bioanal Chem ; 411(17): 3789-3800, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31161320

RESUMO

MicroRNAs (miRNAs) in a blood sample are usually measured by quantitative reverse transcription PCR (qRT-PCR), microarray, and next-generation sequencing (NGS) which requires time-consuming pre-treatment, manual operation, and a stand-alone instrument. To overcome these disadvantages, miRNA testing has been developed using the automated analyzers routinely used in clinical laboratories. An isothermal DNA amplification reaction was adapted to a fully automated immunoassay analyzer that conducts extraction, amplification, and detection processes at 37 °C in 44 min. In a reaction vessel, a pre-designed single-stranded signal DNA was amplified in the presence of miRNA, using DNA templates, DNA polymerase, and nicking endonuclease. Then, the amplified signal DNA was hybridized by one DNA probe attached to a magnetic particle and another DNA probe labeled with acridinium ester. After the chemiluminescence reaction, luminescence intensity was automatically measured. The automated assays of cancer-related miRNAs were implemented on the analyzer with throughput of 66 tests per hour. In the assays with one-step amplification, three miRNAs (miR-21-5p, miR-18a-5p, and miR-500a-3p) at concentrations lower than 100 fM were automatically detected and the cross reactivity for miR-21-5p with fifteen similar miRNAs was not higher than 0.02%. In the assay with two-step amplification, detection sensitivity and amplification rate for miR-21-5p were 3 fM and 103-fold, respectively. The coefficient of variations (CVs) in the measurement at the target concentrations from 5 fM to 1000 pM were less than 8%. Furthermore, we also achieved automated nucleic acid detection in human serum. The proposed fully automated miRNA assays showed high sensitivity, low cross reactivity, and reproducibility suitable for clinical use. Graphical abstract.


Assuntos
Imunoensaio/métodos , MicroRNAs/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Automação , Humanos , Luminescência
17.
J Biol Regul Homeost Agents ; 33(3): 946-956, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31190512

RESUMO

Female fertility plays a decisive role in the reproduction of mammals, with related issues that include oocyte or embryo quality, establishment of pregnancy, and the physiology of the tissues that contribute to reproduction and metabolic disorders associated with reproductive failure. Although reproductive failure may be attributed to various factors in different species, female infertility is largely controlled by a number of molecular signals that can be regulated in a cycle- and tissue-dependent manner.


Assuntos
Líquido Folicular/química , MicroRNAs/análise , Técnicas de Reprodução Assistida , Feminino , Humanos , Infertilidade Feminina/terapia , Oócitos , Gravidez
18.
J Biochem ; 166(3): 271-279, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31157375

RESUMO

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide. miR-484 is previously reported to be a crucial modulator during the process from precancerous lesion to cancer. Tumour suppressor candidate 5 (TUSC5) is a potential tumour suppressor, but its expression and function in HCC are obscure. In this study, we aimed to explore the roles of miR-484 and TUSC5 in HCC, and clarify the relationship between them. We demonstrated that miR-484 was significantly up-regulated in HCC, while TUSC5 was down-regulated. TUSC5 was validated as the target gene of miR-484 and both of them were associated with the prognosis of HCC patients. miR-484 mimics markedly promoted the malignant phenotypes while TUSC5 plasmid had the opposite effect. In conclusion, miR-484/TUSC5 is potential diagnostic biomarkers and therapy targets for HCC.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas de Membrana/metabolismo , MicroRNAs/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/diagnóstico , Linhagem Celular Tumoral , Feminino , Humanos , Neoplasias Hepáticas/diagnóstico , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/genética , MicroRNAs/análise , MicroRNAs/genética , Pessoa de Meia-Idade , Proteínas Supressoras de Tumor/análise , Proteínas Supressoras de Tumor/genética
19.
Talanta ; 202: 342-348, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171193

RESUMO

A molecular beacons (MBs) loaded on molybdenum disulfide (MoS2) nanosheets as fluorescence probes for sensitive and versatile detection of microRNAs (miRNAs) through hybridization chain reaction (HCR) has been designed. MoS2 was used as a adsorbent to capture the MBs and a selective fluorescence quencher to reduce the background signal. In the absence of miRNAs, HCR could not be triggered due to the stability of MB probes. The probes attached to the MoS2 surface, efficiently quenching fluorescence of the G-quadruplex/Thioflavin T. However, the presence of target miRNAs triggers the HCR process to generate large amount of HCR products. Meanwhile, the HCR products of long nanowires chain with abundant G-quadruplexes could not be adsorbed on the surface of MoS2, and therefore detach from the MoS2. Consequently, Thioflavin T could be embedded in G-quadruplexes and produced strong fluorescence signal. This fluorescence emission signal could achieve detection of miRNA as low as 4.2 pM and a wide linear ranges from 0.1 to 100 nM. In addition, a versatile fluorescence probe has been developed for detection of miRNA-21 by changing the miRNA-recognition domain of MB. Thus, the fluorescent probe would be a potential alternative tool for biomedical research and clinical molecular diagnostics.


Assuntos
Dissulfetos/química , Corantes Fluorescentes/química , Quadruplex G , MicroRNAs/análise , Sondas Moleculares/química , Molibdênio/química , Hibridização de Ácido Nucleico , Humanos
20.
Talanta ; 202: 349-353, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171194

RESUMO

Studies have shown that microRNAs affect the development of tumors. In many cases, they can be applied as biomarkers for the diagnosis of cancer; therefore, simple and sensitive analytical methods for detection of miRNAs are necessary. In this study, miR-141, which is used to diagnose several types of cancer, was detected in water and serum samples using a biosensor designed based on streptavidin-coated magnetic beads (SMBs), complementary sequences of miR-141 and PicoGreen as the fluorescent dye. The method is relatively fast and simple. Briefly, in the presence of miR-141, the complementary sequence forms a DNA:RNA double-strand on the surface of SMBs with intercalated PicoGreen. Upon attachment of the PicoGreen, the fluorescence intensity increased significantly (1000-fold). In the absence of a target, only single-stranded DNA (complementary strand of miR-141) existed on the surface of the SMBs. The fluorescence of the PicoGreen was low. The results revealed that the detection limits of the biosensor for miR-141 were 70 and 113.8 nmol L-1 in deionized water and serum samples, respectively.


Assuntos
Fluorescência , Corantes Fluorescentes/química , MicroRNAs/análise , Humanos , Compostos Orgânicos/química
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