MicroRNAs (miRNAs) are small non-coding RNAs (18-22 nucleotides) that regulate gene expression and are associated with various diseases, including Laryngeal Cancer (LCa), which has a high mortality rate due to late diagnosis. Traditional methods for miRNA detection present several drawbacks (time-consuming steps, high cost and high false positive rate). Early-stage diagnosis and selective detection of miRNAs remain challenging. This study proposes a 3D flexible biosensor that combines nanofibers (NFs), gold nanoparticles (AuNPs), and an inverse molecular sentinel (iMS) for enzyme-free, SERS-based detection of miRNA-223-3p, evaluated as a potential LCa biomarker. The electrospun flexible nanofibers decorated with AuNPs enhance Raman signal. Selective detection of miRNA-223-3p is achieved by immobilizing an iMS-DNA probe labeled with a Raman reporter (Cyanine 3) on the AuNPs. The iMS distinctive stem-and-loop structure undergoes a conformational change upon interaction with the miRNA-223-3p, producing an "on to off" SERS signal. The proposed sensor demonstrated a linear detection range from 10 to 250 fM, with a limit of detection (LOD) of 19.50 ± 0.05 fM. The sensor selectivity was confirmed by analyzing the SERS signal behaviour in the presence of both Non-complementary miRNA and miRNA with three mismatched base pairs. This easily fabricable sensor requires no amplification and offers key advantages, including sensitivity, flexibility, and cost-effectiveness.

• MeSH
• biosenzitivní techniky * metody   MeSH
• časná detekce nádoru metody   MeSH
• kovové nanočástice * chemie   MeSH
• lidé MeSH
• limita detekce MeSH
• mikro RNA * analýza   genetika   MeSH
• nádory hrtanu * diagnóza   genetika   MeSH
• nanovlákna * chemie   MeSH
• Ramanova spektroskopie * metody   MeSH
• zlato * chemie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

miRNAs are promising biomarkers but methods for their measurement are not clear. We therefore examined three miRNA detection technologies and considered the analytical characteristics essential for clinical utilization. TaqMan assays, SplintR-qPCR and miREIA were compared for their absolute quantification bias, conformity and robustness. Absolute concentrations of miR-142-5p, miR-23a-3p and miR-93-5p were measured with all three methods using 30 samples. Robustness was evaluated by measurement of miR-21-5p in five uniform experiments. Correlations were miRNA-specific, but we observed a different absolute concentration range in RT-qPCR (fmol/μl) and methods evading the RT process (amol/μl). Consistently, RT-less methods reported better robustness (CV 8-19%) than RT-qPCR (CV 39-50%). The calibration curve in TaqMan Advanced assay was influenced by dilution media. Methods avoiding RT seem to be a promising future alternative for miRNA measurement.

• MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• lidé MeSH
• mikro RNA analýza   genetika   MeSH
• nádorové biomarkery analýza   genetika   MeSH
• nádory genetika   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

MicroRNAs (miRNAs) are becoming a very important group of molecules especially since their connection to numerous diseases has been revealed. The potential in gene therapy as well as in diagnostics is being widely investigated leading to the demand of sensitive, selective and simple methods of isolation and detection. The combined advantages of magnetic particle-based separation with sensitive electrochemical detection may offer a very valuable tool for these purposes. In this study, the miR‑124 was targeted as an example analyte for development and optimization of the isolation procedure coupled to the electrochemical detection. The sensitivity of the method was demonstrated by the limit of detection at the level of nanomolar concentration (4 nM). To verify the applicability of the procedure to the real samples, miR‑124 was isolated from the human embryonic kidney cells naturally expressing this miRNA molecule and the results were compared to the amount of miR‑124 isolated from the cells transfected by the pENTR-miR‑124 plasmid leading to the overexpression of miR‑124.

• MeSH
• biosenzitivní techniky * MeSH
• lidé MeSH
• limita detekce MeSH
• messenger RNA biosyntéza   genetika   MeSH
• mikro RNA genetika   izolace a purifikace   MeSH
• nádorové buněčné linie MeSH
• regulace genové exprese u nádorů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

This paper reports a simple electrochemical strategy for the determination of microRNAs (miRNAs) using a commercial His-Tag-Zinc finger protein (His-Tag-ZFP) that binds preferably (but non-sequence specifically) RNA hybrids over ssRNAs, ssDNAs, and dsDNAs. The strategy involves the use of magnetic beads (His-Tag-Isolation-MBs) as solid support to capture the conjugate formed in homogenous solution between His-Tag-ZFP and the dsRNA homohybrid formed between the target miRNA (miR-21 selected as a model) and a biotinylated synthetic complementary RNA detector probe (b-RNA-Dp) further conjugated with a streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The electrochemical detection is carried out by amperometry at disposable screen-printed carbon electrodes (SPCEs) (- 0.20 V vs Ag pseudo-reference electrode) upon magnetic capture of the resultant magnetic bioconjugates and H2O2 addition in the presence of hydroquinone (HQ). The as-prepared biosensor exhibits a dynamic concentration range from 3.0 to 100 nM and a detection limit (LOD) of 0.91 nM for miR-21 in just ~ 2 h. An acceptable discrimination was achieved between the target miRNA and other non-target nucleic acids (ssDNA, dsDNA, ssRNA, DNA-RNA, miR-122, miR-205, and single central- or terminal-base mismatched sequences). The biosensor was applied to the analysis of miR-21 from total RNA (RNAt) extracted from epithelial non-tumorigenic and adenocarcinoma breast cells without target amplification, pre-concentration, or reverse transcription steps. The versatility of the methodology due to the ZFP's non-sequence-specific binding behavior makes it easily extendable to determine any target RNA only by modifying the biotinylated detector probe.

• MeSH
• biosenzitivní techniky přístrojové vybavení   MeSH
• buněčné linie MeSH
• elektrochemické techniky přístrojové vybavení   MeSH
• lidé MeSH
• limita detekce MeSH
• mikro RNA analýza   MeSH
• nádorové buněčné linie MeSH
• zinkové prsty * MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH

MicroRNAs (miRNAs) are small regulatory RNAs, which mediate selective repression of gene expression. miRNAs play important roles in many natural and pathological processes. Numerous tools were developed for their detection and functional analysis. There are many excellent articles covering different areas of miRNA biology in detail. At the same time, I think there are many colleagues who face a miRNA-related research problem and would appreciate having an introductory general overview of tools for miRNA analysis, which would help them in considering available options. Accordingly, this review provides an elementary roadmap to navigate among available tools for miRNA analysis. The most common problems and errors observed in miRNA research are also discussed.

• MeSH
• lidé MeSH
• mikro RNA fyziologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH

Identifikace disproporcionálně exprimovaných miRNA v periferních krevních buňkách a ve svalových buňkách u pacientů s polymyozitidou a dermatomyozitidou za účelem zjištění jejich podílu na patogenezi onemocnění. Využití kvantitativního stanovení abnormálně produkovaných miRNA k hodnocení onemocnění, jeho aktivity a prognózy.; Identification of disproportionally expressed miRNAs in peripheral blood and muscle cells in patients suffering from polymyositis and dermatomyositis; investigation of their role in pathogenesis of the disease. Usage of quantitative measurements of abnormally expressed miRNAs as markers for evaluation of disease activity and prognosis.

• MeSH
• časná diagnóza MeSH
• diagnostické techniky molekulární MeSH
• genetická predispozice k nemoci MeSH
• interpretace statistických dat MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• mikro RNA analýza   MeSH
• mikročipová analýza MeSH
• myozitida diagnóza   genetika   MeSH
• regulace genové exprese MeSH

• Konspekt
• Biochemie. Molekulární biologie. Biofyzika
• NLK Obory
• biologie
• revmatologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu IGA MZ ČR

Přestože se spektrum biomarkerů užívaných v onkologii neustále rozšiřuje, zůstává stanovení morfologie nádorů rozhodujícím kritériem diagnostiky. U karcinomů prsu bylo nedávno zjištěno, že stupeň diferenciace je provázen změnami exprese miRNA. Není však nic známo o tom, zda popsané změny souvisí se změnou morfologie a zda mají individuální nebo obecný charakter. Cílem navrhovaného projektu je použít zatím unikátní přístup a srovnat expresní profil miRNA z různých částí téhož nádoru s rozdílnou morfologickou charakteristikou i nádorů se stejnou morfologií od různých pacientek a zároveň zjistit, zda je možné tyto specifické miRNA detekovat v krvi. Pro řešení navrhujeme použít mikrodisekční metody izolace buněk z parafínových bloků TNBC a mikročipovou analýzu miRNA. Tkáňové a sérové hladiny vybraných miRNA budou sledovány pomocí qPCR v rozšířeném validačním souboru a jejich funkce bude studována pomocí experimentů na buněčných liniích. Projekt by mohl přispět k odhalení nových principů řídících morfogenezi v nádorech a rovněž k identifikaci nových diagnostických a prognostických znaků.; Although the spectrum of cancer biomarkers is constantly expanding, determining the morphology is a decisive criterion for diagnosis. Recent research in breast cancer has shown that the degree of differentiation is accompanied by changes in miRNA expression. However, nothing is known about relationship between the described changes and tumor morphology or whether these changes have an individual or general character. We suggest to compare the miRNA expression profiles from different parts of the same tumor which have different morphology as well as tumors with the same morphology from different patients and analyse whether specific miRNAs are released into blood. Microdissection of cells from paraffin blocks of TNBC and miRNA microarray analysis will be used. Tissue and serum levels of selected miRNAs will be monitored by qPCR in an extended validation set and their functions will be studied by experiments on cell lines. The project could contribute to the discovery of new principles governing morphogenesis in tumors as well as identify novel diagnostic and prognostic markers.

• MeSH
• časná detekce nádoru MeSH
• exprese genu MeSH
• lidé MeSH
• mikro RNA MeSH
• mikročipová analýza MeSH
• mikrodisekce MeSH
• morfogeneze MeSH
• nádorové biomarkery analýza   MeSH
• prognóza MeSH
• triple-negativní karcinom prsu diagnóza   MeSH
• Check Tag
• lidé MeSH
• ženské pohlaví MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• genetika, lékařská genetika
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

The microRNA (miRNA) belongs to short noncoding RNA molecules (generally 20-25 nt in length) which are able of specific gene regulation expression 1. These regulatory molecules have significant influence on different biological processes like cell proliferation, development, differentiation, apoptosis and metabolism 2. The aberrant expression of various miRNAs is involved in the cell pathological status formation. The abnormal miRNA expression levels were found at number diseases like inflammatory and autoimmune diseases, diabetes, cardiovascular and neurodegenerative diseases and cancer 3-6. Specific miRNAs expression profiles were identified at various tumors so miRNA are interesting diagnostic biomarkers. Moreover their detection might be helpful to find more about cancer staging, prognosis and/or response to treatment 7. Different studies have shown that miR-150 is upregulated in lung cancer tissue and lupus nephritis, downregulated at patients with heart failure, acute myeloblastic leukemia, colorectal cancer and systemic sclerosis 8-14. Because of miRNAs importance the simple and fast detection method is needed. Therefore, the sensitive electrochemical analysis was combined with specific magnetic particles-based isolation.

• Klíčová slova
• voltammetry,
• MeSH
• biologické markery MeSH
• elektrochemické techniky * metody   statistika a číselné údaje   využití   MeSH
• kalibrace MeSH
• magnetické jevy MeSH
• magnetismus * MeSH
• mikro RNA * izolace a purifikace   MeSH
• velikost částic MeSH
• Publikační typ
• práce podpořená grantem MeSH

Lymfomy centrální nervové soustavy (CNS) představují jednu z nejagresivnějších malignit, vyznačující se špatnou prognózou a krátkou dobou přežití. Jejich současná diagnóza je založena na metodách s nízkou citlivostí a specifičností, schopných zachytit až již rozvinutý a hůře léčitelný lymfom. MikroRNA jsou krátké nekódující, nádorově specifické RNA, regulující genovou expresi na post-transkripční úrovni, jejichž specifickou vlastností je vysoká stabilita v tělních tekutinách. Cílem návrhu je vyvinout metodu pro včasnou a citlivou detekci lymfomů CNS na základě měření extracelulární mikroRNA v mozkomíšním moku a plazmě, která by umožnila včasnou a tím efektivnější léčbu nebo CNS profylaxi. V laboratoři 1.LF UK jsme vyvinuli metodu stanovení mikroRNA v mozkomíšním moku a předběžné výsledky ukazují, že jsme identifikovali index specifických nádorových mikroRNA, schopný s vysokou senzitivitou a s několikaměsíčním předstihem detekovat lymfomové postižení CNS, předpovědět jeho progresi či relaps a odrážející účinnost léčby; nahrazující tak nedostatky současných diagnostických metod. ...; Lymphoma involving CNS represents one of the most aggressive malignancies with poor prognosis and high mortality. Currently, their diagnosis suffers from low sensitivity and specificity. More powerful tools for early detection, response evaluation and CNS relapse risk prediction are urgently needed. Lymphomas overexpress short oncogenic regulatory RNAs called microRNAs that influence tumor growth and spreading. Unlike longer species, microRNAs are surprisingly stable and detectable in body fluids including cerebrospinal fluid (CSF). Proposed aim is to develop a method for sensitive and early detection and monitoring of CNS lymphoma, based on measurement of extracellular microRNAs in CSF and plasma that can serve as criteria for more effective early treatment or CNS prophylaxis. Our preliminary data indicate that we identified specific set of oncogenic microRNAs that can with high sensitivity and several months in advance identify CNS lymphoma involvement, reflects therapy efficiency or predict progression or relapse, thus substituting the weakness of current diagnostic methods.

• MeSH
• časná detekce nádoru MeSH
• exprese genu MeSH
• krevní plazma MeSH
• lidé MeSH
• lymfom diagnóza   genetika   MeSH
• mikro RNA krev   mozkomíšní mok   MeSH
• mozkomíšní mok MeSH
• nemoci centrálního nervového systému diagnóza   genetika   MeSH
• recidiva MeSH
• Check Tag
• lidé MeSH

• Konspekt
• Patologie. Klinická medicína
• NLK Obory
• onkologie
• genetika, lékařská genetika
• neurologie
• NLK Publikační typ
• závěrečné zprávy o řešení grantu AZV MZ ČR

miRNA expression in triple-negative breast cancers (TNBC) has mainly been studied from a methodological viewpoint. However, it has not been considered that miRNA expression profile may be associated with a specific morphological entity inside every tumor. The verification of this hypothesis on a set of 25 TNBCs was the subject of our previous work, where we confirmed specific expression of the studied miRNAs in 82 samples of different morphologies including inflammatory infiltrate, spindle cell, clear cell, and metastases after RNA extraction and purification as well as microchip and biostatistical analysis. In the current work, we demonstrate a low suitability of in situ hybridization method for miRNA detection compared to RT-qPCR, and in detail discuss the biological role of 8 miRNAs with the most significant changes of expression.

• MeSH
• lidé MeSH
• mikro RNA * genetika   metabolismus   MeSH
• triple-negativní karcinom prsu * genetika   patologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH