small RNA sequencing
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Marine oomycetes have recently been shown to be concurrently infected by (-)ssRNA viruses of the order Bunyavirales. In this work, even higher virus variability was found in a single isolate of Phytophthora condilina, a recently described member of Phytophthora phylogenetic Clade 6a, which was isolated from brackish estuarine waters in southern Portugal. Using total and small RNA-seq the full RdRp of 13 different potential novel bunya-like viruses and two complete toti-like viruses were detected. All these viruses were successfully confirmed by reverse transcription polymerase chain reaction (RT-PCR) using total RNA as template, but complementarily one of the toti-like and five of the bunya-like viruses were confirmed when dsRNA was purified for RT-PCR. In our study, total RNA-seq was by far more efficient for de novo assembling of the virus sequencing but small RNA-seq showed higher read numbers for most viruses. Two main populations of small RNAs (21 nts and 25 nts-long) were identified, which were in accordance with other Phytophthora species. To the best of our knowledge, this is the first study using small RNA sequencing to identify viruses in Phytophthora spp.
- MeSH
- fylogeneze MeSH
- genom virový MeSH
- malá nekódující RNA genetika MeSH
- otevřené čtecí rámce MeSH
- Phytophthora virologie MeSH
- RNA virová genetika MeSH
- RNA-viry klasifikace genetika izolace a purifikace MeSH
- sekvenční analýza DNA MeSH
- sekvenční analýza RNA * MeSH
- sekvenování transkriptomu MeSH
- virové nemoci virologie MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Portugalsko MeSH
This study describes the application of high-throughput sequencing of small RNA analysis of the efficacy of using Ribavirin to eliminate Grapevine leafroll-associated virus 1, Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus from Vitis vinifera cv. Riesling. The original plant used for sanitation by Ribavirin treatment was one naturally infected with all the viruses mentioned above as confirmed by RT-PCR. A tissue cultures of the plant were established and plantlets obtained were sanitized using Ribavirin. Three years after sanitation, a small RNA sequencing method for virus detection, targeting 21, 22 and 24 nt-long viral small RNAs (vsRNAs), was used to analyze both the mother plant and the sanitized plants. The results showed that the mother plant was infected by the three mentioned viruses and additionally by two viroids - Hop stunt viroid and Grapevine yellow speckle viroid 1. After Ribavirin treatment, the plants contained only the two viroids, with the complete elimination of all the viruses previously present.
- MeSH
- nemoci rostlin prevence a kontrola virologie MeSH
- ribavirin farmakologie MeSH
- RNA virová genetika MeSH
- rostlinné viry účinky léků genetika MeSH
- sekvenční analýza DNA MeSH
- Vitis virologie MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Small RNA-sequencing (RNA-Seq) is being increasingly used for profiling of circulating microRNAs (miRNAs), a new group of promising biomarkers. Unfortunately, small RNA-Seq protocols are prone to biases limiting quantification accuracy, which motivated development of several novel methods. Here, we present comparison of all small RNA-Seq library preparation approaches that are commercially available for quantification of miRNAs in biofluids. Using synthetic and human plasma samples, we compared performance of traditional two-adaptor ligation protocols (Lexogen, Norgen), as well as methods using randomized adaptors (NEXTflex), polyadenylation (SMARTer), circularization (RealSeq), capture probes (EdgeSeq), or unique molecular identifiers (QIAseq). There was no single protocol outperforming others across all metrics. Limited overlap of measured miRNA profiles was documented between methods largely owing to protocol-specific biases. Methods designed to minimize bias largely differ in their performance, and contributing factors were identified. Usage of unique molecular identifiers has rather negligible effect and, if designed incorrectly, can even introduce spurious results. Together, these results identify strengths and weaknesses of all current methods and provide guidelines for applications of small RNA-Seq in biomarker research.
Celogenomové sekvenační analýzy odhalily, že převážná část lidského genomu je transkribována, a identifikovaly tisíce protein nekódujících transkriptů. Nekódující RNA (ncRNA) se dělí na dvě hlavní skupiny: malé a dlouhé ncRNA. Tento přehledový článek je zaměřen na ncRNA s regulační funkcí, a to především na mikroRNA a dlouhé ncRNA. Tyto ncRNA regulují genovou expresi na transkripční a posttranskripční úrovni. V tomto kontextu ncRNA zasahují do regulace většiny buněčných procesů a jejich deregulace má vážné dopady na fenotyp. Již stovky studií prokázaly zapojení ncRNA do patogeneze mnoha onemocnění, od metabolických poruch přes onemocnění orgánových systémů až po různé typy nádorů. Z klinického hlediska patří ncRNA do nové generace diagnostických a prognostických biomarkerů s velkým potenciálem. Vzhledem k vysoké tkáňové specifciitě a schopnosti regulovat více genů často v rámci jedné signální dráhy představují ncRNA i atraktivní terapeutické cíle. Narůstající poznatky o širokém spektru působení ncRNA ukazují na klíčovou roli těchto transkriptů v regulaci exprese. Řada aspektů z biologie ncRNA ještě není objasněna a jejich pochopení nám poskytne nový pohled na komplexnost regulační sítě.
Whole-genome sequencing analyses revealed that the majority of the human genome is transcribed and identified thousands of protein non-coding transcripts. Non-coding RNAs (ncRNAs) are divided into two main groups: small and long ncRNAs. This review is focused on the regulatory ncRNAs mainly on microRNAs and long ncRNAs. These ncRNAs regulate gene expression at the transcriptional and post-transcriptional levels. In this context, ncRNAs are involved in the regulation of most cellular processes and their deregulation has serious impacts on the phenotype. Hundreds of studies have implicated ncRNAs in the pathogenesis of many diseases ranging from metabolic disorders to diseases of organ systems as well as various types of cancers. Clinically, ncRNAs belong to a new generation of diagnostic and prognostic biomarkers with a great potential. Due to high tissue specificity and ability to regulate multiple genes often within one signaling pathway, ncRNAs represent attractive therapeutic targets. Increasing knowledge about a wide spectrum of ncRNA actions demonstrate a pivotal role of these transcripts in expression regulation. Many aspects of the ncRNA biology are still unclear and their understanding will provide us a new perspective on the complexity of the regulatory network.
Biologická terapie, jejímž mechanizmem účinku je použití monoklonálních protilátek proti nějakému proteinu, je používána v klinické praxi již řadu let. V současné době ale vstupují do klinické praxe nové léky ze skupiny biologické terapie, které účinkují na principu RNA-interference. RNA-interference je proces, kterým buňky všech živých organizmů regulují expresi svých genů a při kterém může být zastaven přenos informace o syntéze konkrétního proteinu mezi DNA a ribosomy. Pro terapeutické účely se tohoto efektu dosahuje podáním umělých syntetizovaných oligonukleotidů s přesně danou sekvencí nukleosidů. Jde buď o krátké úseky dvouvláknové RNA, nebo o jednovláknové oligonukleotidy. Pro klinické využití byla nutná pro zvýšení jejich stability a odstranění některých nežádoucích účinků jejich chemická modifikace, a dále pak vazba na další substance, které umožní jejich cílený transport do požadované tkáně. Celá řada těchto léků je již v pokročilých fázích klinických studií a některé z nich vstupují na farmaceutický trh.
Biological therapy, whose mechanism of action is the use of monoclonal antibodies against a protein, has been used in clinical practice for many years. However, new drugs from the group of biological therapies that act on the principle of RNA interference are now entering clinical practice. RNA interference is the process by which cells in all living organisms regulate the expression of their genes, and in which the transfer of information about the synthesis of a particular protein between DNA and ribosomes can be stopped. For therapeutic purposes, this effect is achieved by administering artificially synthesized oligonucleotides – short chains of RNA with a precise nucleoside sequence. These are either short stretches of double- stranded RNA or single-stranded oligonucleotides. For clinical use, their chemical modification was necessary to increase their stability and remove some of their side effects, and then binding to other substances to allow their targeted transport to the desired tissue. A number of these drugs are already in advanced stages of clinical trials, and some are entering the pharmaceutical market.
The 6C RNA family is a class of small RNAs highly conserved in Actinobacteria, including the genera Mycobacterium, Streptomyces and Corynebacterium whose physiological function has not yet been elucidated. We found that strong transcription of the cgb_03605 gene, which encodes 6C RNA in C. glutamicum, was driven by the SigA- and SigB-dependent promoter Pcgb_03605. 6C RNA was detected at high level during exponential growth phase (180 to 240 molcules per cell) which even increased at the entry of the stationary phase. 6C RNA level did not decrease within 240 min after transcription had been stopped with rifampicin, which suggests high 6C RNA stability. The expression of cgb_03605 further increased approximately twofold in the presence of DNA-damaging mitomycin C (MMC) and nearly threefold in the absence of LexA. Deletion of the 6C RNA gene cgb_03605 resulted in a higher sensitivity of C. glutamicum toward MMC and UV radiation. These results indicate that 6C RNA is involved in the DNA damage response. Both 6C RNA level-dependent pausing of cell growth and branched cell morphology in response to MMC suggest that 6C RNA may also be involved in a control of cell division.
- MeSH
- bakteriální RNA genetika MeSH
- Corynebacterium glutamicum genetika růst a vývoj metabolismus MeSH
- genetická transkripce MeSH
- malá nekódující RNA chemie genetika MeSH
- promotorové oblasti (genetika) MeSH
- regulace genové exprese u bakterií MeSH
- sekvence nukleotidů MeSH
- sigma faktor metabolismus MeSH
- SOS odpověď (genetika) genetika MeSH
- stabilita RNA MeSH
- vazba proteinů MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
... AND ANALYSIS -- Fractionation of small RNA species using HPLC 29 -- Fractionation using anion-exchange ... ... gels 90 -- Recovery of RNA from agarose gels 91 -- Use of sequencing to determine the size of RNA 92 ... ... -- Sequencing genes 92 -- Sequencing RNA 93 -- References 93 -- 4. ... ... Determination of RNA sequences 95 -- An overview of methods for determining RNA sequences 95 -- Methods ... ... fragments on polyacrylamide gels 115 -- Direct sequencing methods for RNA 116 -- Sequencing RNA using ...
xi, 196 stran : ilustrace, tabulky ; 24 cm
Bylo zjištěno, že RNA interference je účinným nástrojem pro inhibici určité sekvence pro expresi genu. Tento děj využívá krátkou dvouvláknovou RNA, hlavně malou interferující RNA a krátkou nekódující RNA, zvanou mikroRNA. Tyto RNA zasahují do exprese genů tím, že je „utišují". Mechanismus RNA interference má obrovský potenciál stát se použitelným v boji proti různým nemocem.
RNA interference has been recognized to be an efficient mechanism for the sequence specific inhibition of gene-expression. This mechanism utilizes short double-stranded RNA, especially small-interfering RNA and short non-coding RNA called microRNA. These RNAs interfere with the expression of the genes in order to silence it. RNA interference mechanism has a great potential to be used as therapeutics against several disease.
The mechanism of action of various viruses has been the primary focus of many studies. Yet, the data on RNA modifications in any type of virus are scarce. Methods for the sensitive analysis of RNA modifications have been developed only recently and they have not been applied to viruses. In particular, the RNA composition of HIV-1 virions has never been determined with sufficiently exact methods. Here, we reveal that the RNA of HIV-1 virions contains surprisingly high amount of the 1-methyladenosine. We are the first to use a liquid chromatography-mass spectrometry analysis (LC/MS) of virion RNA, which we combined with m1A profiling and deep sequencing. We found that m1A was present in the tRNA, but not in the genomic HIV-1 RNA and the abundant 7SL RNA. We were able to calculate that an HIV-1 virion contains per 2 copies of genomic RNA and 14 copies of 7SL RNA also 770 copies of tRNA, which is approximately 10 times more than thus far expected. These new insights into the composition of the HIV-1 virion can help in future studies to identify the role of nonprimer tRNAs in retroviruses. Moreover, we present a promising new tool for studying the compositions of virions.
- MeSH
- adenosin analogy a deriváty metabolismus MeSH
- chromatografie kapalinová metody MeSH
- genom virový genetika MeSH
- HIV-1 genetika fyziologie MeSH
- hmotnostní spektrometrie metody MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- RNA malá cytoplazmatická genetika MeSH
- RNA transferová genetika metabolismus MeSH
- RNA virová genetika metabolismus MeSH
- sekvence nukleotidů MeSH
- sestavení viru genetika MeSH
- signál-rozpoznávající částice genetika MeSH
- virion genetika metabolismus MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
PURPOSE: Progress in radiation therapy of head and neck squamous cell carcinomas (HNSCCs) is logically linked to the development of molecular predictors that would help to enhance individually tailored treatment. MicroRNA (miRNA) expression profiles in tumors have repeatedly been tested to optimize the molecular diagnostics of HNSCC. In addition to tumor tissues, miRNAs are stably present in body fluids, including saliva, and can thus be collected non-invasively. The aim of our current study was to evaluate whether salivary miRNAs have potential as response predictors in HNSCC patients treated with intensity modulated radiation therapy (IMRT). METHODS: In total 48 HNSCC patients treated by definitive IMRT were enrolled in our prospective study. To identify predictive salivary miRNAs, we used small RNA sequencing in 14 saliva samples of HNSCC patients and qRT-PCR validation of selected miRNA candidates in an independent set of 34 patients. RESULTS: We found that salivary miR-15a-5p and miR-15b-5p exhibited differential levels between patients with and without complete remission (p = 0.025 and p = 0.028, respectively). Subsequent Kaplan-Meier analysis confirmed that patients with higher levels of miR-15a-5p reached a significantly longer locoregional progression-free survival (LPFS) than those with low levels (p = 0.024). Finally, multivariate Cox regression analysis revealed that miR-15a-5p may serve as an independent predictive biomarker of LPFS in HNSCC patients treated with IMRT (HR 0.104; 95% CI 0.004-0.911; p = 0.04). CONCLUSIONS: We conclude that salivary miR-15a-5p may represent a potential biomarker for individualized treatment decision-making in HNSCC patients.
- MeSH
- doba přežití bez progrese choroby MeSH
- lidé středního věku MeSH
- lidé MeSH
- mikro RNA genetika metabolismus MeSH
- nádory hlavy a krku genetika radioterapie MeSH
- proporcionální rizikové modely MeSH
- radioterapie s modulovanou intenzitou * MeSH
- sekvenční analýza RNA * MeSH
- senioři MeSH
- sliny metabolismus MeSH
- Check Tag
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
