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1.
Medicine (Baltimore) ; 99(31): e21297, 2020 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-32756109

RESUMO

Vitiligo is a chronic skin condition lack of melanocytes. However, researches on the aetiology and pathogenesis of vitiligo are still under debate. This study aimed to explore the key genes and pathways associated with occurrence and development of vitiligo.Weighted gene coexpression network analysis (WGCNA) was applied to reanalyze the gene expression dataset GSE65127 systematically. Functional enrichments of these modules were carried out at gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), gene set variation analysis (GSVA), and gene set enrichment analysis (GSEA). Then, a map of regulatory network was delineated according to pivot analysis and drug prediction. In addition, hub genes and crucial pathways were validated by an independent dataset GSE75819. The expressions of hub genes in modules were also tested by quantitative real-time polymerase chain reaction (qRT-PCR).Eight coexpressed modules were identified by WGCNA based on 5794 differentially expressed genes of vitiligo. Three modules were found to be significantly correlated with Lesional, Peri-Lesional, and Non-Lesional, respectively. The persistent maladjusted genes included 269 upregulated genes and 82 downregulated genes. The enrichments showed module genes were implicated in immune response, p53 signaling pathway, etc. According to GSEA and GSVA, dysregulated pathways were activated incessantly from Non-Lesional to Peri-Lesional and then to Lesional, 4 of which were verified by an independent dataset GSE75819. Finally, 42 transcription factors and 228 drugs were spotted. Focusing on the persistent maladjusted genes, a map of regulatory network was delineated. Hub genes (CACTIN, DCTN1, GPR143, HADH, MRPL47, NKTR, NUF2) and transcription factors (ITGAV, SYK, PDPK1) were validated by an independent dataset GSE75819. In addition, hub genes (CACTIN, DCTN1, GPR143, MRPL47, NKTR) were also confirmed by qRT-PCR.The present study, at least, might provide an integrated and in-depth insight for exploring the underlying mechanism of vitiligo and predicting potential diagnostic biomarkers and therapeutic targets.


Assuntos
Vitiligo/genética , Grupo com Ancestrais do Continente Asiático , Estudos de Casos e Controles , China , Biologia Computacional , Bases de Dados Factuais , Perfilação da Expressão Gênica , Humanos , RNA/análise , Vitiligo/fisiopatologia
4.
PLoS One ; 15(7): e0234993, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32645014

RESUMO

The main functions of the choroid plexus (CP) are the production of cerebral spinal fluid (CSF), the formation of the blood-CSF barrier, and regulation of immune response. This barrier allows for the exchange of specific nutrients, waste, and peripheral immune cells between the blood stream and CSF. Borrelia burgdorferi (Bb), the causative bacteria of Lyme disease, is associated with neurological complications including meningitis-indeed, Bb has been isolated from the CSF of patients. While it is accepted that B. burgdorferi can enter the central nervous system (CNS) of patients, it is unknown how the bacteria crosses this barrier and how the pathogenesis of the disease leads to the observed symptoms in patients. We hypothesize that during infection Borrelia burgdorferi will induce an immune response conducive to the chemotaxis of immune cells and subsequently lead to a pro-inflammatory state with the CNS parenchyma. Primary human choroid plexus epithelial cells were grown in culture and infected with B. burgdorferi strain B31 MI-16 for 48 hours. RNA was isolated and used for RNA sequencing and RT-qPCR validation. Secreted proteins in the supernatant were analyzed via ELISA. Transcriptome analysis based on RNA sequencing determined a total of 160 upregulated genes and 98 downregulated genes. Pathway and biological process analysis determined a significant upregulation in immune and inflammatory genes specifically in chemokine and interferon related pathways. Further analysis revealed downregulation in genes related to cell to cell junctions including tight and adherens junctions. These results were validated via RT-qPCR. Protein analysis of secreted factors showed an increase in inflammatory chemokines, corresponding to our transcriptome analysis. These data further demonstrate the role of the CP in the modulation of the immune response in a disease state and give insight into the mechanisms by which Borrelia burgdorferi may disseminate into, and act upon, the CNS. Future experiments aim to detail the impact of B. burgdorferi on the blood-CSF-barrier (BCSFB) integrity and inflammatory response within animal models.


Assuntos
Borrelia burgdorferi/patogenicidade , Plexo Corióideo/patologia , Células Epiteliais/patologia , Doença de Lyme/microbiologia , Barreira Hematoencefálica , Borrelia burgdorferi/imunologia , Células Cultivadas , Plexo Corióideo/imunologia , Plexo Corióideo/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Inflamação/metabolismo , Doença de Lyme/imunologia , Doença de Lyme/patologia , Proteínas/análise , RNA/análise
5.
PLoS One ; 15(7): e0235793, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32634162

RESUMO

Extracellular vesicles (EVs) are small vesicles secreted from cells. They have crucial biological functions in intercellular communications and may even be biomarkers for cancer. The various methods used to isolate EVs from body fluid and cell culture supernatant have been compared in prior studies, which determined that the component yield and physical properties of isolated EVs depend largely on the isolation method used. Several novel and combined methods have been recently developed, which have not yet been compared to the established methods. Therefore, the purpose of this study is to compare the physical and functional differences in EVs isolated using a differential centrifugation method, the precipitation-based Invitrogen kit, the ExoLutE kit, and the Exodisc, of which the latter two were recently developed. We investigated the properties of EVs isolated from non-infected and Kaposi's sarcoma-associated herpesvirus-infected human umbilical vein endothelial cells using each method and determined the yields of DNA, RNA, and proteins using quantitative polymerase chain reaction and bicinchoninic acid assays. Additionally, we determined whether the biological activity of EVs correlated with the quantity or physical properties of the EVs isolated using different methods. We found that Exodisc was the most suitable method for obtaining large quantities of EVs, which might be useful for biomarker investigations, and that the EVs separated using Exodisc exhibited the highest complement activation activity. However, we also found that the functional properties of EVs were best maintained when differential centrifugation was used. Effective isolation is necessary to study EVs as tools for diagnosing cancer and our findings may have relevant implications in the field of oncology by providing researchers with data to assist their selection of a suitable isolation method.


Assuntos
Fracionamento Celular/métodos , Células Endoteliais/química , Vesículas Extracelulares/química , Biomarcadores/análise , Centrifugação/métodos , Precipitação Química , DNA/análise , Células Endoteliais/virologia , Vesículas Extracelulares/virologia , Infecções por Herpesviridae/virologia , Herpesvirus Humano 8/isolamento & purificação , Células Endoteliais da Veia Umbilical Humana , Humanos , Proteínas/análise , RNA/análise
6.
Nat Commun ; 11(1): 2704, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483174

RESUMO

Index hopping is the main cause of incorrect sample assignment of sequencing reads in multiplexed pooled libraries. We introduce a statistical model for estimating the sample index-hopping rate in multiplexed droplet-based single-cell RNA-seq data and for probabilistic inference of the true sample of origin of hopped reads. We analyze several datasets and estimate the sample index hopping probability to range between 0.003-0.009, a small number that counter-intuitively gives rise to a large fraction of phantom molecules - the fraction of phantom molecules exceeds 8% in more than 25% of samples and reaches as high as 85% in low-complexity samples. Phantom molecules lead to widespread complications in downstream analyses, including transcriptome mixing across cells, emergence of phantom copies of cells from other samples, and misclassification of empty droplets as cells. We demonstrate that our approach can correct for these artifacts by accurately purging the majority of phantom molecules from the data.


Assuntos
Algoritmos , Artefatos , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Modelos Estatísticos , RNA/análise , Análise de Célula Única/métodos , Simulação por Computador , Sequenciamento de Nucleotídeos em Larga Escala/normas , Humanos , RNA/genética , Reprodutibilidade dos Testes , Análise de Célula Única/normas
7.
Nat Methods ; 17(7): 689-693, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32541852

RESUMO

We present split-FISH, a multiplexed fluorescence in situ hybridization method that leverages a split-probe design to achieve enhanced specificity. Split-FISH reduces off-target background fluorescence, decreases false positives and enables accurate RNA profiling in uncleared tissues. We demonstrate the efficacy of split-FISH on various mouse tissues by quantifying the distribution and abundance of 317 genes in single cells and reveal diverse localization patterns for spatial regulation of the transcriptome in complex tissues.


Assuntos
Hibridização in Situ Fluorescente/métodos , RNA/análise , Animais , Células Cultivadas , Humanos , Camundongos , Análise de Célula Única , Transcriptoma
8.
Nat Biotechnol ; 38(6): 708-714, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32518404

RESUMO

Large-scale sequencing of RNA from individual cells can reveal patterns of gene, isoform and allelic expression across cell types and states1. However, current short-read single-cell RNA-sequencing methods have limited ability to count RNAs at allele and isoform resolution, and long-read sequencing techniques lack the depth required for large-scale applications across cells2,3. Here we introduce Smart-seq3, which combines full-length transcriptome coverage with a 5' unique molecular identifier RNA counting strategy that enables in silico reconstruction of thousands of RNA molecules per cell. Of the counted and reconstructed molecules, 60% could be directly assigned to allelic origin and 30-50% to specific isoforms, and we identified substantial differences in isoform usage in different mouse strains and human cell types. Smart-seq3 greatly increased sensitivity compared to Smart-seq2, typically detecting thousands more transcripts per cell. We expect that Smart-seq3 will enable large-scale characterization of cell types and states across tissues and organisms.


Assuntos
Perfilação da Expressão Gênica/métodos , RNA/análise , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Alelos , Animais , Humanos , Camundongos , RNA/genética , Isoformas de RNA/análise , Isoformas de RNA/genética , Sensibilidade e Especificidade , Transcriptoma/genética
9.
J Vis Exp ; (160)2020 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-32568231

RESUMO

Gene expression analysis by RNA sequencing (RNA-seq) enables unique insights into clinical samples that can potentially lead to mechanistic understanding of the basis of various diseases as well as resistance and/or susceptibility mechanisms. However, FFPE tissues, which represent the most common method for preserving tissue morphology in clinical specimens, are not the best sources for gene expression profiling analysis. The RNA obtained from such samples is often degraded, fragmented, and chemically modified, which leads to suboptimal sequencing libraries. In turn, these generate poor quality sequence data that may not be reliable for gene expression analysis and mutation discovery. In order to make the most of FFPE samples and obtain the best possible data from low quality samples, it is important to take certain precautions while planning experimental design, preparing sequencing libraries, and during data analysis. This includes the use of appropriate metrics for precise sample quality control (QC), identifying the best methods for various steps during the sequencing library generation, and careful library QC. In addition, applying correct software tools and parameters for sequence data analysis is critical in order to identify artifacts in RNA-seq data, filter out contamination and low quality reads, assess uniformity of gene coverage, and measure the reproducibility of gene expression profiles among biological replicates. These steps can ensure high accuracy and reproducibility for profiling of very heterogeneous RNA samples. Here we describe the various steps for sample QC, library preparation and QC, sequencing, and data analysis that can help to increase the amount of useful data obtained from low quality RNA, such as that obtained from FFPE-RNA tissues.


Assuntos
Inclusão em Parafina , Estabilidade de RNA , RNA/análise , Análise de Sequência de RNA/métodos , Fixação de Tecidos , Análise de Dados , Perfilação da Expressão Gênica , Biblioteca Gênica , Genoma , Humanos , Controle de Qualidade , RNA/genética , Reprodutibilidade dos Testes , Transcriptoma
10.
PLoS One ; 15(5): e0233651, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32469980

RESUMO

Transference of RNAs and ribosomes from Schwann cell-to-axon was demonstrated in normal and regenerating peripheral nerves. Previously, we have shown that RNAs transfer is dependent on F-actin cytoskeleton and Myosin Va. Here, we explored the contribution of microtubules to newly synthesized RNAs transport from Schwann cell nuclei up to nodal microvilli in sciatic nerves. Results using immunohistochemistry and quantitative confocal FRET analysis indicate that Schwann cell-derived RNAs co-localize with microtubules in Schwann cell cytoplasm. Additionally, transport of Schwann cell-derived RNAs is nocodazole and colchicine sensitive demonstrating its dependence on microtubule network integrity. Moreover, mRNAs codifying neuron-specific proteins are among Schwann cell newly synthesized RNAs population, and some of them are associated with KIF1B and KIF5B microtubules-based motors.


Assuntos
Axônios/metabolismo , Microtúbulos/metabolismo , RNA/metabolismo , Células de Schwann/metabolismo , Nervo Isquiático/metabolismo , Animais , Masculino , Bainha de Mielina/metabolismo , Regeneração Nervosa , RNA/análise , Transporte de RNA , Ratos , Ratos Sprague-Dawley
11.
Proc Natl Acad Sci U S A ; 117(21): 11744-11752, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404418

RESUMO

Auditory experience drives neural circuit refinement during windows of heightened brain plasticity, but little is known about the genetic regulation of this developmental process. The primary auditory cortex (A1) of mice exhibits a critical period for thalamocortical connectivity between postnatal days P12 and P15, during which tone exposure alters the tonotopic topography of A1. We hypothesized that a coordinated, multicellular transcriptional program governs this window for patterning of the auditory cortex. To generate a robust multicellular map of gene expression, we performed droplet-based, single-nucleus RNA sequencing (snRNA-seq) of A1 across three developmental time points (P10, P15, and P20) spanning the tonotopic critical period. We also tone-reared mice (7 kHz pips) during the 3-d critical period and collected A1 at P15 and P20. We identified and profiled both neuronal (glutamatergic and GABAergic) and nonneuronal (oligodendrocytes, microglia, astrocytes, and endothelial) cell types. By comparing normal- and tone-reared mice, we found hundreds of genes across cell types showing altered expression as a result of sensory manipulation during the critical period. Functional voltage-sensitive dye imaging confirmed GABA circuit function determines critical period onset, while Nogo receptor signaling is required for its closure. We further uncovered previously unknown effects of developmental tone exposure on trajectories of gene expression in interneurons, as well as candidate genes that might execute tonotopic plasticity. Our single-nucleus transcriptomic resource of developing auditory cortex is thus a powerful discovery platform with which to identify mediators of tonotopic plasticity.


Assuntos
Córtex Auditivo , Núcleo Celular/metabolismo , RNA , Análise de Célula Única/métodos , Transcriptoma/genética , Animais , Córtex Auditivo/crescimento & desenvolvimento , Córtex Auditivo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Camundongos , Receptores Nogo/genética , Receptores Nogo/metabolismo , RNA/análise , RNA/genética , RNA/metabolismo , Análise de Sequência de RNA/métodos
12.
Zhonghua Gan Zang Bing Za Zhi ; 28(4): 365-368, 2020 Apr 20.
Artigo em Chinês | MEDLINE | ID: mdl-32403892

RESUMO

RNAscope is a new generation of in situ hybridization technology and with the advantage of new probe design, it is now being gradually applied to a wide range of research fields, and its research scope is constantly expanding. Our country has large number of liver disease patients, so there is a great demand for histological testing and research evaluation based upon biological information. This technology has unique specificity and sensitivity in situ level, which makes up for the technical defects of immunohistochemistry and traditional in situ hybridization. Realizing the multiplicity and monomolecular level detection, the qualitative and quantitative analysis of nucleic acid level can be carried out in many aspects of the field of liver disease, and the visual evaluation can be achieved by combining it with tissue in situ to highlight the value of "gold standard". This article summarizes the recent year's application of RNAscope technology carried out in liver histology field.


Assuntos
Imuno-Histoquímica , Hibridização In Situ , Hepatopatias/diagnóstico , RNA/análise , Humanos , Sensibilidade e Especificidade
13.
Nucleic Acids Res ; 48(11): 5859-5872, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32421779

RESUMO

Subcellular organization of RNAs and proteins is critical for cell function, but we still lack global maps and conceptual frameworks for how these molecules are localized in cells and tissues. Here, we introduce ATLAS-Seq, which generates transcriptomes and proteomes from detergent-free tissue lysates fractionated across a sucrose gradient. Proteomic analysis of fractions confirmed separation of subcellular compartments. Unexpectedly, RNAs tended to co-sediment with other RNAs in similar protein complexes, cellular compartments, or with similar biological functions. With the exception of those encoding secreted proteins, most RNAs sedimented differently than their encoded protein counterparts. To identify RNA binding proteins potentially driving these patterns, we correlated their sedimentation profiles to all RNAs, confirming known interactions and predicting new associations. Hundreds of alternative RNA isoforms exhibited distinct sedimentation patterns across the gradient, despite sharing most of their coding sequence. These observations suggest that transcriptomes can be organized into networks of co-segregating mRNAs encoding functionally related proteins and provide insights into the establishment and maintenance of subcellular organization.


Assuntos
Fracionamento Celular , Microambiente Celular , Espaço Intracelular/química , RNA/análise , RNA/metabolismo , Análise de Sequência de RNA , Transcriptoma , Animais , Extratos Celulares/química , Centrifugação com Gradiente de Concentração , Feminino , Fígado/citologia , Fígado/metabolismo , Espectrometria de Massas , Camundongos , RNA/química , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Ribossomos/química , Sacarose
14.
Braz Oral Res ; 34: e015, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32130362

RESUMO

We sought to compare the characteristics and clinical significance of neutrophil extracellular traps in gingival samples from patients with periodontitis and those with gingivitis. The clinical indexes of gingival samples from patients with periodontitis and gingivitis were measured; the expression of TNF-alpha and IL-8 was measured by real-time fluorescent quantitative PCR; and the expression of TLR-8 and MMP-9 was measured by western blotting assays. Chemotaxis, phagocytosis and phagocytic activity of neutrophils were measured. Compared with the healthy group, the expression of TNF-α and IL-8 in the periodontitis group and the gingivitis group increased significantly (p < 0.05), and TNF-α in the gingivitis group was significantly lower than that in the healthy group (p < 0.05). The expression of IL-8 in the periodontitis group was significantly higher than that in the periodontitis group (p < 0.05). Furthermore, the expression of TLR-8 and MMP-9 in the periodontitis group was different from that in the gingivitis group and the healthy group, and the expression of TLR-8 and MMP-9 in the gingivitis group was significantly different from that in the healthy group (p < 0.05). In addition, the neutrophil mobility index in healthy people was 3.02 ± 0.53, that in the periodontitis group was 2.21 ± 0.13, and that in the gingivitis group was 2.31 ± 0.12. In conclusion, the chemotaxis of neutrophils in gingival samples of patients with periodontitis and gingivitis was decreased, the phagocytotic ability and activity of neutrophils were reduced, and the release of the extracellular trap-releasing inducible factors TNF-alpha and IL-8 also declined.


Assuntos
Armadilhas Extracelulares , Gengivite/patologia , Neutrófilos/patologia , Periodontite/patologia , Actinas/análise , Adulto , Western Blotting , Estudos de Casos e Controles , Eletroforese em Gel de Ágar , Feminino , Humanos , Interleucina-8/análise , Masculino , Metaloproteinase 9 da Matriz/análise , Pessoa de Meia-Idade , Índice Periodontal , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real , Valores de Referência , Receptor 8 Toll-Like/análise , Fator de Necrose Tumoral alfa/análise , Adulto Jovem
16.
Chem Commun (Camb) ; 56(16): 2483-2486, 2020 Feb 25.
Artigo em Inglês | MEDLINE | ID: mdl-32002523

RESUMO

Through a rational construction of an RNA toehold switch sensor, the glucometer-based detection of nucleic acids was innovatively simplified into a completely homogeneous and label-free process. Compared with traditional strategies that rely on multiple operations such as chemical conjugation and bead separation, this new strategy is more robust, user-friendly, reagent-saving, and reproducible, and can be universally adapted for use on extensive target species, e.g. herein, the real-world pathogen genes.


Assuntos
Técnicas Biossensoriais , DNA/análise , RNA/análise , Técnicas de Amplificação de Ácido Nucleico
17.
PLoS One ; 15(2): e0229423, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32084228

RESUMO

RNA quality and quantity are important factors for ensuring the accuracy of gene expression analysis and other RNA-based downstream applications. Thus far, only a limited number of methodological studies have compared sample storage and RNA extraction procedures for human cells. We compared three commercially available RNA extraction kits, i.e., (NucliSENS) easyMAG, RNeasy (Mini Kit) and RiboPure (RNA Purification Kit-blood). In addition, additional conditions, such as storage medium and storage temperature of human peripheral blood mononuclear cells were evaluated, i.e., 4 °C for RNAlater or -80 °C for QIAzol and for the respective cognate lysis buffers; easyMAG, RNeasy or RiboPure. RNA was extracted from aliquots that had been stored for one day (Run 1) or 83 days (Run 2). After DNase treatment, quantity and quality of RNA were assessed by means of a NanoDrop spectrophotometer, 2100 Bioanalyzer and RT-qPCR for the ACTB reference gene. We observed that high-quality RNA can be obtained using RNeasy and RiboPure, regardless of the storage medium, whereas samples stored in RNAlater resulted in the least amount of RNA extracted. In addition, RiboPure combined with storage of samples in its cognate lysis buffer yielded twice as much RNA as all other procedures. These results were supported by RT-qPCR and by the reproducibility observed for two independent extraction runs.


Assuntos
Leucócitos Mononucleares/metabolismo , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos , Actinas/genética , Humanos , RNA/análise , RNA/genética , Kit de Reagentes para Diagnóstico , Reação em Cadeia da Polimerase em Tempo Real/normas , Padrões de Referência , Reprodutibilidade dos Testes
18.
Biochemistry (Mosc) ; 85(2): 147-166, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32093592

RESUMO

Recently, there has been a rapid progress in the development of techniques for isothermal amplification of nucleic acids as an alternative to polymerase chain reaction (PCR). The advantage of these methods is that the nucleic acids amplification can be carried out at constant temperature, unlike PCR, which requires cyclic temperature changes. Moreover, isothermal amplification can be conducted directly in living cells. This review describes the principles of isothermal amplification techniques and demonstrates their high efficiency in designing new highly sensitive detection methods of nucleic acids and enzymes involved in their modifications. The data on successful application of isothermal amplification methods for the analysis of cells and biomolecules with the use of DNA/RNA aptamers are presented.


Assuntos
DNA/análise , DNA/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA/análise , RNA/genética , Temperatura , Animais , DNA/metabolismo , Humanos , Reação em Cadeia da Polimerase , RNA/metabolismo
19.
J Chromatogr A ; 1618: 460875, 2020 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-31982098

RESUMO

Large RNAs including messenger RNAs (mRNAs) are promising candidates for development of new drug products and vaccines. Development of high resolution methods for direct analysis of large RNAs, especially for purity in general and size or length in particular, is critical to support new drug development and manufacture. However, resolution based on size or length for large RNAs is limited even by capillary electrophoresis (CE), which is one of the most efficient separation methods for nucleic acids in general. This paper presents a capillary gel electrophoresis (CGE) method for separating large RNA molecules by size or length under strongly denaturing, non-aqueous conditions. We believe that our work constitutes the first time that a gel suitable for CGE prepared with high molecular weight polymers and using only formamide as solvent has been successfully employed to analyze large RNAs on the basis of their size or length with high resolution. With an eye toward application for mRNAs in particular, separation conditions in this work were optimized for RNAs approximately 2000 nucleotides (nt) in length. As compared to a standard CGE method using an aqueous gel, resolution for commercially-available RNA ladder components at 1500 and 2000 nt is increased approximately 6-fold. The impacts of polymer type, molecular weight of the polymer, and polymer concentration on the separation were studied and optimized. Analysis of the results presented here also provides guidance for optimization of separation conditions for RNAs with different sizes as needed for particular applications in the future.


Assuntos
Química Farmacêutica/métodos , Eletroforese Capilar , RNA/isolamento & purificação , Peso Molecular , Polímeros/química , RNA/análise , Solventes/química
20.
Nature ; 577(7790): 392-398, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31915380

RESUMO

More than twelve morphologically and physiologically distinct subtypes of primary somatosensory neuron report salient features of our internal and external environments1-4. It is unclear how specialized gene expression programs emerge during development to endow these subtypes with their unique properties. To assess the developmental progression of transcriptional maturation of each subtype of principal somatosensory neuron, we generated a transcriptomic atlas of cells traversing the primary somatosensory neuron lineage in mice. Here we show that somatosensory neurogenesis gives rise to neurons in a transcriptionally unspecialized state, characterized by co-expression of transcription factors that become restricted to select subtypes as development proceeds. Single-cell transcriptomic analyses of sensory neurons from mutant mice lacking transcription factors suggest that these broad-to-restricted transcription factors coordinate subtype-specific gene expression programs in subtypes in which their expression is maintained. We also show that neuronal targets are involved in this process; disruption of the prototypic target-derived neurotrophic factor NGF leads to aberrant subtype-restricted patterns of transcription factor expression. Our findings support a model in which cues that emanate from intermediate and final target fields promote neuronal diversification in part by transitioning cells from a transcriptionally unspecialized state to transcriptionally distinct subtypes by modulating the selection of subtype-restricted transcription factors.


Assuntos
Neurogênese , Neurônios/fisiologia , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Camundongos , Fator de Crescimento Neural/metabolismo , Neurônios/citologia , RNA/análise , RNA/genética , Análise de Célula Única , Fator de Transcrição Brn-3B/genética , Fator de Transcrição Brn-3B/metabolismo , Fator de Transcrição Brn-3C/genética , Fator de Transcrição Brn-3C/metabolismo
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