Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 11.409
Filtrar
1.
Biochemistry (Mosc) ; 84(9): 1093-1106, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31693469

RESUMO

Differential expression of 30,003 genes was studied in the liver of female Wistar rats fed with isocaloric diets with the excess of fat, fructose, or cholesterol, or their combinations for 62 days using the method of whole-transcriptome profiling on a microchip. Relative mRNA expression levels of the Asah2, Crot, Crtc2, Fmo3, GSTA2, LOC1009122026, LOC102551184, NpY, NqO1, Prom1, Retsat, RGD1305464, Tmem104, and Whsc1 genes were also determined by RT-qPCR. All the tested diets affected differently the key metabolic pathways (KEGGs). Significant changes in the expression of steroid metabolism gene were observed in the liver of animals fed with the tested diets (except the high-fat high fructose diet). Both high-fat and high-fructose diets caused a significant decrease in the expression of squalene synthase (FDFT1 gene) responsible for the initial stage of cholesterol synthesis. On the contrary, in animals fed with the high-cholesterol diet (0.5% cholesterol), expression of the FDFT1 gene did not differ from the control group; however, these animals were characterized by changes in the expression of glucose and glycogen synthesis genes, which could lead to the suppression of glycogen synthesis and gluconeogenesis. At the same time, this group demonstrated different liver tissue morphology in comparison with the animals fed with the high-fructose high-fat diet, manifested as the presence of lipid vacuoles of a smaller size in hepatocytes. The high-fructose and high-fructose high-fat diets affected the metabolic pathways associated with intracellular protein catabolism (endocytosis, phagocytosis, proteasomal degradation, protein processing in the endoplasmic reticulum), tight junctions and intercellular contacts, adhesion molecules, and intracellular RNA transport. Rats fed with the high-fructose high-fat or high-cholesterol diets demonstrated consistent changes in the expression of the Crot, Prom1, and RGD1305464 genes, which reflected a coordinated shift in the regulation of lipid and carbohydrate metabolisms.


Assuntos
Colesterol/farmacologia , Gorduras na Dieta/farmacologia , Açúcares da Dieta/farmacologia , Frutose/farmacologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , RNA/genética , Transcriptoma/efeitos dos fármacos , Animais , Colesterol/administração & dosagem , Colesterol/metabolismo , Biologia Computacional , Gorduras na Dieta/administração & dosagem , Gorduras na Dieta/metabolismo , Açúcares da Dieta/administração & dosagem , Açúcares da Dieta/metabolismo , Feminino , Frutose/administração & dosagem , Frutose/metabolismo , Perfilação da Expressão Gênica , Fígado/citologia , RNA/análise , RNA/isolamento & purificação , Ratos , Ratos Wistar , Transcriptoma/genética
2.
DNA Cell Biol ; 38(11): 1223-1232, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31566423

RESUMO

To date, the clinical course of idiopathic membranous nephropathy (iMN) remains unclear and lacks direct and effective diagnostic methods. To better understand the host gene expression changes involved in the iMN process and identify the potential signatures for clinical diagnosis, we performed a whole genome-wide transcriptome profile of peripheral blood cells (PBC) from patients with iMN and healthy controls (HCs). A total of 188 differentially expressed genes (DEGs) were detected in patients with iMN versus HCs. Gene ontology (GO) functional enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that these DEGs were mainly correlated with protein targeting, ion homeostasis GO terms, and ribosome and phagosome pathways. The top 10 differentially expressed protein-coding genes with >2-fold changes and high expression levels were validated using quantitative real-time PCR, and showed high consistency with the high-throughput sequencing results. HLA-C, S100A8, and FTH1 genes were selected for further validation and showed the most significant difference between the iMN and HC group, indicating that they could be used as potential clinical diagnostic biomarkers. Our results provide novel potential diagnostic signatures for iMN and have important implications for better understanding the pathogenesis of iMN.


Assuntos
Biomarcadores/sangue , Células Sanguíneas/metabolismo , Glomerulonefrite Membranosa , Transcriptoma , Biomarcadores/análise , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Glomerulonefrite Membranosa/sangue , Glomerulonefrite Membranosa/diagnóstico , Glomerulonefrite Membranosa/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA/análise , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
3.
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
5.
Nat Methods ; 16(8): 743-749, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31363225

RESUMO

Cellular behavior is controlled by the interplay of diverse biomolecules. Most experimental methods, however, can only monitor a single molecule class or reaction type at a time. We developed an in vitro nuclear magnetic resonance spectroscopy (NMR) approach, which permitted dynamic quantification of an entire 'heterotypic' network-simultaneously monitoring three distinct molecule classes (metabolites, proteins and RNA) and all elementary reaction types (bimolecular interactions, catalysis, unimolecular changes). Focusing on an eight-reaction co-transcriptional RNA folding network, in a single sample we recorded over 35 time points with over 170 observables each, and accurately determined five core reaction constants in multiplex. This reconstruction revealed unexpected cross-talk between the different reactions. We further observed dynamic phase-separation in a system of five distinct RNA-binding domains in the course of the RNA transcription reaction. Our Systems NMR approach provides a deeper understanding of biological network dynamics by combining the dynamic resolution of biochemical assays and the multiplexing ability of 'omics'.


Assuntos
Redes Reguladoras de Genes , Metaboloma , Ressonância Magnética Nuclear Biomolecular/métodos , Proteínas/análise , RNA/análise , Células HEK293 , Humanos , Conformação de Ácido Nucleico , Conformação Proteica , Proteínas/química , RNA/química , Dobramento de RNA
6.
Anal Chim Acta ; 1077: 30-66, 2019 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-31307723

RESUMO

This review summarizes progress in electroanalysis of organic compounds and biomacromolecules by means of bare BDD-based electrodes for the period of 2009-2018. New trends, which have emerged in the reported decade and which have improved their performance in batch voltammetric and amperometric methods and electrochemical detection in liquid flow techniques are commented. Importance of BDD surface termination, effect of boron doping level, and utilization of adsorption of analytes on BDD surfaces enabling development of adsorptive voltammetric techniques are addressed. Further, possibilities of simultaneous determination of analytes by means of voltammetric techniques utilizing computational approaches and multiple-pulse amperometric detection are discussed. Strategies leading to enhancement of sensitivity such as nanostructuring of the BDD surface, fabrication of BDD-based composite materials or new approaches in construction of microelectrodes and microelectrode arrays for biosensing represent another area of interest. Attention is paid to possibilities in detection of amino acids, peptides and proteins, nucleobases, nucleos(t)ides and DNA/RNA.


Assuntos
Boro/química , Diamante/química , Microeletrodos , Compostos Orgânicos/análise , DNA/análise , Técnicas Eletroquímicas/instrumentação , Técnicas Eletroquímicas/métodos , Peptídeos/análise , Proteínas/análise , RNA/análise
7.
Nature ; 571(7765): 419-423, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31292545

RESUMO

Single-cell RNA sequencing (scRNA-seq) has highlighted the important role of intercellular heterogeneity in phenotype variability in both health and disease1. However, current scRNA-seq approaches provide only a snapshot of gene expression and convey little information on the true temporal dynamics and stochastic nature of transcription. A further key limitation of scRNA-seq analysis is that the RNA profile of each individual cell can be analysed only once. Here we introduce single-cell, thiol-(SH)-linked alkylation of RNA for metabolic labelling sequencing (scSLAM-seq), which integrates metabolic RNA labelling2, biochemical nucleoside conversion3 and scRNA-seq to record transcriptional activity directly by differentiating between new and old RNA for thousands of genes per single cell. We use scSLAM-seq to study the onset of infection with lytic cytomegalovirus in single mouse fibroblasts. The cell-cycle state and dose of infection deduced from old RNA enable dose-response analysis based on new RNA. scSLAM-seq thereby both visualizes and explains differences in transcriptional activity at the single-cell level. Furthermore, it depicts 'on-off' switches and transcriptional burst kinetics in host gene expression with extensive gene-specific differences that correlate with promoter-intrinsic features (TBP-TATA-box interactions and DNA methylation). Thus, gene-specific, and not cell-specific, features explain the heterogeneity in transcriptomes between individual cells and the transcriptional response to perturbations.


Assuntos
Regulação da Expressão Gênica/genética , Análise de Sequência de RNA/métodos , Análise de Célula Única , Transcrição Genética/genética , Alquilação , Animais , Ciclo Celular , Citomegalovirus/fisiologia , Metilação de DNA , Fibroblastos/metabolismo , Fibroblastos/virologia , Cinética , Camundongos , Regiões Promotoras Genéticas/genética , RNA/análise , RNA/química , Compostos de Sulfidrila/química
8.
Neoplasma ; 66(5): 736-745, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31169020

RESUMO

Bladder cancer remains a very challenging disease to treat with the high rates of recurrence and progression associated with current therapies. Although the association between bladder cancer pathology and circRNAs remains undetermined, circRNAs signatures may be useful as prognostic and predictive factors and clinical tools for assessing disease state and outcome. This study investigates if these circRNAs can be used as biomarkers for bladder cancer diagnosis. Using bioinformatics method to analysis GEO databases (GSE37815, GSE39093, GSE97239, and GSE92675) for differentially expressed RNAs in bladder cancer and normal bladder tissues were screened from. The related volcanic maps and the interaction network maps of differentially expressed RNAs were drawn, and the mRNA-miRNA and miRNA-circRNA interaction were predicted to establish mRNA-miRNA-circRNA competitive endogenous RNA (ceRNA) network. The differential circRNAs related to prognosis of bladder cancer patients were screened based on the influence of miRNA interacting with the circRNA above on survival rate. The expression of miRNA (hsa-mir-214), circRNA (hsa_circ_0076704, hsa_circ_0081963, hsa_circ_0001361) in bladder cancer tissues, adjacent tissues, bladder cancer cells and normal bladder epithelial cells were validated by qRT-PCR. Kaplan Meier curve analysis confirmed the relationship between circRNA (hsa_circ_0076704) and overall survival and prognosis of bladder cancer patients. Through database screening and analysis, we found 19231 differentially expressed genes, 847 differentially expressed miRNAs, 7282 differentially expressed circRNAs. The establishment of ceRNA network consisted of 28 DERNAs (differentially- expressed RNAs), 12 Demi-RNAs and 12 DEcircRNAs. Further prognostic analysis showed that circRNA interacted miRNA hsa-miR-106b, hsa-miR-145 and hsa-miR-214 were associated with overall survival in patients with bladder cancer (P < 0.05). Among them, hsa_circ_0076704, hsa_circ_0081963 and hsa_circ_0001361 are potential circRNA related to OS in bladder cancer and expressed in bladder cancer. The expression of hsa-mir-214 was contrary. Further Kaplan Meier survival analysis showed that hsa_circ_0076704 had significant prognostic value (P < 0.05). In conclusion, hsa_circ_0076704 is independent prognostic factor for bladder cancer.


Assuntos
Biomarcadores , RNA , Neoplasias da Bexiga Urinária , Biomarcadores/análise , Humanos , Prognóstico , RNA/análise , RNA Mensageiro/metabolismo , Neoplasias da Bexiga Urinária/diagnóstico
9.
Histochem Cell Biol ; 152(3): 239-247, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31197457

RESUMO

Histological and immunohistochemical studies require high-quality paraffin blocks, where proper fixation of tissue samples with formalin is a key point. However, in some cases, the possibility to preserve biological samples prior to the formalin fixation or to use deposited tissues from biobanks is important. RNA-stabilizing reagent RNALater represents a potential option, but its suitability for pathological and immunohistochemical studies remains underinvestigated. Here, comparative study of formalin-fixed tissues and those had undergone preservation with RNALater was performed for different SCID mice tissues (brain, liver, kidney, and lung) using histological staining (hematoxylin-eosin and Weigert-van Gieson) or immunostaining for b-actin, glial fibrillary acidic protein, and glycosaminoglycan chondroitin sulfate. It was shown that RNALater preservation for 7-14 days was suitable for histological characterisation of mouse lung tissue, whereas all other tissues demonstrated some changes. Immunoreactivity of all the studied tissues was affected to a different extent, and the observed changes were detected at the 7th day already and continued to get worse by the 14th day. Overall, RNALater preservation affects immunogenicity of normal mouse tissues (brain, liver, kidney, and lung) making them unsuitable for immunohistochemistry. Some tissues retain their morphology (lung tissue) or demonstrate moderate changes (brain, liver, kidney), suggesting a restricted suitability of the RNALater-preserved tissues for histological analysis.


Assuntos
Imuno-Histoquímica/métodos , RNA/análise , RNA/química , Preservação de Tecido/métodos , Animais , Indicadores e Reagentes , Camundongos , Camundongos SCID , Soluções
10.
Anal Chim Acta ; 1068: 96-103, 2019 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-31072482

RESUMO

Herein, we combined toehold exchange with ligation-free rolling circle amplification (RCA) by programming nucleolytic conversion of hairpin probe into sensors, allowed for both high specific recognition and universal signal amplification for RNA detection. The rational engineered HP ensured highly specific recognition based on toehold exchange and allowed the pre-formed circular template for RCA to be shared for different RNAs detection. Generally, detecting different RNA requires different circular template for signal amplification. In this paper, the circular template for RCA was independent of the sequences of the target, so the signal amplification system was an universal one for different RNAs detection. Taking miRNA let-7d as a model target, this method showed a wide linear range from 1 fM to 1 nM with a detection limit of 0.46 fM and exhibited a remarkable selectivity even in distinguishing homologous miRNAs with 1-nt or 2-nt difference. To evaluate the potential of the method, it was applied to analysis the let-7d concentration in human serum, total RNA, and cell lysates. In conclusion, we believe this method exhibits potential for both specific discrimination and universal signal amplification for RNA analysis in complex matrices.


Assuntos
Técnicas Biossensoriais , Técnicas de Amplificação de Ácido Nucleico , Sondas RNA/química , RNA/análise , Células A549 , Linhagem Celular , Humanos , Conformação de Ácido Nucleico , RNA/genética , Espectrometria de Fluorescência
11.
Nat Protoc ; 14(6): 1734-1755, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31053798

RESUMO

R-loops are prevalent three-stranded non-B DNA structures composed of an RNA-DNA hybrid and a single strand of DNA. R-loops are implicated in various basic nuclear processes, such as class-switch recombination, transcription termination and chromatin patterning. Perturbations in R-loop metabolism have been linked to genomic instability and have been implicated in human disorders, including cancer. As a consequence, the accurate mapping of these structures has been of increasing interest in recent years. Here, we describe two related immunoprecipitation-based methods for mapping R-loop structures: basic DRIP-seq (DNA-RNA immunoprecipitation followed by high-throughput DNA sequencing), an easy, robust, but resolution-limited technique; and DRIPc-seq (DNA-RNA immunoprecipitation followed by cDNA conversion coupled to high-throughput sequencing), a high-resolution and strand-specific iteration of the method that permits accurate R-loop mapping genome wide. Briefly, after gentle DNA extraction and restriction digestion with a cocktail of enzymes, R-loop structures are immunoprecipitated with the anti-RNA-DNA hybrid S9.6 antibody. Compared with DRIP-seq, in which the immunoprecipitated DNA is directly sequenced, DRIPc-seq permits the recovery of the RNA moiety of R-loops, and these RNA strands are subjected to strand-specific RNA sequencing (RNA-seq) analysis. DRIPc-seq can be performed in 5 d and can be applied to any cell type, provided sufficient starting material can be collected. Accurately mapping R-loop distribution in various cell lines and under varied conditions is essential to understanding the formation, roles and dynamic resolution of these important structures.


Assuntos
DNA/análise , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Imunoprecipitação/métodos , RNA/análise , Animais , Anticorpos/química , Anticorpos Monoclonais/química , DNA/genética , Humanos , Camundongos , Conformação de Ácido Nucleico , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase/métodos , RNA/genética
12.
Nat Methods ; 16(6): 533-544, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31110282

RESUMO

Fluorescence in situ hybridization (FISH) reveals the abundance and positioning of nucleic acid sequences in fixed samples. Despite recent advances in multiplexed amplification of FISH signals, it remains challenging to achieve high levels of simultaneous amplification and sequential detection with high sampling efficiency and simple workflows. Here we introduce signal amplification by exchange reaction (SABER), which endows oligonucleotide-based FISH probes with long, single-stranded DNA concatemers that aggregate a multitude of short complementary fluorescent imager strands. We show that SABER amplified RNA and DNA FISH signals (5- to 450-fold) in fixed cells and tissues. We also applied 17 orthogonal amplifiers against chromosomal targets simultaneously and detected mRNAs with high efficiency. We then used 10-plex SABER-FISH to identify in vivo introduced enhancers with cell-type-specific activity in the mouse retina. SABER represents a simple and versatile molecular toolkit for rapid and cost-effective multiplexed imaging of nucleic acid targets.


Assuntos
DNA/análise , Corantes Fluorescentes/metabolismo , Hibridização in Situ Fluorescente/métodos , Oligonucleotídeos/química , Imagem Óptica/métodos , RNA/análise , Retina/metabolismo , Animais , Células Cultivadas , DNA/genética , DNA de Cadeia Simples/química , Humanos , Camundongos , RNA/genética , Retina/diagnóstico por imagem
13.
Biosens Bioelectron ; 136: 76-83, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31039490

RESUMO

We investigated the feasibility of simultaneous detection of multiple environmentally- and biomedically-relevant RNA biomarker target sequences on a single newly fabricated 384-ch sensor array chip aiming at practical application. The individual sensor is composed of a photolithographically-fabricated Au/Cr-based electrode modified with peptide nucleic acid (PNA) probes. The sensor array chips showed sequence-specific responses upon hybridization of the probes with target sequences complementary to the probes in contrast to mismatch versions. The target oligonucleotides have 15-22 mer sequences from messenger RNAs for estrogen-responsive genes and microRNAs for lung cancer biomarkers. The dependence on target concentrations of sensor responses was observed by using a single chip on which experiments for detection of several target concentrations proceeded simultaneously, with the detection limit of 7.33 × 10-8 M. As more realistic samples, oligonucleotide samples amplified by PCR from a synthesized template sequence were applied to the chip. They showed sequence-specific responses, revealing the potential for fabricated sensor array chips to be utilized to analyze PCR samples. Unlike complicated and expensive chips that require nanofabrication, our sensor array chips based on glass coated with gold thin films are simple and can be fabricated from inexpensive and readily available materials.


Assuntos
Técnicas Biossensoriais/métodos , Oligonucleotídeos/análise , Ácidos Nucleicos Peptídicos/química , RNA/análise , Cromo/química , Eletrodos , Biomarcadores Ambientais , Ouro/química , Humanos , Neoplasias/diagnóstico , Reação em Cadeia da Polimerase
14.
Nucleic Acids Res ; 47(11): 5892-5905, 2019 06 20.
Artigo em Inglês | MEDLINE | ID: mdl-30957850

RESUMO

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.


Assuntos
Mycobacterium tuberculosis/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA/análise , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Complexos Multienzimáticos , Mycobacterium smegmatis/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/genética , Proteoma , Proteômica , RNA/química , RNA Helicases/metabolismo , Estabilidade de RNA , RNA Bacteriano/metabolismo , Ribonuclease III/metabolismo , Ribonucleases/metabolismo , Tiouridina/química , Transcriptoma
15.
Methods Mol Biol ; 1979: 379-392, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31028649

RESUMO

Simultaneous detection of both RNA and protein in individual single cells offers a powerful tool for genotype-to-phenotype investigations. Proximity extension assay (PEA) is a quantitative, sensitive, and multiplex protein detection system that has superb utility in single-cell omic analysis. We implemented PEA using the flexible microfluidic workflow of the Fluidigm® C1™ system followed by real-time quantitative polymerase chain reaction (RT-qPCR) on the Fluidigm Biomark™ HD system. With this workflow, targeted quantification of RNAs and proteins within individual cells is readily conducted.


Assuntos
Perfilação da Expressão Gênica/métodos , Técnicas Analíticas Microfluídicas/métodos , Proteínas/análise , RNA/análise , Análise de Célula Única/métodos , Animais , Humanos , Proteínas/genética , RNA/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fluxo de Trabalho
16.
Nat Protoc ; 14(5): 1489-1508, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30962605

RESUMO

Non-coding RNA (ncRNA) molecules have been shown to play a variety of cellular roles; however, the contributions of different types of RNA to specific phenomena are often hard to dissect. To study the role of RNA in the assembly of DNA damage response (DDR) foci, we developed the RNase A treatment and reconstitution (RATaR) method, in which cells are mildly permeabilized, incubated with recombinant RNase A and subsequently reconstituted with different RNA species, under conditions of RNase A inactivation and inhibition of endogenous transcription. The block of transcription right after RNase A removal represents a key innovation of RATaR, preventing potential contributions of endogenously neo-synthesized transcripts to the phenotypes studied. A critical aspect of this technique is the balance between sufficient permeabilization of membranes to allow enzyme/RNA access into the cell nucleus and cell viability. Here, we present our protocol for RNA-dependent DDR foci disassembly and reassembly using fluorescent DDR RNAs (DDRNAs) in NIH2/4 cells, an engineered NIH3T3-derived cell line. The use of sequence-specific, fluorescent RNA molecules permits the concomitant determination of their subcellular localization and biological functions. We also outline adaptations of RATaR when implemented in different cell lines exposed to various genotoxic treatments, such as γ-radiation, restriction enzymes and telomere deprotection. In all these cases, the entire procedure can be completed within 2 h without the need for special equipment or uncommon skills. We believe this technique will prove useful for investigating the contribution of RNA to a variety of relevant cellular processes.


Assuntos
Dano ao DNA , Reparo do DNA , RNA não Traduzido , Ribonuclease Pancreático/metabolismo , Animais , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Técnicas Genéticas , Células HeLa , Humanos , Camundongos , Células NIH 3T3 , RNA/análise , RNA/genética , RNA/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/fisiologia
17.
Methods Mol Biol ; 1973: 261-279, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31016708

RESUMO

Molecular beacons composed of the artificial serinol nucleic acid (SNA) have demonstrated utility as novel fluorescence probes for visualization of RNA in fixed cells using both conventional fluorescence in situ hybridization (FISH) and wash-free FISH protocols. The SNA molecular beacons have higher affinity for target RNA and greater sensitivity than molecular beacons composed of DNA. Here we describe facile synthesis of the SNA using a conventional DNA synthesizer and protocols for purification by PAGE and HPLC as well as methods for use of the SNA molecular beacon in FISH.


Assuntos
Corantes Fluorescentes/química , Hibridização in Situ Fluorescente/métodos , Ácidos Nucleicos/química , Sondas de Oligonucleotídeos/química , Propanolaminas/química , Propilenoglicóis/química , RNA/análise , Hibridização de Ácido Nucleico
18.
Nat Protoc ; 14(6): 1708-1733, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31028373

RESUMO

Understanding the structure-function relationships between diverse cell types in a complex organ environment requires detailed in situ reconstruction of cell-associated molecular properties in the context of 3D, macro-scale tissue architecture. We recently developed clearing-enhanced 3D (Ce3D), a simple and effective method for tissue clearing that achieves excellent transparency; preserves cell morphology, tissue architecture, and reporter molecule fluorescence; and is robustly compatible with direct immunolabeling. These characteristics permit high-quality multiplex fluorescence microscopy of large tissue volumes, as well as image analysis using advanced platforms such as volumetric histocytometry, collectively allowing quantitative characterization of cells with respect to their spatial positioning within tissues on the basis of phenotypic and functional markers. Ce3D clearing is fast, achieving robust transparency of most tissues within 24 h, albeit still necessitating additional time for staining, imaging, and analysis (1-2 weeks). Here, we provide detailed procedures for tissue clearing using Ce3D, including optimized workflows for tissue processing and staining, as well as treatment of difficult-to-clear organs such as the brain. We also describe a new procedure for RNA detection in Ce3D-treated tissues, as well as provide additional details for the volumetric histocytometry data processing steps. Finally, we discuss limitations and work-around strategies for improving antibody-based tissue immunolabeling, fluorophore multiplexing, large-volume microscopy, and computational analysis of large image datasets. Together, these detailed procedures and solutions for high-resolution volumetric microscopy with Ce3D enable quantitative visualization of cells and tissues at a high level of detail, allowing exploration of cellular spatial relationships in a variety of tissue settings.


Assuntos
Imagem Tridimensional/métodos , Microscopia Confocal/métodos , Microscopia de Fluorescência/métodos , Animais , Química Encefálica , Feminino , Imuno-Histoquímica/métodos , Masculino , Camundongos , RNA/análise , Coloração e Rotulagem/métodos , Fixação de Tecidos/métodos
19.
Leukemia ; 33(8): 2022-2033, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30992504

RESUMO

Monitoring tumour burden and therapeutic response through analyses of circulating cell-free tumour DNA (ctDNA) and extracellular RNA (exRNA) in multiple myeloma (MM) patients were performed in a Phase Ib trial of 24 relapsed/refractory patients receiving oral azacitidine in combination with lenalidomide and dexamethasone. Mutational characterisation of paired BM and PL samples at study entry identified that patients with a higher number of mutations or a higher mutational fractional abundance in PL had significantly shorter overall survival (OS) (p = 0.005 and p = 0.018, respectively). A decrease in ctDNA levels at day 5 of cycle 1 of treatment (C1D5) correlated with superior progression-free survival (PFS) (p = 0.017). Evaluation of exRNA transcripts of candidate biomarkers indicated that high CRBN levels coupled with low levels of SPARC at baseline were associated with shorter OS (p = 0.000003). IKZF1 fold-change <0.05 at C1D5 was associated with shorter PFS (p = 0.0051) and OS (p = 0.0001). Furthermore, patients with high baseline CRBN coupled with low fold-change at C1D5 were at the highest risk of progression (p = 0.0001). In conclusion, this exploratory analysis has provided the first demonstration in MM of ctDNA for predicting disease outcome and of the utility of exRNA as a biomarker of therapeutic response.


Assuntos
DNA Tumoral Circulante/análise , Mieloma Múltiplo/tratamento farmacológico , RNA/análise , Efeitos Psicossociais da Doença , Genes p53 , Humanos , Fator de Transcrição Ikaros/análise , Mieloma Múltiplo/sangue , Mieloma Múltiplo/genética , Mieloma Múltiplo/mortalidade , Mutação , Peptídeo Hidrolases/análise , Prognóstico
20.
J Fish Biol ; 95(2): 393-400, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31017661

RESUMO

The partial cDNA sequences of eight reference genes (actb, tuba1, gapdh58, gapdh59, eef1a1, RNA 18 s, pabpc1, ube2I) were cloned from largemouth bass Micropterus salmoides. The expression levels of these eight genes were compared in the various tissues (eye, spleen, kidney, gill, muscle, brain, liver, heart, gut and gonad) of M. salmoides fed on forage fish. The results showed that the candidate genes exhibited tissue-specific expression to various degrees and the stability ranking order was eef1a1 > tuba1 > RNA 18 s > pabpc1 > ube2I > actb > gapdh58 > gapdh59 among tissue types. Four candidate genes eef1a1, tuba1, RNA 18 s and actb were used to analyse the stability in liver tissues of largemouth bass between the forage-fish group and the formulated-feed group. The candidate genes also showed some changes in expression levels in the livers, while eef1a1 and tuba1 had the most stable expression in livers of fish fed on alternative diets within 10 candidates. So eef1a1 and tuba1 were recommended as optimal reference gene in quantitative real-time PCR analysis to normalise the expression levels of target genes in tissues and lives of the M. salmoides fed on alternative diets. In livers, the expression levels of gck normalised by eef1a1 and tuba1 showed the significant up-regulation in formulated feed group (P < 0.05) than those in forage-fish group. While sex difference has no significant effects on the expression levels of gck in both groups.


Assuntos
Bass/genética , Dieta/veterinária , Animais , Bass/anatomia & histologia , Bass/fisiologia , DNA Complementar/biossíntese , DNA Complementar/química , Bases de Dados de Ácidos Nucleicos , Expressão Gênica , Instabilidade Genômica , Glucoquinase/genética , Fígado/metabolismo , RNA/análise , RNA/isolamento & purificação , RNA/normas , Reação em Cadeia da Polimerase em Tempo Real
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA