RESUMO
In eukaryotes, capped RNAs include long transcripts such as messenger RNAs and long noncoding RNAs, as well as shorter transcripts such as spliceosomal RNAs, small nucleolar RNAs, and enhancer RNAs. Long capped transcripts can be profiled using cap analysis gene expression (CAGE) sequencing and other methods. Here, we describe a sequencing library preparation protocol for short capped RNAs, apply it to a differentiation time course of the human cell line THP-1, and systematically compare the landscape of short capped RNAs to that of long capped RNAs. Transcription initiation peaks associated with genes in the sense direction have a strong preference to produce either long or short capped RNAs, with one out of six peaks detected in the short capped RNA libraries only. Gene-associated short capped RNAs have highly specific 3' ends, typically overlapping splice sites. Enhancers also preferentially generate either short or long capped RNAs, with 10% of enhancers observed in the short capped RNA libraries only. Enhancers producing either short or long capped RNAs show enrichment for GWAS-associated disease SNPs. We conclude that deep sequencing of short capped RNAs reveals new families of noncoding RNAs and elucidates the diversity of transcripts generated at known and novel promoters and enhancers.
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Gene expression profiles in homologous tissues have been observed to be different between species, which may be due to differences between species in the gene expression program in each cell type, but may also reflect differences in cell type composition of each tissue in different species. Here, we compare expression profiles in matching primary cells in human, mouse, rat, dog, and chicken using Cap Analysis Gene Expression (CAGE) and short RNA (sRNA) sequencing data from FANTOM5. While we find that expression profiles of orthologous genes in different species are highly correlated across cell types, in each cell type many genes were differentially expressed between species. Expression of genes with products involved in transcription, RNA processing, and transcriptional regulation was more likely to be conserved, while expression of genes encoding proteins involved in intercellular communication was more likely to have diverged during evolution. Conservation of expression correlated positively with the evolutionary age of genes, suggesting that divergence in expression levels of genes critical for cell function was restricted during evolution. Motif activity analysis showed that both promoters and enhancers are activated by the same transcription factors in different species. An analysis of expression levels of mature miRNAs and of primary miRNAs identified by CAGE revealed that evolutionary old miRNAs are more likely to have conserved expression patterns than young miRNAs. We conclude that key aspects of the regulatory network are conserved, while differential expression of genes involved in cell-to-cell communication may contribute greatly to phenotypic differences between species.
Assuntos
Evolução Molecular , Transcriptoma , Animais , Galinhas/genética , Cães , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Humanos , Camundongos , MicroRNAs/metabolismo , Motivos de Nucleotídeos , Análise de Componente Principal , Regiões Promotoras Genéticas , Ratos , Especificidade da Espécie , Fatores de Transcrição/metabolismoRESUMO
MicroRNAs (miRNAs) are short non-coding RNAs with key roles in cellular regulation. As part of the fifth edition of the Functional Annotation of Mammalian Genome (FANTOM5) project, we created an integrated expression atlas of miRNAs and their promoters by deep-sequencing 492 short RNA (sRNA) libraries, with matching Cap Analysis Gene Expression (CAGE) data, from 396 human and 47 mouse RNA samples. Promoters were identified for 1,357 human and 804 mouse miRNAs and showed strong sequence conservation between species. We also found that primary and mature miRNA expression levels were correlated, allowing us to use the primary miRNA measurements as a proxy for mature miRNA levels in a total of 1,829 human and 1,029 mouse CAGE libraries. We thus provide a broad atlas of miRNA expression and promoters in primary mammalian cells, establishing a foundation for detailed analysis of miRNA expression patterns and transcriptional control regions.
Assuntos
Perfilação da Expressão Gênica/métodos , MicroRNAs/genética , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Animais , Células Cultivadas , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Camundongos , MicroRNAs/metabolismoRESUMO
Mammals are composed of hundreds of different cell types with specialized functions. Each of these cellular phenotypes are controlled by different combinations of transcription factors. Using a human non islet cell insulinoma cell line (TC-YIK) which expresses insulin and the majority of known pancreatic beta cell specific genes as an example, we describe a general approach to identify key cell-type-specific transcription factors (TFs) and their direct and indirect targets. By ranking all human TFs by their level of enriched expression in TC-YIK relative to a broad collection of samples (FANTOM5), we confirmed known key regulators of pancreatic function and development. Systematic siRNA mediated perturbation of these TFs followed by qRT-PCR revealed their interconnections with NEUROD1 at the top of the regulation hierarchy and its depletion drastically reducing insulin levels. For 15 of the TF knock-downs (KD), we then used Cap Analysis of Gene Expression (CAGE) to identify thousands of their targets genome-wide (KD-CAGE). The data confirm NEUROD1 as a key positive regulator in the transcriptional regulatory network (TRN), and ISL1, and PROX1 as antagonists. As a complimentary approach we used ChIP-seq on four of these factors to identify NEUROD1, LMX1A, PAX6, and RFX6 binding sites in the human genome. Examining the overlap between genes perturbed in the KD-CAGE experiments and genes with a ChIP-seq peak within 50 kb of their promoter, we identified direct transcriptional targets of these TFs. Integration of KD-CAGE and ChIP-seq data shows that both NEUROD1 and LMX1A work as the main transcriptional activators. In the core TRN (i.e., TF-TF only), NEUROD1 directly transcriptionally activates the pancreatic TFs HSF4, INSM1, MLXIPL, MYT1, NKX6-3, ONECUT2, PAX4, PROX1, RFX6, ST18, DACH1, and SHOX2, while LMX1A directly transcriptionally activates DACH1, SHOX2, PAX6, and PDX1. Analysis of these complementary datasets suggests the need for caution in interpreting ChIP-seq datasets. (1) A large fraction of binding sites are at distal enhancer sites and cannot be directly associated to their targets, without chromatin conformation data. (2) Many peaks may be non-functional: even when there is a peak at a promoter, the expression of the gene may not be affected in the matching perturbation experiment.
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Next-generation sequencing experiments have shown that microRNAs (miRNAs) are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched miRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by 1 nt at their 3' end, and moreover that the 3' end of miR-21 is posttranscriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small-RNA sequencing suggested that PARN degrades miR-21 in the 3'-to-5' direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a down-regulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the noncancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncogenic miRNA miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.
Assuntos
Adenina/metabolismo , MicroRNAs/metabolismo , Neoplasias/genética , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA , Sequência de Bases , Citosina/metabolismo , Exorribonucleases/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Células MCF-7 , MicroRNAs/química , MicroRNAs/genética , Modelos Biológicos , Dados de Sequência Molecular , Neoplasias/patologia , Conformação de Ácido Nucleico , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ribonuclease III/metabolismoRESUMO
Mast cells (MCs) mature exclusively in peripheral tissues, hampering research into their developmental and functional programs. Here, we employed deep cap analysis of gene expression on skin-derived MCs to generate the most comprehensive view of the human MC transcriptome ever reported. An advantage is that MCs were embedded in the FANTOM5 project, giving the opportunity to contrast their molecular signature against a multitude of human samples. We demonstrate that MCs possess a unique and surprising transcriptional landscape, combining hematopoietic genes with those exclusively active in MCs and genes not previously reported as expressed by MCs (several of them markers of unrelated tissues). We also found functional bone morphogenetic protein receptors transducing activatory signals in MCs. Conversely, several immune-related genes frequently studied in MCs were not expressed or were weakly expressed. Comparing MCs ex vivo with cultured counterparts revealed profound changes in the MC transcriptome in in vitro surroundings. We also determined the promoter usage of MC-expressed genes and identified associated motifs active in the lineage. Befitting their uniqueness, MCs had no close relative in the hematopoietic network (also only distantly related with basophils). This rich data set reveals that our knowledge of human MCs is still limited, but with this resource, novel functional programs of MCs may soon be discovered.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mastócitos/citologia , Análise de Sequência de DNA/métodos , Transcriptoma , Motivos de Aminoácidos , Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Linhagem da Célula , Análise por Conglomerados , Bases de Dados Factuais , Marcadores Genéticos , Hematopoese/genética , Células-Tronco Hematopoéticas/citologia , Humanos , Sistema Imunitário , Família Multigênica , Regiões Promotoras Genéticas , Pele/metabolismoRESUMO
Regulated transcription controls the diversity, developmental pathways and spatial organization of the hundreds of cell types that make up a mammal. Using single-molecule cDNA sequencing, we mapped transcription start sites (TSSs) and their usage in human and mouse primary cells, cell lines and tissues to produce a comprehensive overview of mammalian gene expression across the human body. We find that few genes are truly 'housekeeping', whereas many mammalian promoters are composite entities composed of several closely separated TSSs, with independent cell-type-specific expression profiles. TSSs specific to different cell types evolve at different rates, whereas promoters of broadly expressed genes are the most conserved. Promoter-based expression analysis reveals key transcription factors defining cell states and links them to binding-site motifs. The functions of identified novel transcripts can be predicted by coexpression and sample ontology enrichment analyses. The functional annotation of the mammalian genome 5 (FANTOM5) project provides comprehensive expression profiles and functional annotation of mammalian cell-type-specific transcriptomes with wide applications in biomedical research.
Assuntos
Atlas como Assunto , Anotação de Sequência Molecular , Regiões Promotoras Genéticas/genética , Transcriptoma/genética , Animais , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Sequência Conservada/genética , Regulação da Expressão Gênica/genética , Redes Reguladoras de Genes/genética , Genes Essenciais/genética , Genoma/genética , Humanos , Camundongos , Fases de Leitura Aberta/genética , Especificidade de Órgãos , RNA Mensageiro/análise , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Sítio de Iniciação de Transcrição , Transcrição Gênica/genéticaRESUMO
Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.
Assuntos
Atlas como Assunto , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica/genética , Anotação de Sequência Molecular , Especificidade de Órgãos , Linhagem Celular , Células Cultivadas , Análise por Conglomerados , Predisposição Genética para Doença/genética , Células HeLa , Humanos , Polimorfismo de Nucleotídeo Único/genética , Regiões Promotoras Genéticas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Iniciação da Transcrição GenéticaRESUMO
Organic anion-transporting polypeptide (OATP) 1B1, encoded by the solute carrier organic anion transporter family member 1B1 (SLCO1B1) gene, mediates the active uptake of various organic anions into hepatocytes and determines their hepatic clearances as the first step in the detoxification pathway. Previous reports indicated that alterations in its function by drug-drug interactions or genetic polymorphisms affect the pharmacokinetics of the substrate drugs. In the present study, we developed a method to genotype SLCO1B1 388A>G (rs2306283) and 521>C (rs4149056), which significantly affect the clinical pharmacokinetics and subsequent side effects such as myopathy caused by statins, OATP1B1 substrates in humans. We used a small aliquot of blood and the isothermal Smart Amplification Process version 2 (SmartAmp-2), which could complete the genotyping of 388A>G and 521T>C within 60 min. The genotypes of 101 genomic DNA samples and blood samples assessed by SmartAmp-2 matched perfectly to those determined previously by the conventional PCR-SSCP method. The SmartAmp-2 method enables the rapid identification of the 388A>G and 521T>C genotypes, saving time and effort in the genomic DNA preparation in clinical practice. This method will be useful for evaluating and predicting altered pharmacological and toxicological effects of substrate drugs caused by SLCO1B1 polymorphisms.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , Transportadores de Ânions Orgânicos/genética , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único , Primers do DNA , Genótipo , Humanos , Transportador 1 de Ânion Orgânico Específico do Fígado , Transportadores de Ânions Orgânicos/sangue , Análise de Sequência de DNA , Fatores de TempoRESUMO
BACKGROUND: In 2009, a pandemic (pdm) influenza A(H1N1) virus infection quickly circulated globally resulting in about 18,000 deaths around the world. In Japan, infected patients accounted for 16% of the total population. The possibility of human-to-human transmission of highly pathogenic novel influenza viruses is becoming a fear for human health and society. METHODOLOGY: To address the clinical need for rapid diagnosis, we have developed a new method, the "RT-SmartAmp assay", to rapidly detect the 2009 pandemic influenza A(H1N1) virus from patient swab samples. The RT-SmartAmp assay comprises both reverse transcriptase (RT) and isothermal DNA amplification reactions in one step, where RNA extraction and PCR reaction are not required. We used an exciton-controlled hybridization-sensitive fluorescent primer to specifically detect the HA segment of the 2009 pdm influenza A(H1N1) virus within 40 minutes without cross-reacting with the seasonal A(H1N1), A(H3N2), or B-type (Victoria) viruses. RESULTS AND CONCLUSIONS: We evaluated the RT-SmartAmp method in clinical research carried out in Japan during a pandemic period of October 2009 to January 2010. A total of 255 swab samples were collected from outpatients with influenza-like illness at three hospitals and eleven clinics located in the Tokyo and Chiba areas in Japan. The 2009 pdm influenza A(H1N1) virus was detected by the RT-SmartAmp assay, and the detection results were subsequently compared with data of current influenza diagnostic tests (lateral flow immuno-chromatographic tests) and viral genome sequence analysis. In conclusion, by the RT-SmartAmp assay we could detect the 2009 pdm influenza A(H1N1) virus in patients' swab samples even in early stages after the initial onset of influenza symptoms. Thus, the RT-SmartAmp assay is considered to provide a simple and practical tool to rapidly detect the 2009 pdm influenza A(H1N1) virus.
Assuntos
Vírus da Influenza A Subtipo H1N1/isolamento & purificação , Vírus da Influenza A Subtipo H1N1/patogenicidade , Influenza Humana/epidemiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Pandemias , DNA Polimerase Dirigida por RNA/metabolismo , Idoso , Criança , Primers do DNA/genética , Farmacorresistência Viral , Feminino , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Humanos , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Influenza Humana/virologia , Oseltamivir/farmacologia , RNA Viral/genética , RNA Viral/isolamento & purificação , Fatores de TempoRESUMO
BACKGROUND: Mesothelioma is a highly malignant tumor that is primarily caused by occupational or environmental exposure to asbestos fibers. Despite worldwide restrictions on asbestos usage, further cases are expected as diagnosis is typically 20-40 years after exposure. Once diagnosed there is a very poor prognosis with a median survival rate of 9 months. Considering this the development of early pre clinical diagnostic markers may help improve clinical outcomes. METHODOLOGY: Microarray expression arrays on mesothelium and other tissues dissected from mice were used to identify candidate mesothelial lineage markers. Candidates were further tested by qRTPCR and in-situ hybridization across a mouse tissue panel. Two candidate biomarkers with the potential for secretion, uroplakin 3B (UPK3B), and leucine rich repeat neuronal 4 (LRRN4) and one commercialized mesothelioma marker, mesothelin (MSLN) were then chosen for validation across a panel of normal human primary cells, 16 established mesothelioma cell lines, 10 lung cancer lines, and a further set of 8 unrelated cancer cell lines. CONCLUSIONS: Within the primary cell panel, LRRN4 was only detected in primary mesothelial cells, but MSLN and UPK3B were also detected in other cell types. MSLN was detected in bronchial epithelial cells and alveolar epithelial cells and UPK3B was detected in retinal pigment epithelial cells and urothelial cells. Testing the cell line panel, MSLN was detected in 15 of the 16 mesothelioma cells lines, whereas LRRN4 was only detected in 8 and UPK3B in 6. Interestingly MSLN levels appear to be upregulated in the mesothelioma lines compared to the primary mesothelial cells, while LRRN4 and UPK3B, are either lost or down-regulated. Despite the higher fraction of mesothelioma lines positive for MSLN, it was also detected at high levels in 2 lung cancer lines and 3 other unrelated cancer lines derived from papillotubular adenocarcinoma, signet ring carcinoma and transitional cell carcinoma.
Assuntos
Células Epiteliais/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Anticorpos Antineoplásicos/imunologia , Biomarcadores/metabolismo , Linhagem da Célula , Células Cultivadas , Células Epiteliais/patologia , Epitélio/metabolismo , Regulação da Expressão Gênica , Humanos , Imuno-Histoquímica , Hibridização In Situ , Pulmão/citologia , Pulmão/metabolismo , Masculino , Proteínas de Membrana/genética , Mesotelina , Mesotelioma/genética , Mesotelioma/imunologia , Mesotelioma/patologia , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Análise de Sequência com Séries de Oligonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Uroplaquina III/genética , Uroplaquina III/metabolismoRESUMO
BACKGROUND: Genetic polymorphisms of the human CYP2A6 gene are considered to be a determinant of smoking behavior and tobacco-related lung cancer risk in male Japanese smokers. We developed a SmartAmp-based genotyping method to detect whole deletion of the CYP2A6 gene directly from blood samples without DNA isolation. METHODS: We validated the new method using CYP2A plasmids, 48 genomic DNA samples and 25 blood samples by utilizing the SmartAmp method, a unique isothermal DNA amplification process. RESULTS: This method could discriminate the CYP2A6 gene from highly homologous CYP2A7 and CYP2A13 genes. CYP2A6*1 (wild-type) and CYP2A6*4 (whole gene deletion) were determined by the new method in perfect accordance with sequence analysis data. CONCLUSIONS: A SmartAmp assay for genotyping the CYP2A6 gene was developed, and the reliability of the method was validated using the conventional PCR method.
Assuntos
Hidrocarboneto de Aril Hidroxilases/sangue , Hidrocarboneto de Aril Hidroxilases/genética , Deleção de Genes , Técnicas de Amplificação de Ácido Nucleico/métodos , Hidrocarboneto de Aril Hidroxilases/deficiência , Citocromo P-450 CYP2A6 , DNA/sangue , DNA/genética , Primers do DNA/genética , Genótipo , Temperatura Alta , Humanos , Fatores de TempoRESUMO
The application of isothermal amplification technologies is rapidly expanding and currently covers different areas such as infectious disease, genetic disorder and drug dosage adjustment. Meanwhile, many of such technologies have complex reaction processes and often require a fine-tuned primer set where existing primer design tools are not sufficient. We have developed a primer selection system for one important primer, the turn-back primer (TP), which is commonly used in loop-mediated amplification (LAMP) and smart amplification process (SmartAmp). We chose 78 parameters related to the primer and target sequence, and explored their relationship to amplification speed using experimental data for 1344 primer combinations. We employed the least absolute shrinkage and selection operator (LASSO) method for parameter selection and estimation of their numerical coefficients. We subsequently evaluated our prediction model using additional independent experiments and compared to the LAMP primer design tool, Primer Explorer version4 (PE4). The evaluation showed that our approach yields a superior primer design in isothermal amplification and is robust against variations in the experimental setup. Our LASSO regression analysis revealed that availability of the 3'- and 5'-end of the primer are particularly important factors for efficient isothermal amplification. Our computer script is freely available at: http://gerg.gsc.riken.jp/TP_optimization/.
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Primers do DNA/química , Técnicas de Amplificação de Ácido Nucleico , Humanos , Software , TemperaturaRESUMO
Clinical studies have suggested that a defect in both glutathione S-transferase (GST) M1 and GSTT1 increases the risk of drug-induced hepatotoxicity. The present study developed the method that enables genotyping of GSTM1 and GSTT1 directly using a small aliquot of blood samples based on an isothermal Smart amplification process version 2 (SmartAmp-2). SmartAmp-2 reaction could complete the genotyping of GSTM1 and GSTT1 within 40 min. The frequency of wild-type, GSTM1 null, GSTT1 null, and double null was 24, 21, 35, and 19%, respectively, consistent with previous reports in the Japanese population. The genotypes of 94 human genomic DNA samples determined by SmartAmp-2 were identical to those determined by the conventional polymerase chain reaction method. SmartAmp-2 was able to determine the genotypes of GSTM1 and GSTT1 even when human blood specimens were used. The SmartAmp-2 method is a rapid and accurate means of identifying the GSTM1 and GSTT1 genotypes, making it less time and more labor efficient in clinical practice than conventional methods requiring preparation of genomic DNA and electrophoresis. This will contribute to evaluate the susceptibility of disease and adverse reactions to drugs caused by deletion of GSTM1 and GSTT1.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/sangue , Glutationa Transferase/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Doença Hepática Induzida por Substâncias e Drogas/enzimologia , Doença Hepática Induzida por Substâncias e Drogas/genética , DNA/genética , Deleção de Genes , Frequência do Gene , Genótipo , Glutationa Transferase/sangue , Humanos , Japão , Polimorfismo GenéticoRESUMO
Most commonly used intercalating fluorescent dyes in DNA detection are lacking any sequence specificity, whereas so-called Exciton Primers can overcome this limitation by functioning as "sequence-specific dyes." After hybridization to complementary sequences, the fluorescence of Exciton Primers provides sequence-specific signals for real-time monitoring of amplification reactions. Applied to the SmartAmp2 mutation detection process, Exciton Primers show high signal strength with low background leading to a superior specificity and sensitivity compared to SYBR Green I. Signal strength can be further enhanced using multiple dyes within one Exciton Primer or use of multiple Exciton Primers in the same amplification reaction. Here we demonstrate the use of Exciton Primers for genotyping a single nucleotide polymorphism (SNP) in the VKORC1 locus (-1639G>A) relevant for Warfarin dosing as an example for Exciton Primers mediated genotyping by SmartAmp2. The genotyping assay can use only one labeled Exciton Primer for endpoint detection, or simultaneously by real-time monitoring detect wild-type and mutant alleles in a one-tube reaction using two Exciton Primers having different dyes. Working directly from blood samples, Exciton Primer mediated genotyping by SmartAmp2 offers superior solutions for rapid point-of-care testing.
Assuntos
Primers do DNA/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Benzotiazóis , Diaminas , Corantes Fluorescentes/química , Genótipo , Humanos , Oxigenases de Função Mista/genética , Compostos Orgânicos/metabolismo , Quinolinas , Vitamina K Epóxido RedutasesRESUMO
Quantitative features of the proteome are extremely useful for studying cellular processes at a molecular level. In this study, we attempted to construct quantitative reference proteome maps of the mouse spleen and lymph node based on 2-DE followed by protein identification using MS. We analyzed more than 1000 spots on the 2-DE images and consequently were able to determine that 919 spots were derived from 328 different genes. To obtain statistically reliable information of the protein levels from these 2-DE images, we measured the volumes of the respective spots on 2-DE images obtained by four to six independent experimental runs. These measurements were used to calculate the variability of the volumes of the respective spots on 2-DE following subcellular fractionation, which enabled us to discriminate differentially produced proteins from those within the range of intrinsic variability. More importantly, while the 2-DE data have been traditionally collected in a gel image-based manner, the resultant quantitative 2-DE data could be analyzed using the same procedure as that for mRNA expression profiles. This greatly assists in bridging the gap between the analyses of transcriptomes and proteomes and enables the integration of this data on the same informational platform.