RESUMEN
We developed a procedure for the preparation of whole transcriptome cDNA libraries depleted of ribosomal RNA from only 1 microg of total RNA. The method relies on a collection of short, computationally selected oligonucleotides, called 'not-so-random' (NSR) primers, to obtain full-length, strand-specific representation of nonribosomal RNA transcripts. In this study we validated the technique by profiling human whole brain and universal human reference RNA using ultra-high-throughput sequencing.
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Encéfalo/metabolismo , ADN Complementario/genética , Perfilación de la Expresión Génica/métodos , Biblioteca de Genes , Clonación Molecular , Humanos , ARN/genética , ARN/metabolismoRESUMEN
The Rhodopseudomonas palustris transcriptional regulator RpaR responds to the RpaI-synthesized quorum-sensing signal p-coumaroyl-homoserine lactone (pC-HSL). Other characterized RpaR homologs respond to fatty acyl-HSLs. We show here that RpaR functions as a transcriptional activator, which binds directly to the rpaI promoter. We developed an RNAseq method that does not require a ribosome depletion step to define a set of transcripts regulated by pC-HSL and RpaR. The transcripts include several noncoding RNAs. A footprint analysis showed that purified His-tagged RpaR (His(6)-RpaR) binds to an inverted repeat element centered 48.5 bp upstream of the rpaI transcript start site, which we mapped by S1 nuclease protection and primer extension analyses. Although pC-HSL-RpaR bound to rpaI promoter DNA, it did not bind to the promoter regions of a number of RpaR-regulated genes not in the rpaI operon. This indicates that RpaR control of these other genes is indirect. Because the RNAseq analysis allowed us to track transcript strand specificity, we discovered that there is pC-HSL-RpaR-activated antisense transcription of rpaR. These data raise the possibility that this antisense RNA or other RpaR-activated noncoding RNAs mediate the indirect activation of genes in the RpaR-controlled regulon.
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4-Butirolactona/análogos & derivados , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Rhodopseudomonas/fisiología , Transducción de Señal , Factores de Transcripción/metabolismo , 4-Butirolactona/metabolismo , Huella de ADN , ADN Bacteriano/metabolismo , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
BACKGROUND: Identifying associations between genotypes and gene expression levels using microarrays has enabled systematic interrogation of regulatory variation underlying complex phenotypes. This approach has vast potential for functional characterization of disease states, but its prohibitive cost, given hundreds to thousands of individual samples from populations have to be genotyped and expression profiled, has limited its widespread application. RESULTS: Here we demonstrate that genomic regions with allele-specific expression (ASE) detected by sequencing cDNA are highly enriched for cis-acting expression quantitative trait loci (cis-eQTL) identified by profiling of 500 animals in parallel, with up to 90% agreement on the allele that is preferentially expressed. We also observed widespread noncoding and antisense ASE and identified several allele-specific alternative splicing variants. CONCLUSION: Monitoring ASE by sequencing cDNA from as little as one sample is a practical alternative to expression genetics for mapping cis-acting variation that regulates RNA transcription and processing.
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Perfilación de la Expresión Génica/métodos , Expresión Génica , Variación Genética , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ADN/métodos , Alelos , Empalme Alternativo , Animales , Elementos sin Sentido (Genética)/genética , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN no Traducido/genética , Transcripción GenéticaRESUMEN
BACKGROUND: DNA copy number variations occur within populations and aberrations can cause disease. We sought to develop an improved lab-automatable, cost-efficient, accurate platform to profile DNA copy number. RESULTS: We developed a sequencing-based assay of nuclear, mitochondrial, and telomeric DNA copy number that draws on the unbiased nature of next-generation sequencing and incorporates techniques developed for RNA expression profiling. To demonstrate this platform, we assayed UMC-11 cells using 5 million 33 nt reads and found tremendous copy number variation, including regions of single and homogeneous deletions and amplifications to 29 copies; 5 times more mitochondria and 4 times less telomeric sequence than a pool of non-diseased, blood-derived DNA; and that UMC-11 was derived from a male individual. CONCLUSION: The described assay outputs absolute copy number, outputs an error estimate (p-value), and is more accurate than array-based platforms at high copy number. The platform enables profiling of mitochondrial levels and telomeric length. The assay is lab-automatable and has a genomic resolution and cost that are tunable based on the number of sequence reads.
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Tumor Carcinoide/genética , Variaciones en el Número de Copia de ADN , Neoplasias Pulmonares/genética , Mitocondrias/genética , Telómero , Animales , Línea Celular Tumoral , Femenino , Humanos , Masculino , Ratones , Análisis de Secuencia de ADN/métodosRESUMEN
microRNAs (miRNAs) are abundant, approximately 21-nucleotide, noncoding regulatory RNAs. Each miRNA may regulate hundreds of mRNA targets, but the identities of these targets and the processes they regulate are poorly understood. Here we have explored the use of microarray profiling and functional screening to identify targets and biological processes triggered by the transfection of human cells with miRNAs. We demonstrate that a family of miRNAs sharing sequence identity with miRNA-16 (miR-16) negatively regulates cellular growth and cell cycle progression. miR-16-down-regulated transcripts were enriched with genes whose silencing by small interfering RNAs causes an accumulation of cells in G(0)/G(1). Simultaneous silencing of these genes was more effective at blocking cell cycle progression than disruption of the individual genes. Thus, miR-16 coordinately regulates targets that may act in concert to control cell cycle progression.
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Ciclo Celular/genética , Ciclo Celular/fisiología , MicroARNs/clasificación , MicroARNs/genética , Familia de Multigenes/genética , Transcripción Genética , Línea Celular , Regulación hacia Abajo , Perfilación de la Expresión Génica , Humanos , Neoplasias/genética , Neoplasias/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , FenotipoRESUMEN
UltraPrep is an open-source, two-step method for purification of cell-free DNA that entails extraction of total DNA followed by size-selective enrichment of the smaller fragments that are characteristic of DNA originating from fragmentation between nucleosome. The advantages of the two related protocols that are described are that they can easily accommodate a wide range of sample input volumes, they rely on simple, magnetic bead-based technology, the yields of cfDNA are directly comparable to the most popular methods for cfDNA purification, and they dramatically reduce the cost of cfDNA isolation relative to currently available commercial methods. We provide a framework for physical and molecular quality analysis of purified cfDNA and demonstrate that the cfDNA generated by UltraPrep meets or exceeds the quality metrics of the most commonly used procedure. In addition, our method removes high molecular weight genomic DNA (hmwgDNA) that can interfere with downstream assay results, thereby addressing one of the primary concerns for preanalytical collection of blood samples.
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Ácidos Nucleicos Libres de Células/aislamiento & purificación , Extracción en Fase Sólida/métodos , Ácidos Nucleicos Libres de Células/sangre , Humanos , Biopsia Líquida , Magnetismo , Nucleosomas/genética , Dióxido de Silicio/química , Extracción en Fase Sólida/economíaRESUMEN
Genomic analysis of circulating, cell-free DNA (cfDNA) is being used extensively for molecular diagnostics. Many approaches rely on the construction of cfDNA genomic libraries, targeted retrieval of specific genomic regions and analysis by next-generation DNA sequencing. Several steps during sample preparation require isolation of DNA fragments within a particular size range. In this Benchmark article, two related methods for size-selective DNA fragment enrichment are described.
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Ácidos Nucleicos Libres de Células/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Biopsia Líquida , Ácidos Nucleicos Libres de Células/química , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , HumanosRESUMEN
BACKGROUND: Liquid biopsy for plasma circulating tumor DNA (ctDNA) next-generation sequencing (NGS) is commercially available and increasingly adopted in clinical practice despite a paucity of prospective data to support its use. METHODS: Patients with advanced lung cancers who had no known oncogenic driver or developed resistance to current targeted therapy (n = 210) underwent plasma NGS, targeting 21 genes. A subset of patients had concurrent tissue NGS testing using a 468-gene panel (n = 106). Oncogenic driver detection, test turnaround time (TAT), concordance, and treatment response guided by plasma NGS were measured. All statistical tests were two-sided. RESULTS: Somatic mutations were detected in 64.3% (135/210) of patients. ctDNA detection was lower in patients who were on systemic therapy at the time of plasma collection compared with those who were not (30/70, 42.9% vs 105/140, 75.0%; OR = 0.26, 95% CI = 0.1 to 0.5, P < .001). The median TAT of plasma NGS was shorter than tissue NGS (9 vs 20 days; P < .001). Overall concordance, defined as the proportion of patients for whom at least one identical genomic alteration was identified in both tissue and plasma, was 56.6% (60/106, 95% CI = 46.6% to 66.2%). Among patients who tested plasma NGS positive, 89.6% (60/67; 95% CI = 79.7% to 95.7%) were also concordant on tissue NGS and 60.6% (60/99; 95% CI = 50.3% to 70.3%) vice versa. Patients who tested plasma NGS positive for oncogenic drivers had tissue NGS concordance of 96.1% (49/51, 95% CI = 86.5% to 99.5%), and directly led to matched targeted therapy in 21.9% (46/210) with clinical response. CONCLUSIONS: Plasma ctDNA NGS detected a variety of oncogenic drivers with a shorter TAT compared with tissue NGS and matched patients to targeted therapy with clinical response. Positive findings on plasma NGS were highly concordant with tissue NGS and can guide immediate therapy; however, a negative finding in plasma requires further testing. Our findings support the potential incorporation of plasma NGS into practice guidelines.
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Carcinoma de Pulmón de Células no Pequeñas/genética , ADN Tumoral Circulante/genética , Neoplasias Pulmonares/genética , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma de Pulmón de Células no Pequeñas/sangre , Carcinoma de Pulmón de Células no Pequeñas/terapia , ADN Tumoral Circulante/sangre , Femenino , Técnicas de Genotipaje , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Biopsia Líquida , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/terapia , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , Mutación , Medicina de Precisión , Estudios ProspectivosRESUMEN
INTRODUCTION: Patients with SCLC have a poor prognosis and limited treatment options. Because access to longitudinal tumor samples is very limited in patients with this disease, we chose to focus our studies on the characterization of plasma cell-free DNA (cfDNA) for rapid, noninvasive monitoring of disease burden. METHODS: We developed a liquid biopsy assay that quantifies somatic variants in cfDNA. The assay detects single nucleotide variants, copy number alterations, and insertions or deletions in 14 genes that are frequently mutated in SCLC, including tumor protein p53 gene (TP53), retinoblastoma 1 gene (RB1), BRAF, KIT proto-oncogene receptor tyrosine kinase gene (KIT), notch 1 gene (NOTCH1), notch 2 gene (NOTCH2), notch 3 gene (NOTCH3), notch 4 gene (NOTCH4), phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha gene (PIK3CA), phosphatase and tensin homolog gene (PTEN), fibroblast growth factor receptor 1 gene (FGFR1), v-myc avian myelocytomatosis viral oncogene homolog gene (MYC), v-myc avian myelocytomatosis viral oncogene lung carcinoma derived homolog gene (MYCL1), and v-myc avian myelocytomatosis viral oncogene neuroblastoma derived homolog gene (MYCN). RESULTS: Over the course of 26 months of peripheral blood collection, we examined 140 plasma samples from 27 patients. We detected disease-associated mutations in 85% of patient samples with mutant allele frequencies ranging from 0.1% to 87%. In our cohort, 59% of the patients had extensive-stage disease, and the most common mutations occurred in TP53 (70%) and RB1 (52%). In addition to mutations in TP53 and RB1, we detected alterations in 10 additional genes in our patient population (PTEN, NOTCH1, NOTCH2, NOTCH3, NOTCH4, MYC, MYCL1, PIK3CA, KIT, and BRAF). The observed allele frequencies and copy number alterations tracked closely with treatment responses. Notably, in several cases analysis of cfDNA provided evidence of disease relapse before conventional imaging. CONCLUSIONS: These results suggest that liquid biopsies are readily applicable in patients with SCLC and can potentially provide improved monitoring of disease burden, depth of response to treatment, and timely warning of disease relapse in patients with this disease.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Biomarcadores de Tumor/sangre , Ácidos Nucleicos Libres de Células/sangre , Neoplasias Pulmonares/patología , Mutación , Recurrencia Local de Neoplasia/patología , Carcinoma Pulmonar de Células Pequeñas/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/genética , Ácidos Nucleicos Libres de Células/genética , Femenino , Estudios de Seguimiento , Humanos , Estudios Longitudinales , Neoplasias Pulmonares/sangre , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia/sangre , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/genética , Estudios Prospectivos , Proto-Oncogenes Mas , Carcinoma Pulmonar de Células Pequeñas/sangre , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genética , Tasa de Supervivencia , Resultado del TratamientoRESUMEN
The breadth of diagnostic procedures that utilize cell free DNA (cfDNA) from human plasma has increased dramatically in recent years. Here, we confirm that tumor-derived cfDNA fragments are similar in size distribution to cfDNA derived from normal tissues. Therefore, collection procedures optimized with healthy donor specimens are likely to be applicable to the diagnosis and monitoring of many different cancer types. We verify that the distribution and DNA sequences of fragmentation sites in cfDNA from both normal-germline and tumor-derived cfDNA are non-random. A broad survey of cfDNA from healthy donors suggests that average individuals possess ~6 ng of cfDNA per mL of plasma. Importantly, the cfDNA present in plasma samples that were initially collected as whole blood in K2-EDTA tubes and subsequently processed by centrifugation is stable for several days at ambient temperatures. This observation has the potential to significantly reduce the cost and logistical complexity of shipping clinical samples from the site of collection to centers proficient in diagnostic analysis. Finally, plasma samples collected with high-volume plasma collection devices possess abundant quantities of cfDNA. Since the quantity of analyzed cfDNA is directly proportional to sensitivity of diagnostic assays, this method of plasma collection, where available, could enable highly sensitive post-treatment disease monitoring and early detection of cancer in at-risk individuals.
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Recolección de Muestras de Sangre/métodos , ADN/sangre , ADN/genética , Genómica , Neoplasias/sangre , Neoplasias/genética , Estudios de Casos y Controles , Sistema Libre de Células/metabolismo , Fragmentación del ADN , Humanos , Nucleosomas/genéticaAsunto(s)
Antineoplásicos Inmunológicos/uso terapéutico , Transformación Celular Neoplásica/patología , Resistencia a Antineoplásicos , Mutación , Receptor de Muerte Celular Programada 1/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Carcinoma Pulmonar de Células Pequeñas/patología , Adenocarcinoma del Pulmón/tratamiento farmacológico , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/secundario , Anciano , Transformación Celular Neoplásica/efectos de los fármacos , Transformación Celular Neoplásica/genética , Femenino , Humanos , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundario , Pronóstico , Carcinoma Pulmonar de Células Pequeñas/tratamiento farmacológico , Carcinoma Pulmonar de Células Pequeñas/genéticaRESUMEN
Malaria caused by Plasmodium falciparum results in approximately 1 million annual deaths worldwide, with young children and pregnant mothers at highest risk. Disease severity might be related to parasite virulence factors, but expression profiling studies of parasites to test this hypothesis have been hindered by extensive sequence variation in putative virulence genes and a preponderance of host RNA in clinical samples. We report here the application of RNA sequencing to clinical isolates of P. falciparum, using not-so-random (NSR) primers to successfully exclude human ribosomal RNA and globin transcripts and enrich for parasite transcripts. Using NSR-seq, we confirmed earlier microarray studies showing upregulation of a distinct subset of genes in parasites infecting pregnant women, including that encoding the well-established pregnancy malaria vaccine candidate var2csa. We also describe a subset of parasite transcripts that distinguished parasites infecting children from those infecting pregnant women and confirmed this observation using quantitative real-time PCR and mass spectrometry proteomic analyses. Based on their putative functional properties, we propose that these proteins could have a role in childhood malaria pathogenesis. Our study provides proof of principle that NSR-seq represents an approach that can be used to study clinical isolates of parasites causing severe malaria syndromes as well other blood-borne pathogens and blood-related diseases.
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Regulación de la Expresión Génica , Malaria/parasitología , Plasmodium falciparum/metabolismo , Transcripción Genética , Animales , Niño , Cartilla de ADN/genética , Cartilla de ADN/metabolismo , Femenino , Perfilación de la Expresión Génica , Humanos , Malaria/genética , Espectrometría de Masas/métodos , Embarazo , Complicaciones Parasitarias del Embarazo , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , RiesgoRESUMEN
Non-coding RNAs (ncRNAs) are an essential class of molecular species that have been difficult to monitor on high throughput platforms due to frequent lack of polyadenylation. Using a polyadenylation-neutral amplification protocol and next-generation sequencing, we explore ncRNA expression in eleven human tissues. ncRNAs 7SL, U2, 7SK, and HBII-52 are expressed at levels far exceeding mRNAs. C/D and H/ACA box snoRNAs are associated with rRNA methylation and pseudouridylation, respectively: spleen expresses both, hypothalamus expresses mainly C/D box snoRNAs, and testes show enriched expression of both H/ACA box snoRNAs and RNA telomerase TERC. Within the snoRNA 14q cluster, 14q(I-6) is expressed at much higher levels than other cluster members. More reads align to mitochondrial than nuclear tRNAs. Many lincRNAs are actively transcribed, particularly those overlapping known ncRNAs. Within the Prader-Willi syndrome loci, the snoRNA HBII-85 (group I) cluster is highly expressed in hypothalamus, greater than in other tissues and greater than group II or III. Additionally, within the disease locus we find novel transcription across a 400,000 nt span in ovaries. This genome-wide polyA-neutral expression compendium demonstrates the richness of ncRNA expression, their high expression patterns, their function-specific expression patterns, and is publicly available.
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Genoma Humano/genética , ARN Nucleolar Pequeño/genética , ARN no Traducido/genética , Perfilación de la Expresión Génica , Humanos , Hipotálamo/metabolismo , Reacción en Cadena de la Polimerasa , Bazo/metabolismoRESUMEN
Studies of the host response to virus infection typically focus on protein-coding genes. However, non-protein-coding RNAs (ncRNAs) are transcribed in mammalian cells, and the roles of many of these ncRNAs remain enigmas. Using next-generation sequencing, we performed a whole-transcriptome analysis of the host response to severe acute respiratory syndrome coronavirus (SARS-CoV) infection across four founder mouse strains of the Collaborative Cross. We observed differential expression of approximately 500 annotated, long ncRNAs and 1,000 nonannotated genomic regions during infection. Moreover, studies of a subset of these ncRNAs and genomic regions showed the following. (i) Most were similarly regulated in response to influenza virus infection. (ii) They had distinctive kinetic expression profiles in type I interferon receptor and STAT1 knockout mice during SARS-CoV infection, including unique signatures of ncRNA expression associated with lethal infection. (iii) Over 40% were similarly regulated in vitro in response to both influenza virus infection and interferon treatment. These findings represent the first discovery of the widespread differential expression of long ncRNAs in response to virus infection and suggest that ncRNAs are involved in regulating the host response, including innate immunity. At the same time, virus infection models provide a unique platform for studying the biology and regulation of ncRNAs.
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Perfilación de la Expresión Génica , Inmunidad Innata , ARN no Traducido/biosíntesis , Síndrome Respiratorio Agudo Grave/inmunología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Transducción de Señal , Transcripción Genética , Animales , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Ratones , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Síndrome Respiratorio Agudo Grave/patología , Síndrome Respiratorio Agudo Grave/virologíaRESUMEN
Acetyl-CoA carboxylases ACC1 and ACC2 catalyze the carboxylation of acetyl-CoA to malonyl-CoA, regulating fatty-acid synthesis and oxidation, and are potential targets for treatment of metabolic syndrome. Expression of ACC1 in rodent lipogenic tissues and ACC2 in rodent oxidative tissues, coupled with the predicted localization of ACC2 to the mitochondrial membrane, have suggested separate functional roles for ACC1 in lipogenesis and ACC2 in fatty acid oxidation. We find, however, that human adipose tissue, unlike rodent adipose, expresses more ACC2 mRNA relative to the oxidative tissues muscle and heart. Human adipose, along with human liver, expresses more ACC2 than ACC1. Using RT-PCR, real-time PCR, and immunoprecipitation we report a novel isoform of ACC2 (ACC2.v2) that is expressed at significant levels in human adipose. The protein generated by this isoform has enzymatic activity, is endogenously expressed in adipose, and lacks the N-terminal sequence. Both ACC2 isoforms are capable of de novo lipogenesis, suggesting that ACC2, in addition to ACC1, may play a role in lipogenesis. The results demonstrate a significant difference in ACC expression between human and rodents, which may introduce difficulties for the use of rodent models for development of ACC inhibitors.
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Acetil-CoA Carboxilasa/química , Acetil-CoA Carboxilasa/metabolismo , Tejido Adiposo Blanco/enzimología , Acetil-CoA Carboxilasa/genética , Acetiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , RatasRESUMEN
MicroRNAs (miRNAs) are a class of small noncoding RNAs that have important regulatory roles in multicellular organisms. The public miRNA database contains 321 human miRNA sequences, 234 of which have been experimentally verified. To explore the possibility that additional miRNAs are present in the human genome, we have developed an experimental approach called miRNA serial analysis of gene expression (miRAGE) and used it to perform the largest experimental analysis of human miRNAs to date. Sequence analysis of 273,966 small RNA tags from human colorectal cells allowed us to identify 200 known mature miRNAs, 133 novel miRNA candidates, and 112 previously uncharacterized miRNA* forms. To aid in the evaluation of candidate miRNAs, we disrupted the Dicer locus in three human colorectal cancer cell lines and examined known and novel miRNAs in these cells. These studies suggest that the human genome contains many more miRNAs than currently identified and provide an approach for the large-scale experimental cloning of novel human miRNAs in human tissues.
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Neoplasias Colorrectales/genética , MicroARNs/genética , ARN Neoplásico/genética , Secuencia de Bases , Línea Celular Tumoral , Colon/metabolismo , ARN Helicasas DEAD-box , Endorribonucleasas/genética , Expresión Génica , Marcación de Gen , Genoma Humano , Humanos , MicroARNs/química , Conformación de Ácido Nucleico , ARN Helicasas/genética , ARN Neoplásico/química , Recto/metabolismo , Ribonucleasa IIIRESUMEN
There has been a surge of interest in the biology of microRNAs and the technology of RNA interference. We describe a simple, robust, inexpensive assay for quantitative analysis of microRNAs and short-interfering RNAs. The method relies on primer extension conversion of RNA to cDNA by reverse transcription followed by quantitative, real-time PCR. Technical parameters critical to the success of the assay are presented. Measurements of microRNA levels are sensitive, with most assays allowing measurements in the femtomolar range, which corresponds to tens of copies per cell or less. The assay has a high dynamic range and provides linear readout over differences in microRNA concentrations that span 6-7 orders of magnitude. The assay is capable of discriminating between related microRNA family members that differ by subtle sequence differences. We used the method for quantitative analysis of six microRNAs across 12 tissue samples. The data confirm striking variation in the patterns of expression of these noncoding regulatory RNAs.
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MicroARNs/análisis , Reacción en Cadena de la Polimerasa/métodos , ARN Interferente Pequeño/análisis , Cartilla de ADN , ADN Complementario/metabolismo , Perfilación de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Interferente Pequeño/genética , Transcripción Reversa , Distribución TisularRESUMEN
Allelic variation in codons that specify amino acids that line the peptide-binding pockets of HLA's Class II antigen-presenting proteins is superimposed on strikingly few deeply diverged haplotypes. These haplotypes appear to have been evolving almost independently for tens of millions of years. By complete resequencing of 20 haplotypes across the approximately 100-kbp region that spans the HLA-DQA1, -DQB1, and -DRB1 genes, we provide a detailed view of the way in which the genome structure at this locus has been shaped by the interplay of selection, gene-gene interaction, and recombination.
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Genes MHC Clase II , Alelos , Animales , Evolución Molecular , Variación Genética , Genoma Humano , Gorilla gorilla/genética , Gorilla gorilla/inmunología , Antígenos HLA-DQ/genética , Cadenas alfa de HLA-DQ , Cadenas beta de HLA-DQ , Antígenos HLA-DR/genética , Cadenas HLA-DRB1 , Haplotipos , Humanos , Desequilibrio de Ligamiento , Modelos Genéticos , Datos de Secuencia Molecular , Pan troglodytes/genética , Pan troglodytes/inmunología , Filogenia , Polimorfismo de Nucleótido Simple , Recombinación Genética , Selección GenéticaRESUMEN
Currently, challenges exist to acquire long-range (hundreds of kilobase pairs) phase-discriminated sequence across substantial numbers of individuals. We have developed a straightforward method for isolating and characterizing specific genomic regions in a haplospecific manner. Real-time PCR is carried out to STS content map and genotype pools of fosmid clones arrayed in 384-well microtiter plates. Single-nucleotide polymorphisms, microsatellite markers, and insertion-deletion polymorphisms are used to differentiate the target region into haplotype-specific tiling paths. DNA of clones from these tiling paths is retrieved from the library and either sequenced by standard shotgun methods or amplified in vitro and sequenced by a primer-based, directed method. This approach provides convenient access to complete, haplotype-resolved resequencing data from multiple individuals across tens to hundreds of thousands of basepairs. We illustrate its implementation with a detailed example of more than 400 kbp from the human CFTR region, across 15 individuals, and summarize our experience applying it to many other human loci.
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Genoma Humano/genética , Haplotipos/genética , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Clonación Molecular/métodos , Genotipo , Humanos , Repeticiones de Microsatélite/genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido Simple/genéticaRESUMEN
The homologous recombination pathway in yeast is an ideal tool for the sequence-specific assembly of plasmids. Complementary 80-nucleotide oligonucleotides that overlap a vector and a target fragment were found to serve as efficient recombination linkers for fragment subcloning. Using electroporation, single-stranded 80-mers were adequate for routine plasmid construction. A cycloheximide-based counterselection was introduced to increase the specificity of cloning by homologous recombination relative to nonspecific vector background. Reconstruction experiments suggest this counterselection increased cloning specificity by 100-fold. Cycloheximide counterselection was used in conjunction with 80-bp linkers to subclone targeted regions from bacterial artificial chromosomes. This technology may find broad application in the final stages of completing the Human Genome Sequencing Project and in applications of BAC clones to the functional analysis of complex genomes.