Showing papers by "Applied Biosystems published in 2013"

Single-cell RNA-Seq profiling of human preimplantation embryos and embryonic stem cells

Abstract: Measuring gene expression in individual cells is crucial for understanding the gene regulatory network controlling human embryonic development. Here we apply single-cell RNA sequencing (RNA-Seq) analysis to 124 individual cells from human preimplantation embryos and human embryonic stem cells (hESCs) at different passages. The number of maternally expressed genes detected in our data set is 22,687, including 8,701 long noncoding RNAs (lncRNAs), which represents a significant increase from 9,735 maternal genes detected previously by cDNA microarray. We discovered 2,733 novel lncRNAs, many of which are expressed in specific developmental stages. To address the long-standing question whether gene expression signatures of human epiblast (EPI) and in vitro hESCs are the same, we found that EPI cells and primary hESC outgrowth have dramatically different transcriptomes, with 1,498 genes showing differential expression between them. This work provides a comprehensive framework of the transcriptome landscapes of human early embryos and hESCs.

A tripartite transcription factor network regulates primordial germ cell specification in mice

Abstract: Surani and colleagues use single-cell transcriptomics analysis in a model of mouse primordial germ cell specification to analyse the collaboration between three transcription factors, BLIMP1, PRDM14 and AP2γ, in determining germ cell fate. They find that BLIMP1 binds directly to repress somatic and cell proliferation genes, and at the same time induces AP2γ, which acts together with PRDM14. The three factors are sufficient for specification and form a tripartite interdependent transcriptional network.

Inhibition of lung metastasis by chemokine CCL17-mediated in vivo silencing of genes in CCR4+ Tregs

Abstract: Despite significant attractiveness of antisense oligonucleotide/RNAi technology, its clinical application has been precluded by a lack of methods for targeted delivery and transduction of primary immune cells in vivo. Here, we devised a chemokine CCL17-based strategy (TARC-arp) that transiently silences expression of genes in immune cells by delivering inhibitory oligonucleotides through their chemokine receptors. In modeling studies using mice with established 4T1.2 breast cancer, we show that IL10 produced by CCR4 cells, in particular FoxP3 regulatory T cells (Tregs), plays an important role in lung metastasis. As such, TARC-arp-mediated silencing of IL10 or FoxP3 in CCR4 Tregs is sufficient to block lung metastasis. Thus, we provide a simple solution that circumvents the problems of RNAi use in vivo, indicating that a disease outcome can be successfully controlled by delivering inhibitory oligonucleotides with chemokines to inactivate a selective subset of immune cells.

Method of automated calibration and diagnosis of laboratory instruments

Abstract: Method and system providing an automated workflow for installing and/or calibrating laboratory equipment. The workflow empowers an end user to perform installation and calibration thereby reducing the costs associated with such activities. The automated workflow taught herein, can greatly reduce the incidence of calibration error by providing for verification of certain events during the calibration process.

Differential Proteomic Analysis of Acute Contusive Spinal Cord Injury in Rats Using iTRAQ Reagent Labeling and LC–MS/MS

Abstract: In this experimental study, differential labeling with isobaric tags for relative and absolute quantitation (iTRAQ) reagents followed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS) proteomic approach was used to investigate differences in the proteome of rat spinal cord at 24 h following a moderate contusion injury. Spinal cord protein samples from the injury epicenter (or from sham controls) were trypsinized and differentially labeled with iTRAQ isotopic reagents. The differentially labeled samples were then combined into one sample mixture, separated by LC, and analyzed using MS/MS. Proteins were quantified by comparing the peak areas of iTRAQ reporter fragment ions in MS/MS spectra. The outcome of this analysis revealed that proteins involved in ubiquitination, endocytosis and exocytosis, energy metabolism, inflammatory response, oxidative stress, cytoskeletal disruption, and vascular damage were significantly altered at 24 h following spinal cord injury (SCI). This study demonstrates the utility of the iTRAQ method in proteomic studies and provides further insights into secondary events that occur during acute times following SCI.

Sample preparation for in situ nucleic acid analysis, methods and compositions therefor

Abstract: Sample preparation processes for in situ RNA or DNA analysis, methods and compositions therefor are provided. Processes provided herein allow DNA or RNA analysis to be carried out in the same tube or on an aliquot of the prepared sample without centrifugation or extraction. The preparation process can be carried out at room temperature in as little as seven minutes and is amenable to high throughput processing using manual or robotic platforms.

Chimeric oligonucleotides for ligation-enhanced nucleic acid detection, methods and compositions therefor

Abstract: Ligation-enhanced nucleic acid detection assay embodiments for detection of RNA or DNA are described. The assay embodiments rely on ligation of chimeric oligonucleotide probes to generate a template for amplification and detection. The assay embodiments are substantially independent of the fidelity of a polymerase for copying compromised nucleic acid. Very little background amplification is observed and as few as 1000 copies of target nucleic acid can be detected. Method embodiments are particularly adept for detection of RNA from compromised samples such as formalin-fixed and paraffin-embedded samples. Heavily degraded and cross-linked nucleic acids of compromised samples, in which classic quantitative real time PCR assays typically fail to adequately amplify signal, can be reliably detected and quantified.

Devices, methods, and systems for nucleic acid amplification

Abstract: A microfluidic device comprising a first surface portion and a first sample retaining element, which have differing affinities to a fluid, and a method comprising supplying a sample to such a device. In some embodiments, the differing affinity is a result of plasma, ion embedding, surface charging, chemical, optical, electronic and/or electromagnetic treatment. Also, a microfluidic device comprising at least one microcapillary device having a sample retaining element, at least one surface of which exhibits hydrophobicity, hydrophilicity, electromagnetic force exertion and electrostatic force exertion. Also, a microfluidic device comprising a first element having a hydrophilic pattern comprising at least a first sample retaining element. Also, a method comprising supplying a sample to a channel between a first element and a second element, and inducing in the first element at least one hydrophilic pattern by electrets or by internal or external electrodes to provide a charged surface.

Methods for bead-based sequencing

Abstract: The present invention provides methods for determining a nucleic acid sequence by performing successive cycles of duplex extension along a single stranded template The cycles comprise steps of extension, ligation, and, preferably, cleavage In certain embodiments the methods make use of extension probes containing phosphorothiolate linkages and employ agents appropriate to cleave such linkages The invention provides methods of determining information about a sequence using at least two distinguishably labeled probe families In certain embodiments the methods acquire less than 2 bits of information from each of a plurality of nucleotides in the template in each cycle In certain embodiments the sequencing reactions are performed on templates attached to beads The invention further provides sets of labeled extension probes containing phosphorothiolate linkages In addition, the invention includes performing multiple sequencing reactions on a single template by removing initializing oligonucleotides and extended strands and performing subsequent reactions using different initializing oligonucleotides