| 1. |
- Barisic, Ivan, et al.
(författare)
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Multiplex detection of antibiotic resistance genes using padlock probes
- 2013
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Ingår i: Diagnostic microbiology and infectious disease. - : Elsevier BV. - 0732-8893 .- 1879-0070. ; 77:2, s. 118-125
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Tidskriftsartikel (refereegranskat)abstract
- The elucidation of resistance mechanisms is of central importance to providing and maintaining efficient medical treatment. However, molecular detection methods covering the complete set of resistance genes with a single test are still missing. Here, we present a novel 100-plex assay based on padlock probes in combination with a microarray that allows the simultaneous large-scale identification of highly diverse beta-lactamases. The specificity of the assay was performed using 70 clinical bacterial isolates, recovering 98% of the beta-lactamase nucleotide sequences present. Additionally, the sensitivity was evaluated with PCR products and genomic bacterial DNA, revealing a detection limit of 10(4) DNA copies per reaction when using PCR products as the template. Pre-amplification of genomic DNA in a 25-multiplex PCR further facilitated the detection of beta-lactamase genes in dilutions of 10(7) cells/mL. In summary, we present an efficient, highly specific, and highly sensitive multiplex detection method for any gene.
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| 2. |
- Ke, Rongqin, et al.
(författare)
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Fourth Generation of Next-Generation Sequencing Technologies : Promise and Consequences
- 2016
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Ingår i: Human Mutation. - : Hindawi Limited. - 1059-7794 .- 1098-1004. ; 37:12, s. 1363-1367
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Forskningsöversikt (refereegranskat)abstract
- In this review, we discuss the emergence of the fourth-generation sequencing technologies that preserve the spatial coordinates of RNA and DNA sequences with up to subcellular resolution, thus enabling back mapping of sequencing reads to the original histological context. This information is used, for example, in two current large-scale projects that aim to unravel the function of the brain. Also in cancer research, fourth-generation sequencing has the potential to revolutionize the field. Cancer Research UK has named Mapping the molecular and cellular tumor microenvironment in order to define new targets for therapy and prognosis one of the grand challenges in tumor biology. We discuss the advantages of sequencing nucleic acids directly in fixed cells over traditional next-generation sequencing (NGS) methods, the limitations and challenges that these new methods have to face to become broadly applicable, and the impact that the information generated by the combination of in situ sequencing and NGS methods will have in research and diagnostics.
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| 3. |
- Ke, Rongqin, et al.
(författare)
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Improving Precision of Proximity Ligation Assay by Amplified Single Molecule Detection
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:7
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Tidskriftsartikel (refereegranskat)abstract
- Proximity ligation assay (PLA) has been proven to be a robust protein detection method. The technique is characterized by high sensitivity and specificity, but the assay precision is probably limited by the PCR readout. To investigate this potential limitation and to improve precision, we developed a digital proximity ligation assay for protein measurement in fluids based on amplified single molecule detection. The assay showed significant improvements in precision, and thereby also detection sensitivity, over the conventional real-time PCR readout.
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| 4. |
- Ke, Rongqin, et al.
(författare)
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In situ sequencing for RNA analysis in preserved tissue and cells
- 2013
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Ingår i: Nature Methods. - : Springer Science and Business Media LLC. - 1548-7091 .- 1548-7105. ; 10:9, s. 857-860
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Tidskriftsartikel (refereegranskat)abstract
- Tissue gene expression profiling is performed on homogenates or on populations of isolated single cells to resolve molecular states of different cell types. In both approaches, histological context is lost. We have developed an in situ sequencing method for parallel targeted analysis of short RNA fragments in morphologically preserved cells and tissue. We demonstrate in situ sequencing of point mutations and multiplexed gene expression profiling in human breast cancer tissue sections.
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| 5. |
- McGinn, Steven, et al.
(författare)
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New Technologies for DNA analysis-A review of the READNA Project.
- 2016
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Ingår i: New Biotechnology. - : Elsevier BV. - 1876-4347 .- 1871-6784.
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Forskningsöversikt (refereegranskat)abstract
- The REvolutionary Approaches and Devices for Nucleic Acid analysis (READNA) project received funding from the European Commission for 4 1/2 years. The objectives of the project revolved around technological developments in nucleic acid analysis. The project partners have discovered, created and developed a huge body of insights into nucleic acid analysis, ranging from improvements and implementation of current technologies to the most promising sequencing technologies that constitute a 3(rd) and 4(th) generation of sequencing methods with nanopores and in situ sequencing, respectively.
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| 6. |
- Zhao, Yansong, et al.
(författare)
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Single Cell RNA Expression Analysis Using Flow Cytometry Based on Specific Probe Ligation and Rolling Circle Amplification
- 2020
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Ingår i: ACS Sensors. - : American Chemical Society (ACS). - 2379-3694. ; 5:10, s. 3031-3036
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Tidskriftsartikel (refereegranskat)abstract
- Conventional flow cytometry has been widely used for high-throughput single-cell gene expression analysis using specific antibody staining. However, this is limited by the availability of high-quality antibodies. We developed a novel flow cytometry RNA detection technique termed RCA-Flow for single-cell RNA expression analysis. We showed that it is able to analyze not only mRNAs but also microRNAs and circular RNAs that are otherwise difficult to analyze by other flow cytometry techniques. The versatility for high-throughput analysis of different types of RNA molecules makes our method possess great potential for both biomedical and clinical applications.
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