| 1. |
- 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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| 2. |
- Mignardi, Marco, et al.
(författare)
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Bridging Histology and Bioinformatics : Computational analysis of spatially resolved transcriptomics
- 2017
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Ingår i: Proceedings of the IEEE. - 0018-9219 .- 1558-2256. ; 105:3, s. 530-541
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Tidskriftsartikel (refereegranskat)abstract
- It is well known that cells in tissue display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment. Traditional methods that analyze gene expression from bulk RNA extracts fail to accurately describe this heterogeneity because of their intrinsic limitation in cellular and spatial resolution. Also, information on histology in the form of tissue architecture and organization is lost in the process. Recently, new transcriptome-wide analysis technologies have enabled the study of RNA molecules directly in tissue samples, thus maintaining spatial resolution and complementing histological information with molecular information important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients. These new methods generally comprise three levels of analysis. At the first level, biochemical techniques are used to generate signals that can be imaged by different means of fluorescence microscopy. At the second level, images are subject to digital image processing and analysis in order to detect and identify the aforementioned signals. At the third level, the collected data are analyzed and transformed into interpretable information by statistical methods and visualization techniques relating them to each other, to spatial distribution, and to tissue morphology. In this review, we describe state-of-the-art techniques used at all three levels of analysis. Finally, we discuss future perspective in this fast-growing field of spatially resolved transcriptomics.
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| 3. |
- Wu, Chenglin, et al.
(författare)
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RollFISH achieves robust quantification of single-molecule RNA biomarkers in paraffin-embedded tumor tissue samples
- 2018
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Ingår i: Communications biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 1
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Tidskriftsartikel (refereegranskat)abstract
- Single-molecule RNA fluorescence in situ hybridization (smFISH) represents a promising approach to quantify the expression of clinically useful biomarkers in tumor samples. However, routine application of smFISH to formalin-fixed, paraffin-embedded (FFPE) samples is challenging due to the low signal intensity and high background noise. Here we present RollFISH, a method combining the specificity of smFISH with the signal boosting of rolling circle amplification. We apply RollFISH to quantify widely used breast cancer biomarkers in cell lines and FFPE samples. Thanks to the high signal-to-noise ratio, we can visualize selected biomarkers at low magnification (20 x) across entire tissue sections, and thus assess their spatial heterogeneity. Lastly, we apply RollFISH to quantify HER2 mRNA in 150 samples on a single tissue microarray, achieving a sensitivity and specificity of detection of HER2-positive samples of similar to 90%. RollFISH is a robust method for quantifying the expression and intratumor heterogeneity of biomarkers in FFPE tissues.
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| 4. |
- 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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| 5. |
- Mignardi, Marco, et al.
(författare)
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Fourth-generation sequencing in the cell and the clinic
- 2014
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Ingår i: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X. ; 6, s. 31-31
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Nearly 40 years ago, DNA was sequenced for the first time. Since then, DNA sequencing has undergone continuous development, passing through three generations of sequencing technology. We are now entering the beginning of a new phase of genomic analysis in which massively parallel sequencing is performed directly in the cell. Two methods have recently been described for in situ RNA sequencing, one targeted and one untargeted, that rely on ligation chemistry. This fourth generation of sequencing technology opens up prospects for transcriptomic analysis, biomarker validation, diagnosis and patient stratification for cancer treatment.
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| 6. |
- Kiflemariam, Sara, et al.
(författare)
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In situ sequencing identifies TMPRSS2-ERG fusion transcripts, somatic point mutations and gene expression levels in prostate cancers
- 2014
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Ingår i: Journal of Pathology. - : Wiley. - 0022-3417 .- 1096-9896. ; 234:2, s. 253-261
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Tidskriftsartikel (refereegranskat)abstract
- Translocations contribute to the genesis and progression of epithelial tumours and in particular to prostate cancer development. To better understand the contribution of fusion transcripts and visualize the clonal composition of multifocal tumours, we have developed a technology for multiplex in situ detection and identification of expressed fusion transcripts. When compared to immunohistochemistry, TMPRSS2-ERG fusion-negative and fusion-positive prostate tumours were correctly classified. The most prevalent TMPRSS2-ERG fusion variants were visualized, identified, and quantitated in human prostate cancer tissues, and the ratio of the variant fusion transcripts could for the first time be directly determined by in situ sequencing. Further, we demonstrate concurrent in situ detection of gene expression, point mutations, and gene fusions of the prostate cancer relevant targets AMACR, AR, TP53, and TMPRSS2-ERG. This unified approach to in situ analyses of somatic mutations can empower studies of intra-tumoural heterogeneity and future tissue-based diagnostics of mutations and translocations.
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| 7. |
- Mignardi, Marco, et al.
(författare)
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Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ.
- 2015
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Ingår i: Nucleic Acids Research. - : Oxford University Press (OUP). - 0305-1048 .- 1362-4962. ; 43:22
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Tidskriftsartikel (refereegranskat)abstract
- In clinical diagnostics a great need exists for targeted in situ multiplex nucleic acid analysis as the mutational status can offer guidance for effective treatment. One well-established method uses padlock probes for mutation detection and multiplex expression analysis directly in cells and tissues. Here, we use oligonucleotide gap-fill ligation to further increase specificity and to capture molecular substrates for in situ sequencing. Short oligonucleotides are joined at both ends of a padlock gap probe by two ligation events and are then locally amplified by target-primed rolling circle amplification (RCA) preserving spatial information. We demonstrate the specific detection of the A3243G mutation of mitochondrial DNA and we successfully characterize a single nucleotide variant in the ACTB mRNA in cells by in situ sequencing of RCA products generated by padlock gap-fill ligation. To demonstrate the clinical applicability of our assay, we show specific detection of a point mutation in the EGFR gene in fresh frozen and formalin-fixed, paraffin-embedded (FFPE) lung cancer samples and confirm the detected mutation by in situ sequencing. This approach presents several advantages over conventional padlock probes allowing simpler assay design for multiplexed mutation detection to screen for the presence of mutations in clinically relevant mutational hotspots directly in situ.
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| 8. |
- Wu, Chengjun, et al.
(författare)
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A mouse mammary epithelial cell line permissive for highly efficient human adenovirus growth
- 2013
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Ingår i: Virology. - : Elsevier BV. - 0042-6822 .- 1096-0341. ; 435:2, s. 363-371
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Tidskriftsartikel (refereegranskat)abstract
- Although a few immunocompetent animal models to study the immune response against human adenoviruses (HAdV) are available, such as Syrian hamsters and cotton rats, HAdV replication is several logs lower compared to human control cells. We have identified a non-transformed mouse epithelial cell line (NMuMG) where HAdV-2 gene expression and progeny formation was as efficient as in the highly permissive human A549 cells. HAdV from species, D and E (HAdV-37 and HAdV-4, respectively) also caused a rapid cytopathic effect in NMuMG cells, while HAdV from species A, B1, B2 and F (HAdV-12, HAdV-3, HAdV-11 and HAdV-41, respectively) failed to do so. NMuMG cells might therefore be useful in virotherapy research and the analysis of antiviral defense mechanisms and the determination of toxicity, biodistribution and specific antitumour activity of oncolytic HAdV vectors.
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| 9. |
- Grundberg, Ida, et al.
(författare)
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In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics
- 2013
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Ingår i: Oncotarget. - : Impact Journals, LLC. - 1949-2553. ; 4:12, s. 2407-2418
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Tidskriftsartikel (refereegranskat)abstract
- Current assays for somatic mutation analysis are based on extracts from tissue sections that often contain morphologically heterogeneous neoplastic regions with variable contents of genetically normal stromal and inflammatory cells, obscuring the results of the assays. We have developed an RNA-based in situ mutation assay that targets oncogenic mutations in a multiplex fashion that resolves the heterogeneity of the tissue sample. Activating oncogenic mutations are targets for a new generation of cancer drugs. For anti-EGFR therapy prediction, we demonstrate reliable in situ detection of KRAS mutations in codon 12 and 13 in colon and lung cancers in three different types of routinely processed tissue materials. High-throughput screening of KRAS mutation status was successfully performed on a tissue microarray. Moreover, we show how the patterns of expressed mutated and wild-type alleles can be studied in situ in tumors with complex combinations of mutated EGFR, KRAS and TP53. This in situ method holds great promise as a tool to investigate the role of somatic mutations during tumor progression and for prediction of response to targeted therapy.
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| 10. |
- 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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