Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
In situ Sequencing: Methods for spatially-resolved transcriptome analysis
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

It is well known that cells in tissues display a large heterogeneity in gene expression due to differences in cell lineage origin and variation in the local environment at different sites in the tissue, a heterogeneity that is difficult to study by analyzing bulk RNA extracts from tissue. Recently, genome-wide transcriptome analysis technologies have enabled the analysis of this variation with single-cell resolution. In order to link the heterogeneity observed at molecular level with the morphological context of tissues, new methods are needed which achieve an additional level of information, such as spatial resolution.

In this thesis I describe the development and application of padlock probes and rolling circle amplification (RCA) as molecular tools for spatially-resolved transcriptome analysis. Padlock probes allow in situ detection of individual mRNA molecules with single nucleotide resolution, visualizing the molecular information directly in the cell and tissue context. Detection of clinically relevant point mutations in tumor samples is achieved by using padlock probes in situ, allowing visualization of intra-tumor heterogeneity. To resolve more complex gene expression patterns, we developed in situ sequencing of RCA products combining padlock probes and next-generation sequencing methods. We demonstrated the use of this new method by, for the first time, sequencing short stretches of transcript molecules directly in cells and tissue. By using in situ sequencing as read-out for multiplexed padlock probe assays, we measured the expression of tens of genes in hundreds of thousands of cells, including point mutations, fusions transcripts and gene expression level.

These molecular tools can complement genome-wide transcriptome analyses adding spatial resolution to the molecular information. This level of resolution is important for the understanding of many biological processes and potentially relevant for the clinical management of cancer patients.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University , 2014. , 49 p.
Keyword [en]
Padlock probes, rolling circle amplification, in situ sequencing, spatially-resolved transcriptomics, molecular diagnostics
National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-110057ISBN: 978-91-7649-066-2 (print)OAI: oai:DiVA.org:su-110057DiVA: diva2:768864
Public defence
2015-01-23, Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B, Stockholm, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Manuscript.

Available from: 2014-12-29 Created: 2014-12-05 Last updated: 2014-12-15Bibliographically approved
List of papers
1. In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics
Open this publication in new window or tab >>In situ mutation detection and visualization of intratumor heterogeneity for cancer research and diagnostics
Show others...
2013 (English)In: OncoTarget, ISSN 1949-2553, E-ISSN 1949-2553, Vol. 4, no 12, 2407-2418 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
Padlock probes, RCA, in situ, KRAS, cancer diagnostics
National Category
Cell Biology
Identifiers
urn:nbn:se:su:diva-100118 (URN)000328936800022 ()
Note

AuthorCount:10;

Available from: 2014-01-27 Created: 2014-01-27 Last updated: 2017-12-06Bibliographically approved
2. In situ sequencing for RNA analysis in preserved tissue and cells
Open this publication in new window or tab >>In situ sequencing for RNA analysis in preserved tissue and cells
Show others...
2013 (English)In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 10, no 9, 857-+ p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-94029 (URN)10.1038/nmeth.2563 (DOI)000323760000020 ()
Note

AuthorCount:7;

Available from: 2013-09-27 Created: 2013-09-24 Last updated: 2017-12-06Bibliographically approved
3. In situ sequencing identifies TMPRSS2-ERG fusion transcripts, somatic point mutations and gene expression levels in prostate cancers
Open this publication in new window or tab >>In situ sequencing identifies TMPRSS2-ERG fusion transcripts, somatic point mutations and gene expression levels in prostate cancers
Show others...
2014 (English)In: Journal of Pathology, ISSN 0022-3417, E-ISSN 1096-9896, Vol. 234, no 2, 253-261 p.Article in journal (Refereed) Published
Abstract [en]

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.

Keyword
TMPRSS2-ERG, padlock probes, in situ sequencing, prostate cancer, somatic mutations
National Category
Cancer and Oncology Biological Sciences
Identifiers
urn:nbn:se:su:diva-109002 (URN)10.1002/path.4392 (DOI)000342976700013 ()
Note

AuthorCount:6;

Available from: 2014-12-05 Created: 2014-11-10 Last updated: 2017-12-05Bibliographically approved
4. Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
Open this publication in new window or tab >>Oligonucleotide gap-fill ligation for mutation detection and sequencing in situ
Show others...
(English)Manuscript (preprint) (Other academic)
National Category
Biomedical Laboratory Science/Technology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-109696 (URN)
Available from: 2014-11-27 Created: 2014-11-27 Last updated: 2016-01-29Bibliographically approved

Open Access in DiVA

fulltext(1154 kB)438 downloads
File information
File name FULLTEXT01.pdfFile size 1154 kBChecksum SHA-512
fdec79ee777e88801f5136e3281e74396d46751c965c5057f1629c227681af2788ebd708992b909936d4759a102a3e541e08b553ab4ddbbe48e8ecaa1bed2ee7
Type fulltextMimetype application/pdf

Search in DiVA

By author/editor
Mignardi, Marco
By organisation
Department of Biochemistry and Biophysics
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Search outside of DiVA

GoogleGoogle Scholar
Total: 438 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 2161 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf