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Factors affecting padlock probe efficiency
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0002-4827-0208
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Stockholm University, Science for Life Laboratory (SciLifeLab).ORCID iD: 0000-0001-9985-0387
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Padlock probes have proved to be extremely versatile and useful molecular tools. They have unique properties that allow them to be used in various applications, ranging from diagnostic assays to spatially resolved transcriptomics. Padlock probes are used for detection of specific DNA or RNA sequences in enzymatic multistep assays. As the assays involve circularization and rolling circle amplification of the padlock probe, different factors play a role in the efficiency of the separate steps. Guidelines for how to design padlock probes have been lacking. We investigated how the length and the secondary structure of the different parts of the padlock probe affected its efficacy in the different steps of the assay as well as the impact on the total assay. The optimal length of the padlock probe is a compromise between a shorter total probe length, which leads to more efficient amplification and longer target specific sequence, which confers more efficient circularization. Complex secondary structure interfering with the detection motif or involving both the target-specific parts of the padlock probe seriously impair the assay efficiency. However, less complex secondary structures can be tolerated without significant efficiency loss. Taken together, the results present important considerations for the design of padlock probes and guidelines for how to improve the general detection efficiency.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:su:diva-177100OAI: oai:DiVA.org:su-177100DiVA, id: diva2:1379511
Available from: 2019-12-17 Created: 2019-12-17 Last updated: 2019-12-17Bibliographically approved
In thesis
1. RNA-based spatial characterization of cell and tissue heterogeneity
Open this publication in new window or tab >>RNA-based spatial characterization of cell and tissue heterogeneity
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Technical advances in cell biology have revolutionized the field of cell biology. With new technology it is now possible to address scientific questions in cell biology at the molecular level. Single-cell RNA-sequencing can reveal transcriptomic information for single cells and spatially resolved transcriptomic technology can visualize thousands or millions of cells and transcripts for spatial molecular profiling. The work in this thesis describes the technological development from traditional in situ hybridization to the current state-of-the-art technology for spatial multiplexed gene expression analysis. This development has enabled RNA-based molecular characterization of cells and tissues with the spatial dimension maintained. The work included in the thesis highlights the potential and the advantages of padlock-probe-based technology for spatial RNA-based profiling of cells and tissues. Furthermore, it demonstrates the possibilities arising from the inherent ability of padlock probes to distinguish between transcripts based on differences in single nucleotides.

The study in paper I investigates the prevalence of Enterovirus species B in patients with Crohn’s disease by a chromogenic in situ hybridization assay combined with immunohistochemistry to detect viral RNA and proteins directly in tissue samples.

In paper II, padlock probes were used to study the spatial gene expression of gene homologs from the X and Y chromosome in human embryonic nervous tissue. Furthermore, a strategy was devised to visualize and evaluate spatial expression patterns.

The padlock probe-based approach for multiplexed spatial transcriptional profiling, in situ sequencing, was applied in paper III to study the regional and cell-type-specific dynamics of A-to-I RNA editing in the developing mouse brain.

In paper IV, a technical characterization of padlock probes was performed with the aim of determining how to design a padlock probe to obtain optimal detection efficiency.

The work in this thesis demonstrates the dramatic shift in how biological questions in cell and tissue biology can be addressed, enabled by the technological evolution of traditional in situ hybridization assays into high-throughput, multiplexed spatial transcription profiling.

Place, publisher, year, edition, pages
Stockholm: Department of Biochemistry and Biophysics, Stockholm University, 2020. p. 65
Keywords
Padlock probes, in situ sequencing, single cell resolution, single nucleotide variant resolution, spatial transcription profiling
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:su:diva-177114 (URN)978-91-7797-958-6 (ISBN)978-91-7797-959-3 (ISBN)
Public defence
2020-02-14, Air & Fire, Science for Life Laboratory, Tomtebodavägen 23 A, Solna, 10:00 (English)
Opponent
Supervisors
Note

At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.

Available from: 2020-01-22 Created: 2019-12-17 Last updated: 2020-01-15Bibliographically approved

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