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Profiling and genotyping individual mRNA molecules through in situ sequencing of super rolling circle amplification products
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. (molecular tools)ORCID iD: 0000-0002-5226-1427
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

We have recently developed a technology for localized sequence library preparation with rolling-circle amplification (RCA) as an approach for in situ sequencing. This method involves generation of clonally amplified and specially confined substrates for next-generation sequencing within the preserved context of cells and tissues. Our approach combines padlock probing, RCA, and sequencing-by-ligation chemistry that can resolve expression profiles of sets of genes and mutations in tissues without loss of histological context. Like other fluorescence-based assays, it can be hindered by high level of background fluorescence. To achieve high signal-to-noise ratios, we now describe a method to boost the amplification generated by RCA of padlock probes in situ by super RCA (sRCA). In this technique, a second padlock probe is hybridized, ligated and amplified on the first RCA product for enhanced, localized amplification. We describe and compare different sRCA strategies where gap-fill ligation was showed to be most efficient. The sRCA products co-localize and have comparable sizes as RCA products but they display at least two fold higher signal intensity. This increase in signal to noise also proved to result in two folds increase in the number of sRCA products detected. By combining sRCA with in situ sequencing for highly multiplex detection in tissue a four-time increase was seen. In summary, we demonstrate that sRCA can significantly increase the performance of padlock-based in situ sequencing for gene expression profiling of tissue sections, enabling detection of low abundant transcripts and the analysis of also highly auto-fluorescent samples. 

Keyword [en]
in situ sequencing; super RCA (sRCA); tissue gene expression profiling, gap-fill padlock probe
National Category
Genetics
Identifiers
URN: urn:nbn:se:uu:diva-331741OAI: oai:DiVA.org:uu-331741DiVA: diva2:1150005
Available from: 2017-10-17 Created: 2017-10-17 Last updated: 2017-10-17
In thesis
1. Molecular Tools for Biomarker Detection
Open this publication in new window or tab >>Molecular Tools for Biomarker Detection
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The advance of biological research promotes the emerging of new methods and solutions to answer the biological questions. This thesis describes several new molecular tools and their applications for the detection of genomic and proteomic information with extremely high sensitivity and specificity or simplify such detection procedures without compromising the performance.

In paper I, we described a general method namely super RCA, for highly specific counting of single DNA molecules. Individual products of a range of molecular detection reactions are magnified to Giga-Dalton levels that are easily detected for counting one by one, using methods such as low-magnification microscopy, flow cytometry, or using a mobile phone camera. The sRCA-flow cytometry readout presents extremely high counting precision and the assay’s coefficient of variation can be as low as 0.5%. sRCA-flow cytometry readout can be applied to detect the tumor mutations down to 1/100,000 in the circulating tumor cell-free DNA.

In paper II, we applied the super RCA method into the in situ sequencing protocol to enhance the amplified mRNA detection tags for better signal-to-noise ratios. The sRCA products co-localize with primary RCA products generated from the gene specific padlock probes and remain as a single individual object in during the sequencing step. The enhanced sRCA products is 100% brighter than regular RCA products and the detection efficiency at least doubled with preserved specificity using sRCA compared to standard RCA.

In paper III, we described a highly specific and efficient molecular switch mechanism namely RCA reporter. The switch will initiate the rolling circle amplification only in the presence of correct target sequences. The RCA reporter mechanism can be applied to recognize single stranded DNA sequences, mRNA sequences and sequences embedded in the RCA products.

In paper IV, we established the solid phase Proximity Ligation Assay against the SOX10 protein using poly clonal antibodies. Using this assay, we found elevated SOX10 in serum at high frequency among vitiligo and melanoma patients. While the healthy donors below the threshold.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1387
Keyword
Rolling circle amplification, padlock probe
National Category
Genetics
Identifiers
urn:nbn:se:uu:diva-331745 (URN)978-91-513-0114-3 (ISBN)
Public defence
2017-12-08, BMC/A1:111a, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2017-11-14 Created: 2017-10-17 Last updated: 2017-11-14

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