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Efficient and scalable serial extraction of DNA and RNA from frozen tissue samples
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
2010 (English)In: Chemical Communications, ISSN 1359-7345, E-ISSN 1364-548X, Vol. 47, no 1, 547-549 p.Article in journal (Refereed) Published
Abstract [en]

Advances in cancer genomics have created a demand for scalable sample processing. We here present a process for serial extraction of nucleic acids from the same frozen tissue sample based on magnetic silica particles. The process is automation friendly with high recoveries of pure DNA and RNA suitable for analysis.

Place, publisher, year, edition, pages
2010. Vol. 47, no 1, 547-549 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-140159DOI: 10.1039/c0cc02248aISI: 000285068300136PubMedID: 21103565OAI: oai:DiVA.org:uu-140159DiVA: diva2:383098
Available from: 2011-01-04 Created: 2011-01-04 Last updated: 2017-12-11Bibliographically approved
In thesis
1. From Tissue to Mutations: Genetic Profiling of Colorectal Cancer
Open this publication in new window or tab >>From Tissue to Mutations: Genetic Profiling of Colorectal Cancer
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Comprehensive characterisation of the mutational landscapes of solid tumours is a multistep process involving the collection of suitable samples, the extraction of nucleic acids and the preparation of these materials for mutational analyses. In this thesis, I aimed to develop a streamlined process for the analysis of colorectal cancer (CRC) patient samples in order to identify novel mutations that hallmark the development of advanced disease.

Papers I and II outline a technique for serial extraction of nucleic acids from frozen tissue that we developed and subsequently implemented on a robotic platform to enable high-throughput processing. The extracted nucleic acids were validated in downstream processes relevant for genetic analyses, including traditional Sanger and next generation sequencing  techniques.

In Paper III, we developed a genotyping method based on multiplex ligation-dependent genome amplification. The method was designed such that InDel polymorphisms of between 30 and 70 % prevalence in a European population were selected and amplified in a multiplex PCR assay. DNA from 24 patient-matched colorectal tumour and normal tissues was genotyped and paired with a high match probability.

In Paper IV, we performed targeted resequencing of 107 primary CRCs, of which approximately half developed metastatic disease or had distant metastases at the time of diagnosis. We chose to analyse 676 genes based on their involvement in key signalling pathways in CRC. We found an enrichment of mutations in the Eph receptor tyrosine kinase gene family in metastatic patients, indicating a potential role for these genes in CRC metastasis.

This thesis outlines a series of procedures that can be employed in a high-throughput setting for the analysis of solid tumours. We applied these methods to the analysis of colorectal tumours and propose a link between novel somatic mutations and metastatic disease.

 

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2014. 47 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1028
Keyword
Nucleic acid extraction, genotyping, targeted sequencing, mutation analysis, colorectal cancer, metastatic disease
National Category
Medical Genetics Cancer and Oncology
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-231508 (URN)978-91-554-9042-3 (ISBN)
Public defence
2014-11-07, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjölds väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2014-10-16 Created: 2014-09-08 Last updated: 2015-01-23

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