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Automation of cDNA Synthesis and Labelling Improves Reproducibility
KTH, School of Biotechnology (BIO), Gene Technology. (Division of Gene Technology)
KTH, School of Biotechnology (BIO), Gene Technology.
KTH, School of Biotechnology (BIO), Gene Technology.
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2009 (English)In: Journal of Biomedicine and Biotechnology, ISSN 1110-7243, E-ISSN 1110-7251, Vol. 2009, 396808- p.Article in journal (Refereed) Published
Abstract [en]

Background. Several technologies, such as in-depth sequencing and microarrays, enable large-scale interrogation of genomes and transcriptomes. In this study, we asses reproducibility and throughput by moving all laboratory procedures to a robotic workstation, capable of handling superparamagnetic beads. Here, we describe a fully automated procedure for cDNA synthesis and labelling for microarrays, where the purification steps prior to and after labelling are based on precipitation of DNA on carboxylic acid-coated paramagnetic beads. Results. The fully automated procedure allows for samples arrayed on a microtiter plate to be processed in parallel without manual intervention and ensuring high reproducibility. We compare our results to a manual sample preparation procedure and, in addition, use a comprehensive reference dataset to show that the protocol described performs better than similar manual procedures. Conclusions. We demonstrate, in an automated gene expression microarray experiment, a reduced variance between replicates, resulting in an increase in the statistical power to detect differentially expressed genes, thus allowing smaller differences between samples to be identified. This protocol can with minor modifications be used to create cDNA libraries for other applications such as in-depth analysis using next-generation sequencing technologies.

Place, publisher, year, edition, pages
2009. Vol. 2009, 396808- p.
Keyword [en]
control maqc project; microarray; gene; platforms; database; ncbi
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:kth:diva-11442DOI: 10.1155/2009/396808ISI: 000271697700001OAI: oai:DiVA.org:kth-11442DiVA: diva2:276123
Note
QC 20100723Available from: 2009-11-10 Created: 2009-11-10 Last updated: 2017-12-12Bibliographically approved
In thesis
1. On Transcriptome Sequencing
Open this publication in new window or tab >>On Transcriptome Sequencing
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

This thesis is about the use of massive DNA sequencing to investigate the transcriptome. During recent decades, several studies have made it clear that the transcriptome comprises a more complex set of biochemical machinery than was previously believed. The majority of the genome can be expressed as transcripts; and overlapping and antisense transcription is widespread. New technologies for the interroga- tion of nucleic acids have made it possible to investigate such cellular phenomena in much greater detail than ever before. For each application, special requirements need to be met. The work presented in this thesis focuses on the transcrip- tome and the development of technology for its analysis. In paper I, we report our development of an automated approach for sample preparation. The procedure was benchmarked against a publicly available reference data set, and we note that our approach outperformed similar manual procedures in terms of reproducibility. In the work reported in papers II-IV, we used different massive sequencing technologies to investigate the transcriptome. In paper II we describe a concatemerization approach that increased throughput by 65% using 454 sequencing,and we identify classes of transcripts not previously described in Populus. Papers III and IV both report studies based on SOLiD sequencing. In the former, we investigated transcripts and proteins for 13% of the human gene and detected a massive overlap for the upper 50% transcriptional levels. In the work described in paper IV, we investigated transcription in non-genic regions of the genome and detected expression from a high number of previ- ously unknown loci.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. 52 p.
Series
Trita-BIO-Report, ISSN 1654-2312 ; 2009:26
Keyword
Transcriptome, RNA-seq, DNA sequencing, gene expression profiling, non-coding RNA, small RNA
National Category
Other Biological Topics Bioinformatics and Systems Biology Genetics Biochemistry and Molecular Biology Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-11446 (URN)978-91-7415-490-0 (ISBN)
Public defence
2009-12-18, Oscar Klein (FR4), Roslagstullsbacken 21, Albanova University Center, Stockholm, 09:00 (Swedish)
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Note
QC 20100723Available from: 2009-12-03 Created: 2009-11-10 Last updated: 2010-07-23Bibliographically approved

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