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Automation of cDNA Synthesis and Labelling Improves Reproducibility

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 be...

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Autores principales: Klevebring, Daniel, Gry, Marcus, Lindberg, Johan, Eidefors, Anna, Lundeberg, Joakim
Formato: Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2009
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763127/
https://www.ncbi.nlm.nih.gov/pubmed/19841682
http://dx.doi.org/10.1155/2009/396808
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author Klevebring, Daniel
Gry, Marcus
Lindberg, Johan
Eidefors, Anna
Lundeberg, Joakim
author_facet Klevebring, Daniel
Gry, Marcus
Lindberg, Johan
Eidefors, Anna
Lundeberg, Joakim
author_sort Klevebring, Daniel
collection PubMed
description 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.
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spelling pubmed-27631272009-10-19 Automation of cDNA Synthesis and Labelling Improves Reproducibility Klevebring, Daniel Gry, Marcus Lindberg, Johan Eidefors, Anna Lundeberg, Joakim J Biomed Biotechnol Methodology Report 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. Hindawi Publishing Corporation 2009 2009-10-15 /pmc/articles/PMC2763127/ /pubmed/19841682 http://dx.doi.org/10.1155/2009/396808 Text en Copyright © 2009 Daniel Klevebring et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Report
Klevebring, Daniel
Gry, Marcus
Lindberg, Johan
Eidefors, Anna
Lundeberg, Joakim
Automation of cDNA Synthesis and Labelling Improves Reproducibility
title Automation of cDNA Synthesis and Labelling Improves Reproducibility
title_full Automation of cDNA Synthesis and Labelling Improves Reproducibility
title_fullStr Automation of cDNA Synthesis and Labelling Improves Reproducibility
title_full_unstemmed Automation of cDNA Synthesis and Labelling Improves Reproducibility
title_short Automation of cDNA Synthesis and Labelling Improves Reproducibility
title_sort automation of cdna synthesis and labelling improves reproducibility
topic Methodology Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763127/
https://www.ncbi.nlm.nih.gov/pubmed/19841682
http://dx.doi.org/10.1155/2009/396808
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