Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis.
To access publisher's full text version of this article click on the hyperlink at the bottom of the page The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrop...
Published in: | Clinical Chemistry |
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Amer Assoc Clinical Chemistry
2013
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Online Access: | http://hdl.handle.net/2336/324522 https://doi.org/10.1373/clinchem.2012.193839 |
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ftlandspitaliuni:oai:www.hirsla.lsh.is:2336/324522 2023-05-15T16:51:49+02:00 Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. Thormar, Hans G Gudmundsson, Bjarki Eiriksdottir, Freyja Kil, Siyoen Gunnarsson, Gudmundur H Magnusson, Magnus Karl Hsu, Jason C Jonsson, Jon J Univ Iceland, Dept Biochem & Mol Biol, IS-101 Reykjavik, Iceland, Lifeind Ehf, Reykjavik, Iceland, Ohio State Univ, Dept Stat, Columbus, OH 43210 USA, Univ Iceland, Dept Pharmacol & Toxicol, IS-101 Reykjavik, Iceland, Landspitali Natl Univ Hosp, Dept Genet & Mol Med, Reykjavik, Iceland 2013 http://hdl.handle.net/2336/324522 https://doi.org/10.1373/clinchem.2012.193839 en eng Amer Assoc Clinical Chemistry http://dx.doi.org/10.1373/clinchem.2012.193839 http://www.clinchem.org/content/59/4/667.full.pdf Clin. Chem. 2013, 59 (4):667-74 1530-8561 23378568 doi:10.1373/clinchem.2012.193839 http://hdl.handle.net/2336/324522 Clinical chemistry Archived with thanks to Clinical chemistry Landspitali Access - LSH-aðgangur DNA kjarnsýra DNA Complementary Electrophoresis Gel Two-Dimensional Oligonucleotide Array Sequence Analysis Reproducibility of Results Article 2013 ftlandspitaliuni https://doi.org/10.1373/clinchem.2012.193839 2022-05-29T08:21:58Z To access publisher's full text version of this article click on the hyperlink at the bottom of the page The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. Technical Development Fund at Rannis/110473-0612 Icelandic Center for Research Funds University of Iceland Research Fund Landspitali Research Fund Article in Journal/Newspaper Iceland Hirsla - Landspítali University Hospital research archive Clinical Chemistry 59 4 667 674 |
institution |
Open Polar |
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Hirsla - Landspítali University Hospital research archive |
op_collection_id |
ftlandspitaliuni |
language |
English |
topic |
DNA kjarnsýra DNA Complementary Electrophoresis Gel Two-Dimensional Oligonucleotide Array Sequence Analysis Reproducibility of Results |
spellingShingle |
DNA kjarnsýra DNA Complementary Electrophoresis Gel Two-Dimensional Oligonucleotide Array Sequence Analysis Reproducibility of Results Thormar, Hans G Gudmundsson, Bjarki Eiriksdottir, Freyja Kil, Siyoen Gunnarsson, Gudmundur H Magnusson, Magnus Karl Hsu, Jason C Jonsson, Jon J Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. |
topic_facet |
DNA kjarnsýra DNA Complementary Electrophoresis Gel Two-Dimensional Oligonucleotide Array Sequence Analysis Reproducibility of Results |
description |
To access publisher's full text version of this article click on the hyperlink at the bottom of the page The causes of imprecision in microarray expression analysis are poorly understood, limiting the use of this technology in molecular diagnostics. Two-dimensional strandness-dependent electrophoresis (2D-SDE) separates nucleic acid molecules on the basis of length and strandness, i.e., double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and RNA·DNA hybrids. We used 2D-SDE to measure the efficiency of cDNA synthesis and its importance for the imprecision of an in vitro transcription-based microarray expression analysis. The relative amount of double-stranded cDNA formed in replicate experiments that used the same RNA sample template was highly variable, ranging between 0% and 72% of the total DNA. Microarray experiments showed an inverse relationship between the difference between sample pairs in probe variance and the relative amount of dsDNA. Approximately 15% of probes showed between-sample variation (P < 0.05) when the dsDNA percentage was between 12% and 35%. In contrast, only 3% of probes showed between-sample variation when the dsDNA percentage was 69% and 72%. Replication experiments of the 35% dsDNA and 72% dsDNA samples were used to separate sample variation from probe replication variation. The estimated SD of the sample-to-sample variation and of the probe replicates was lower in 72% dsDNA samples than in 35% dsDNA samples. Variation in the relative amount of double-stranded cDNA synthesized can be an important component of the imprecision in T7 RNA polymerase-based microarray expression analysis. Technical Development Fund at Rannis/110473-0612 Icelandic Center for Research Funds University of Iceland Research Fund Landspitali Research Fund |
author2 |
Univ Iceland, Dept Biochem & Mol Biol, IS-101 Reykjavik, Iceland, Lifeind Ehf, Reykjavik, Iceland, Ohio State Univ, Dept Stat, Columbus, OH 43210 USA, Univ Iceland, Dept Pharmacol & Toxicol, IS-101 Reykjavik, Iceland, Landspitali Natl Univ Hosp, Dept Genet & Mol Med, Reykjavik, Iceland |
format |
Article in Journal/Newspaper |
author |
Thormar, Hans G Gudmundsson, Bjarki Eiriksdottir, Freyja Kil, Siyoen Gunnarsson, Gudmundur H Magnusson, Magnus Karl Hsu, Jason C Jonsson, Jon J |
author_facet |
Thormar, Hans G Gudmundsson, Bjarki Eiriksdottir, Freyja Kil, Siyoen Gunnarsson, Gudmundur H Magnusson, Magnus Karl Hsu, Jason C Jonsson, Jon J |
author_sort |
Thormar, Hans G |
title |
Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. |
title_short |
Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. |
title_full |
Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. |
title_fullStr |
Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. |
title_full_unstemmed |
Importance of the efficiency of double-stranded DNA formation in cDNA synthesis for the imprecision of microarray expression analysis. |
title_sort |
importance of the efficiency of double-stranded dna formation in cdna synthesis for the imprecision of microarray expression analysis. |
publisher |
Amer Assoc Clinical Chemistry |
publishDate |
2013 |
url |
http://hdl.handle.net/2336/324522 https://doi.org/10.1373/clinchem.2012.193839 |
genre |
Iceland |
genre_facet |
Iceland |
op_relation |
http://dx.doi.org/10.1373/clinchem.2012.193839 http://www.clinchem.org/content/59/4/667.full.pdf Clin. Chem. 2013, 59 (4):667-74 1530-8561 23378568 doi:10.1373/clinchem.2012.193839 http://hdl.handle.net/2336/324522 Clinical chemistry |
op_rights |
Archived with thanks to Clinical chemistry Landspitali Access - LSH-aðgangur |
op_doi |
https://doi.org/10.1373/clinchem.2012.193839 |
container_title |
Clinical Chemistry |
container_volume |
59 |
container_issue |
4 |
container_start_page |
667 |
op_container_end_page |
674 |
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1766041925875400704 |