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Rumlow A, Keunen E, Klein J, et al. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting. PLoS One. 2016;11(9):e0163679doi: 10.1371/journal.pone.0163679.
Rumlow, A., Keunen, E., Klein, J., Pallmann, P., Riemenschneider, A., Cuypers, A., & Papenbrock, J. (2016). Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting. PloS one, 11(9), e0163679. https://doi.org/10.1371/journal.pone.0163679
Rumlow, Annekathrin, et al. "Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting." PloS one vol. 11,9 (2016): e0163679. doi: https://doi.org/10.1371/journal.pone.0163679
Rumlow A, Keunen E, Klein J, Pallmann P, Riemenschneider A, Cuypers A, Papenbrock J. Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting. PLoS One. 2016 Sep 29;11(9):e0163679. doi: 10.1371/journal.pone.0163679. eCollection 2016. PMID: 27685087; PMCID: PMC5042561.
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PLoS One. 2016 Sep 29;11(9):e0163679. doi: 10.1371/journal.pone.0163679. eCollection 2016.

Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting.

PloS one

Annekathrin Rumlow, Els Keunen, Jan Klein, Philip Pallmann, Anja Riemenschneider, Ann Cuypers, Jutta Papenbrock

Affiliations

  1. Institute of Botany, Leibniz University Hannover, Hannover, Germany.
  2. Centre for Environmental Sciences, Hasselt University, Diepenbeek, Belgium.
  3. Department of Mathematics and Statistics, Lancaster University, Lancaster, United Kingdom.

PMID: 27685087 PMCID: PMC5042561 DOI: 10.1371/journal.pone.0163679

Abstract

Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions. The comparison of many different factors influencing plant growth challenges the gene expression analysis for specific gene-targeted experiments, especially with regard to the choice of suitable reference genes. The aim of this study is to compare expression results obtained by Northern blot, semi-quantitative PCR and RT-qPCR, and to identify a reliable set of reference genes for oilseed rape (Brassica napus L.) suitable for comparing gene expression under complex experimental conditions. We investigated the influence of several factors such as sulfur deficiency, different time points during the day, varying light conditions, and their interaction on gene expression in oilseed rape plants. The expression of selected reference genes was indeed influenced under these conditions in different ways. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. After careful comparison of the three methods mentioned above, Northern blot analysis seems to be a reliable and cost-effective alternative for gene expression analysis under a complex growth regime. For using this method in a quantitative way a number of references was validated revealing that for our experiment a set of three references provides an appropriate normalization. Semi-quantitative PCR was prone to many handling errors and difficult to control while RT-qPCR was very sensitive to expression fluctuations of the reference genes.

Conflict of interest statement

The authors have declared that no competing interests exist.

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