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Please use this identifier to cite or link to this item: http://hdl.handle.net/1942/22727

Title: Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting
Authors: Rumlow, Annekathrin
Keunen, Els
Klein, Jan
Pallmann, Philip
Riemenschneider, Anja
Cuypers, Ann
Papenbrock, Jutta
Issue Date: 2016
Citation: PLOS ONE, 11(9)
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.
Notes: [Rumlow, Annekathrin; Klein, Jan; Riemenschneider, Anja; Papenbrock, Jutta] Leibniz Univ Hannover, Inst Bot, Hannover, Germany. [Keunen, Els; Cuypers, Ann] Hasselt Univ, Ctr Environm Sci, Diepenbeek, Belgium. [Pallmann, Philip] Univ Lancaster, Dept Math & Stat, Lancaster, England. [Klein, Jan] Univ Erlangen Nurnberg, Dept Biol, Erlangen, Germany.
URI: http://hdl.handle.net/1942/22727
DOI: 10.1371/journal.pone.0163679
ISI #: 000384328500095
ISSN: 1932-6203
Category: A1
Type: Journal Contribution
Validation: ecoom, 2017
Appears in Collections: Research publications

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