![]() This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.ĭata Availability: All relevant data are within the paper and its Supporting Information files.įunding: The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover. Received: ApAccepted: SeptemPublished: September 29, 2016Ĭopyright: © 2016 Rumlow et al. PLoS ONE 11(9):Įditor: Sara Amancio, Universidade de Lisboa Instituto Superior de Agronomia, PORTUGAL (2016) Quantitative Expression Analysis in Brassica napus by Northern Blot Analysis and Reverse Transcription-Quantitative PCR in a Complex Experimental Setting. 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.Ĭitation: Rumlow A, Keunen E, Klein J, Pallmann P, Riemenschneider A, Cuypers A, et al. 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. 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. Therefore, a recently developed algorithm, called GrayNorm, was applied to validate a set of reference genes for normalizing results obtained by Northern blot analysis. The expression of selected reference genes was indeed influenced under these conditions in different ways. 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 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. 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. Analysis of gene expression is one of the major ways to better understand plant reactions to changes in environmental conditions.
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