Details
| Originalsprache | Englisch |
|---|---|
| Aufsatznummer | e0163679 |
| Fachzeitschrift | PLOS ONE |
| Jahrgang | 11 |
| Ausgabenummer | 9 |
| Publikationsstatus | Veröffentlicht - 29 Sept. 2016 |
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.
ASJC Scopus Sachgebiete
- Biochemie, Genetik und Molekularbiologie (insg.)
- Allgemeine Biochemie, Genetik und Molekularbiologie
- Agrar- und Biowissenschaften (insg.)
- Allgemeine Agrar- und Biowissenschaften
- Allgemein
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in: PLOS ONE, Jahrgang 11, Nr. 9, e0163679, 29.09.2016.
Publikation: Beitrag in Fachzeitschrift › Artikel › Forschung › Peer-Review
}
TY - JOUR
T1 - Quantitative expression analysis in Brassica napus by Northern blot analysis and reverse transcription-quantitative PCR in a complex experimental setting
AU - Rumlow, Annekathrin
AU - Keunen, Els
AU - Klein, Jan
AU - Pallmann, Philip
AU - Riemenschneider, Anja
AU - Cuypers, Ann
AU - Papenbrock, Jutta
N1 - Funding Information: We acknowledge the Deutsche Saatveredelung AG, Lippstadt, Germany, for providing us with seeds of the Brassica napus cultivar. We would like to thank Pamela von Trzebiatowski and Julia Volker for technical assistance and the gardeners Yvonne Leye and Lutz Krüger for taking care of the plants. We are grateful to Miriam Laxa, Ina Horst and Christoph Peterhänsel, Leibniz University Hannover, for valuable suggestions concerning RT-qPCR. The publication of this article was funded by the Open Access Fund of the Leibniz Universität Hannover.
PY - 2016/9/29
Y1 - 2016/9/29
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84991716583&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0163679
DO - 10.1371/journal.pone.0163679
M3 - Article
AN - SCOPUS:84991716583
VL - 11
JO - PLOS ONE
JF - PLOS ONE
SN - 1932-6203
IS - 9
M1 - e0163679
ER -