A Guideline to Family-wide Comparative State-of-the-art qRT-PCR Analysis Exemplified with a Brassicaceae Cross-species Seed Germination Case Study [W][OA]
|Kai Graeber*, Ada Linkies*, Andrew T.A. Wood, Gerhard Leubner-Metzger
* Both authors contributed equally to this work
|University of Freiburg, Faculty of Biology, Institute for Biology II, Botany / Plant Physiology, D-79104 Freiburg, Germany, Web: 'The Seed Biology Place' http://www.seedbiology.de (K.G., A.L., G.L.-M.)
The University of Nottingham, Division of Statistics, School of Mathematical Sciences, University Park, Nottingham NG7 2RD, United Kingdom (A.T.A.W.)
Received February 8, 2011; revised May 6, 2011; accepted May 27, 2011; published June 10, 2011.
|Supplemental Data Online
The following materials are available in the online version of this article.
Supplemental Figures, Tables and Methods (165 KB PDF).
Supplemental Figure 1. Determination of RNA integrity via gel electrophoresis.
Supplemental Table 1. Sequence similarities to Arabidopsis and accession numbers of Lepidium sativum reference gene cDNA sequences.
Supplemental Table 2. Primer sequences used for qRT-PCR.
Supplemental Methods. Detailed selection procedure for identification of putative Brassica napus orthologs of Arabidopsis reference genes
on a Brassica microarray for comparative gene expression stability analysis as shown in Figure 5.
Supplemental Data Set 1 (74 KB Excel file). Transcript expression values of the 15 seed germination reference gene candidates mined from the Lepidium sativum microarrays.
Supplemental Data Set 2 (139 KB Excel file). Arabidopsis seed-related microarray experiments used for expression analysis and GeNORM expression stability rankings of reference genes.
Supplemental Data Set 3 (49 KB Excel file). Expression stability ranking comparisons and ranking distance measures of Lepidium sativum, Arabidopsis and Brassica napus genes.
Hyperlink to Lepidium sativum microarray data sets of Linkies et al. (2009).
Synopsis: Developmental processes like seed germination are characterised by massive transcriptome changes. This study compares seed transcriptome datasets of different Brassicaceae to identify stable expressed reference genes for cross-species qRT-PCR normalisation. A workflow is presented for improving RNA quality, qRT-PCR performance, and normalisation when analysing expression changes across species.