The Plant Cell 23: 2045-2063 (2011)

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' (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.

LARGE-SCALE BIOLOGY ARTICLE (The Plant Cell Front Section Feature)
Abstract. Comparative biology includes the comparison of transcriptome and quantitative real-time RT-PCR (qRT-PCR) datasets in a range of species to detect evolutionarily conserved and divergent processes. Transcript abundance analysis of target genes by qRT-PCR requires a highly accurate and robust workflow. This includes reference genes with high expression stability, i.e. low inter-sample transcript abundance variation, for correct target gene normalisation. Cross-species qRT-PCR for proper comparative transcript quantification requires reference genes suitable for different species. We addressed this issue by using tissue-specific transcriptome datasets of germinating Lepidium sativum seeds to identify new candidate reference genes. We investigated their expression stability in germinating seeds of L. sativum and Arabidopsis thaliana by qRT-PCR, combined with in silico analysis of A. thaliana and Brassica napus microarray datasets. This revealed that reference gene expression stability is higher for a given developmental process between distinct species than for distinct developmental processes within a given single species. The identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of Brassicaceae seed germination. Furthermore using germinating seeds we exemplify optimisation of the qRT-PCR workflow for challenging tissues regarding RNA quality, transcript stability, and tissue abundance. Our work therefore can serve as a guideline for moving beyond A. thaliana by establishing high-quality cross-species qRT-PCR.
Key words: abscisic acid (ABA), Arabidiopsis thaliana, Brassicaceae cross-species work, Brassica napus (oilseed rape), comparative genomics, expression stability, micropylar endosperm (cap), gene expression, GeNORM analysis, Lepidium sativum (garden cress), next-generation sequencing, PCR efficiency, phylotranscriptomics, quantitative real-time RT-PCR (qRT-PCR), radicle, ranking distance analysis, Real-time PCR Miner algorithm (, reverse transcription (RT), RNA quality control, seed germination, reference gene, transcriptome analysis

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.

Financial support: Our work is funded by grants of the Deutsche Forschungsgemeinschaft (grant no. DFG LE720/6 and LE720/7) to G.L.-M., and the ERA-NET Plant Genomics vSEED project (BBG02488X1) to A.T.A.W., which are gratefully acknowledged.

Article in PDF format (1.2 MB)
Supplemental  data file (156 KB)
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