The Seed Biology Place

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' 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.
www.plantcell.org/cgi/doi/10.1105/tpc.111.084103

Figure 6. Comparison of Transcript Expression Studies of UBQ11 During Arabidopsis Seed Germination.

(A) Normalised qRT-PCR results.
Efficiency-corrected transcript abundance of UBQ11 was normalised by the geometric mean of the three best performing reference genes for Arabidopsis seed germination (At1G17210, At2G04660, At2G20000; as indicated in Figure 4). Mean values ± SD, N=3.

(B) Microarray-based results.
Affymetrix GeneChip ATH1 data normalised using GCOS and the MAS5.0 algorithm with a target value (TGT) of 100 for UBQ11 during the Arabidopsis germination timecourse was obtained from the BAR server (Toufighi et al., 2005; Winter et al., 2007).
Note: Microarray expression data compiled from different experiments (see Supplemental Data Set 2) using BAR server eNorthern 'raw' expression values. Mean values ± SD, N=2-4.

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.

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

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Abstract

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Hyperlink to
Supplemental
Datasets 1 to 3

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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]

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]