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 1. Effect of Different PCR Efficiencies on the Measured Transcript Abundance of EF1-α and ACT7 with and without Efficiency Correction.

Different PCR efficiencies were obtained by varying the concentrations of the gene-specific primers as described in Table 2. Lowered primer concentration results in reduction of PCR efficiency recognizable by decreased steepness of the exponential phases in the qRT-PCR fluorescence curves (insets in the upper right corners; shown are individual amplification plots for 3 different primer concentrations, 4 biological replicates each). PCR efficiencies were determined by PCR Miner software. Efficiency-corrected transcript abundance was calculated as describe in methods by using the average efficiency of all samples for each gene and each primer concentration. Non-efficiency corrected transcript abundance was calculated from the CT values determined by PCR Miner assuming 100 % PCR efficiency. Mean values ± SD of 4 biological replicates are presented.

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]