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.

PCR efficiency

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)
Fig. 1     
Fig. 6
Fig. 2     
Tab. 1
Fig. S1
Fig. 3     
Tab. 2
Tab. S1
Fig. 4     
Tab. 3
Tab. S2
Fig. 5     
Tab. 4

Hyperlink to
Datasets 1 to 3
          © American Society of Plant Biologists

The Seed Biology Place
Copyright © Gerhard Leubner 2000

Webdesign Gerhard Leubner 2000
Best viewed with browser version 4 and 800x600 pixel
This page was last updated on 30 July, 2011