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

Table 2. Impact of Gene-specific Primer Concentrations on the PCR Efficiencies of qRT-PCR Reactions for EF1-α and ACT7.

Gene	Primer Concentration [nM]^a	PCR Efficiency^b	Fold Increase compared to 35 nM

EF1-α	35	0.74 ± 0.02	1.0
	70	0.82 ± 0.04	1.1
	140	0.88 ± 0.03	1.2

ACT7	35	0.65 ± 0.02	1.0
	70	0.83 ± 0.01	1.3
	140	0.91 ± 0.03	1.4

(a) cDNA was obtained from RT reactions with 0.3 nmol pentadecamers (R15d in Table 1) and total RNA of combined CAP&RAD tissues dissected from 8h-imbibed Lepidium sativum seeds (Figure 2). QRT-PCR reactions were performed with three different concentrations of gene-specific primers using the same amounts of input cDNA. (b) PCR efficiencies were determined for each reaction using the PCR Miner algorithm (Zhao and Fernald, 2005). A value of 1.0 corresponds to 100 % PCR efficiency, which corresponds to an exact doubling of amplicon numbers in each PCR cycle. Mean PCR efficiency values ± SD are presented for 4 biological replicates.

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]