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.

Table 4. The Effect of Treatments and Tissues on the Average Expression Stability (M) Ranking of Traditional and New Reference Genes for Lepidium sativum Seed Germination.


Subseries 'Treatments',
Combined Tissue (CAP, RAD) Results


Subseries 'Tissues',
Combined Treatment (CON, ABA) Results








LesaG04320 (0.23)
LesaG17210 (0.23)

LesaG04660 (0.46)
LesaG20000 (0.46)


LesaG17210 (0.26)
LesaG20000 (0.26)

LesaG17210 (0.34)
LesaG20000 (0.34)

LesaG20000 (0.39)

LesaG04320 (0.50)


LesaG04320 (0.31)

LesaG04320 (0.39)

CYP5 (0.54)

ACT7 (0.57)


LesaG04660 (0.41)

LesaG04660 (0.42)

EF-1α (0.64)

CYP5 (0.64)


UBQ11 (0.48)

CYP5 (0.46)

ACT7 (0.67)

ACT8 (0.73)


EF-1α (0.53)

ACT7 (0.49)

LesaG04660 (0.70)

CYP1 (0.80)


ACT7 (0.57)

EF-1α (0.50)

CYP1 (0.91)

5.8s rRNA (0.91)


CYP5 (0.60)

CYP1 (0.57)

5.8s rRNA (1.07)

UBQ11 (1.05)


5.8s rRNA (0.71)

ACT8 (0.63)

ACT8 (1.19)

LesaG19980 (1.38)


ACT8 (0.82)

5.8s rRNA (0.70)

UBQ11 (1.35)

LesaG17210 (2.07)


CYP1 (0.91)

LesaG19980 (0.78)

LesaG19980 (1.49)

EF-1α (2.65)


LesaG19980 (1.21)

UBQ11 (0.86)

Distinct subseries of efficiency-corrected qRT-PCR results (Figure 3A) were analysed with GeNORM to obtain average reference genes stability measures M (numbers in brackets). In the subseries 'treatments' the samples from different tissues (CAP and RAD) and times of the series without (CON) and with ABA addition are compared. In the subseries 'tissues' the samples from different treatments (CON and ABA) and times of the CAP and the RAD series are compared. For each treatment or tissue the reference genes are ranked from most stable (top, low M values) to least stable (bottom, high M values). New reference genes derived from the L. sativum transcriptome analysis (Fig. 3 and Fig. 4) are in bold. Note that GeNORM does not discriminate between the two most stable genes.

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
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