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

Supplemental Table 2. Primer sequences used for quantitative PCR.

Primer name	Sequence (5' to 3')
At1g17210-qF2	CTGCTTCATATGAATCACGAG
At1g17210-qR2	TCAACACTATCTGCACGTTGT
At2g04660-qF1	TTCTGGAAGCAGTGGGTGAA
At2g04660-qR1	CTCCACTTCCATCTGTAAGC
At2g19980-F1	AATATTCCACCGACGGAACG
At2g19980-R1	TAAGGCTTCTCCGTAAACCAA
At2g20000-qF1	GTATAGCTCCACCACCACTT
At2g20000-qR1	TCTTCTAGGTGCTTGAAGAGT
AT4g04320-qF1	ACTCGGTATGTGGCTTAGTC
AT4g04320-qR1	TTCTCAAGCAATGAAGCAGGA
Lesa17210-FP2	TCCGCCCTTGTATGGACGAGAAG
Lesa17210-RP2	CTCTGGCACCTGGGAAAGCCA
Lesa04660-FP1	AGCTGGGTCTATTGCACGAAGGG
Lesa04660-RP1	TCGTTTGCTCACTGCTGGTGCTT
Lesa19980-FP1	TGTAGCACAACCAAGCCTAGTCGAT
Lesa19980-RP1	GGGTCCAACCTCAGGACACAATGA
Lesa20000-FP1	TCTGGTCCACGACGGAGCTTG
Lesa20000-RP1	GCGTTGCTCACATTTCCGCTTACT
Lesa04320-FP2	AAGCAATGAAGCAGGATCATCCCAA
Lesa04320-RP2	AGGAGAAACTCGGTATGTGGCTTAGT
Ubi11-qPCR-F2	GATGCAGATCTTCGTTAAGACT
Ubi11-qPCR-R2	CCTTCCTTATCCTGGATCTTG
Ef1a-qPCR-F	TGAGCACGCTCTTCTTGCT
Ef1a-qPCR-R	GTGGCATCCATCTTGTTACA
Cyp1-qPCR-F2	GGATCCTGTCGATGGCGAAC
Cyp1-qPCR-R2	TCCACGACCTGCCCAAACAC
Cyp5-qPCR-F2	CTTCAGAGCTTTGTGCACAGG
Cyp5-qPCR-R2	AAGCTGGGAATGATTCGATG
Act7/8-qPCR-F	GGTCGTACAACCGGTATTGT
Act7-qPCR-R	GATAGCATGTGGAAGTGAGAA
Act8-qPCR-R	GAAGAGCATACCCCTCGTA
5.8S rRNA-For	CTTTGAAGCCAAGTTGCGC
5.8S rRNA-Rev	CGTCCCACACTCGTGAAAAT

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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Fig. S1

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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]