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)
 
Fig. 1     
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Abstract
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Tab. 1
Fig. S1
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Tab. 2
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Hyperlink to
Supplemental
Datasets 1 to 3
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