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]
* Both authors contributed equally to this work
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 1. Impact of Different Reverse Transcription (RT) Priming Methods on the RT Efficiencies of EF1-α and ACT7 Measured as Output Apparent Transcript Abundance of the qRT-PCR Reactions. | |||
| Gene | RT priming methoda | Apparent transcript abundanceb | Fold increase compared to R6 |
| EF1-α | R6 | 4.3 x 10-6 ± 0.7 x 10-6 | 1.0 |
| R15 | 4.1 x 10-5 ± 1.2 x 10-5 | 9.4 | |
| R6+dT | 8.7 x 10-6 ± 2.4 x 10-6 | 2.0 | |
| R15+dT | 3.9 x 10-5 ± 1.3 x 10-5 | 8.9 | |
| R15d | 7.7 x 10-5 ± 1.9 x 10-5 | 17.5 | |
| ACT7 | R6 | 2.6 x 10-7 ± 0.8 x 10-7 | 1.0 |
| R15 | 2.8 x 10-6 ± 1.1 x 10-6 | 10.9 | |
| R6+dT | 8.9 x 10-7 ± 3.5 x 10-7 | 3.4 | |
| R15+dT | 2.7 x 10-6 ± 1.0 x 10-6 | 10.4 | |
| R15d | 9.1 x 10-6 ± 2.7 x 10-6 | 34.9 | |
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| (a) Different
primer combinations and concentrations were used in RT reactions with 5 µg
total RNA from dry Lepidium sativum seeds: R6 = 0.14 nmol random hexamers, R15 = 0.14 nmol random pentadecamers,
R6+dT = 0.14 nmol random hexamers + 0.05 nmol oligo(dT), R15+dT = 0.14 nmol
random pentadecamers + 0.05 nmol oligo(dT), R15d = 0.3 nmol pentadecamers
(amount per reaction). (b) The output apparent transcript abundances were determined as (1+EAverageofReplicates) (-CT) (see methods) and are presented as mean values ± SD from 4 biological replicates. Equal RNA input amounts were used for the RT reactions, but different RT priming efficiencies resulted in different cDNA amounts. Of these, equal input volumes were used in the actual qRT-PCR reactions and generated based on differences in cDNA amounts different output apparent transcript abundances. |
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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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