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. 2007 Jun 8:3:7.
doi: 10.1186/1746-4811-3-7.

A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors

Camila Caldana  1 , Wolf-Rüdiger Scheible, Bernd Mueller-Roeber, Slobodan Ruzicic
Affiliations

A quantitative RT-PCR platform for high-throughput expression profiling of 2500 rice transcription factors

Camila Caldana et al. Plant Methods. .

Abstract

Background: Quantitative reverse transcription - polymerase chain reaction (qRT-PCR) has been demonstrated to be particularly suitable for the analysis of weakly expressed genes, such as those encoding transcription factors. Rice (Oryza sativa L.) is an important crop and the most advanced model for monocotyledonous species; its nuclear genome has been sequenced and molecular tools are being developed for functional analyses. However, high-throughput methods for rice research are still limited and a large-scale qRT-PCR platform for gene expression analyses has not been reported.

Results: We established a qRT-PCR platform enabling the multi-parallel determination of the expression levels of more than 2500 rice transcription factor genes. Additionally, using different rice cultivars, tissues and physiological conditions, we evaluated the expression stability of seven reference genes. We demonstrate this resource allows specific and reliable detection of the expression of transcription factor genes in rice.

Conclusion: Multi-parallel qRT-PCR allows the versatile and sensitive transcriptome profiling of large numbers of rice transcription factor genes. The new platform complements existing microarray-based expression profiling techniques, by allowing the analysis of lowly expressed transcription factor genes to determine their involvement in developmental or physiological processes. We expect that this resource will be of broad utility to the scientific community in the further development of rice as an important model for plant science.

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Figures

Figure 1
Figure 1
Flow-chart of an optimised protocol for qRT-PCR in large-scale expression profiling experiments. The protocol implements multiple optimised and control steps including RNA isolation, digestion of genomic DNA (gDNA), evaluation of cDNA quality, primer design and data analysis. The absence of gDNA was confirmed by quantitative RT-PCR (qRT-PCR) with primer pairs targeting various non-coding regions. The quality of the cDNA was tested using different reference genes, as outlined in the text.
Figure 2
Figure 2
Distribution of calculated PCR efficiencies as determined for two indica rice cultivars. The two distributions were compared using a Kolmogorov-Smirnov test and were found to be significantly different at p = 0.001. Non-parametric comparison of mean values (Mann-Whitney U test) confirmed the presence of statistically significant differences at p = 0.000001. Transformation to expression values revealed that the slightly different PCR efficiencies could lead to a mean difference of maximal 0.3, when the fold change was expressed as log2. Individual primer pairs can thus exhibit slight differences for their target genes in different cultivars. However, this does not significantly affect the overall applicability of the primer platform for expression profiling experiments (Caldana et al., manuscript in preparation).
Figure 3
Figure 3
Linearity and sensitivity of qRT-PCR. cDNA derived from root or shoot RNA was mixed in different ratios (as indicated; total amount of cDNA was 1 ng) and used as template to test transcript abundance of three selected genes (Os03g55610, Os08g38220, and Os12g38200) via qRT-PCR. A linear relationship between root (or shoot) cDNA and expression level of the various genes was observed. Symbols in both panels represent the mean ± SD (n = 3).
Figure 4
Figure 4
Expression stability of selected reference genes as calculated by geNORM. The data sets used for the calculation of the average expression stability (M) values are provided in Additional file 2. 'All tested conditions' includes all probes tested (different cultivars, tissues and physiological conditions); 'Root tissue (100 mM salt stress)' includes all root samples of salt stress-treated plants; 'Root tissue' and 'Shoot tissue' include samples of non-stressed tissues of different cultivars. A lower M value indicates more stable expression. According to the geNORM manual, genes with M values >1.5 are not suitable reference genes for the selected conditions. Gene codes and primers used for the qRT-PCR experiments are given in Table 1.
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