Analysis of the rice genome indicated that 21% of all genes give rise to alternatively spliced transcripts 13. In the case of TFs, splice variants can affect the architecture of the DNA-binding domain and often show tissue-specific expression patterns 14. To distinguish between such variants, splice variant-specific primer pairs were designed for the 5.7 % of all TF loci (131 TF loci) where this was possible. In total, primer pairs for 2508 gene models derived from 2306 loci were designed (Additional file 1). The design of primers followed a set of stringent criteria, as generally suggested in qRT-PCR protocols (e.g. Primer Express Software v2.0 Application Manual, Applied Biosystems). To minimize the risk of amplifying contaminating genomic DNA, primers spanning at least one exon-exon junction, or annealing to different exons, were designed where possible (56% of predicted gene models). However, 35% of genes contained no introns. The specificity of each primer was confirmed by comparing its sequence with all predicted rice coding sequences (CDS) using the BLASTN tool at TIGR 15 to ensure that at least one primer of each pair targets a unique site within the set of predicted rice CDS.

RNA was initially extracted using a phenol-based method from two different tissues (root and shoot) of four rice cultivars, three of which were indica cultivars (Cham, DR2 and Lua man) and the fourth was a japonica cultivar (Nipponbare). This protocol (e.g. as described by Czechowski et al. and Jain et al. 516) gave satisfactory total RNA yield, but the RNA quality was too low for the synthesis of high-quality cDNA [data not shown] 17. We therefore used the guanidinium thiocyanate-based RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) 18. Total RNA extraction was straightforward and provided RNA with high yield and quality from both root and shoot tissue (45–75 μg total RNA/100 mg fresh weight).

RNA preparations are usually contaminated with low amounts of genomic DNA, which can result in non-specific amplification 19. The manufacturer's recommended on-column DNAse treatment was not sufficient to remove interfering genomic DNA. Therefore, we performed a second DNAse incubation on the isolated RNA to eliminate detectable genomic DNA contamination. Genomic DNA was detected by qRT-PCR using 0.125 μg of isolated RNA as template, and three different primer pairs annealing to intergenic regions of chromosomes 1 (AP003727, positions 7366–7426) and 7 (AP006456, positions 27334624–27334684), and an intron of the gene Os01g01840. It is important to select more than a single genomic region to assess genomic DNA contamination because chromosomal sites can be differentially accessible to DNAse I. Omitting the second DNAse digestion always resulted in the amplification of some, but not all, of these genomic regions (data not shown). In most cases the C_T values obtained were >30. Synthesis of cDNA from the isolated RNA was only performed when all three genomic control amplifications scored negative.

We chose a two-step qRT-PCR protocol where reverse transcription and PCR-mediated cDNA amplification are carried out in subsequent steps in separate tubes. The two-step protocol is preferred when SYBR Green is used as a detection dye because it diminishes unwanted primer dimer formation 20. The reverse transcriptase reaction was primed with oligo-(dT) instead of random-sequence primers because the latter preferentially selects for more abundant mRNA species and not for transcripts of weakly expressed genes, such as many TF genes. A flowchart of the protocol is shown in Figure 1.

All 2508 primer pairs were checked by qRT-PCR using cDNAs synthesised from the roots of the rice cultivars, Cham and DR2 kept under control and salt stress (100 mM NaCl) conditions. Additionally, shoot cDNA from DR2 was used to test a smaller set of primer pairs targeting 192 TF genes. Melting curve analysis was performed for all PCR products to confirm the occurrence of specific amplification peaks and the absence of primer-dimer formation. Finally, all 2508 PCR products from the control DR2 root sample were run on 4% agarose gels and photographically documented (available upon request).

Approximately 3% of all TF genes analysed (i.e. 73 out of 2508 genes) did not yield detectable PCR amplicons, indicating no or weak expression under the employed conditions. In most cases (57 TF genes) at least one primer spanned an exon-exon junction thus precluding tests for priming efficiency on genomic DNA template. Only 2.5% of all reactions (61 TF genes) yielded unspecific PCR products as indicated by multiple or incorrectly sized amplicons.

Although the fluorescence during qRT-PCR is primarily determined by the starting abundance of a given cDNA template, its increase during the run is strongly affected by the amplification efficiency. When this efficiency is 100%, the amount of a cDNA targeted by a given primer pair is doubled in every PCR cycle of the exponential phase. However, the priming site, the sequence specificity of the primer, and the unwanted formation of primer-dimers are factors that can significantly affect the efficiency of individual PCR reactions. We used the LinRegPCR software 21 to determine the PCR efficiency of each primer pair of the TF qRT-PCR platform, taking into account all amplification profiles obtained (27588 in total). Firstly, the correlation coefficients (R) assigned to efficiency values were used to evaluate the amplification curves and all reactions with an R < 0.990, reflecting low-quality amplifications, were excluded from further analyses. Subsequently, average PCR efficiencies were computed for each individual primer pair across all analyzed samples. This showed that 8% of the 2508 primer pairs (200 TF genes) displayed PCR efficiencies greater than 1.90, and 87% of the primer pairs (2182 TF genes) had efficiencies of 1.51–1.90. Only 5% of the primer pairs (125 TF genes) had PCR efficiencies with mean values below 1.4, and these mostly represented reactions that lacked detectable fragment amplification (C_T > 40) or that generated unspecific PCR products. The efficiencies of reference genes tested in this work ranged from 1.73 to 1.96 (see below; Table 1).

The TF qRT-PCR platform was developed primarily using the nuclear genome sequence of the japonica cultivar Nipponbare (TIGR annotation) 15, which might affect its general applicability for experiments involving indica cultivars. SNP (single nucleotide polymorphism) variation among the two rice subspecies was found to be low (<0.4%) 22, and these mostly occur in intergenic regions which contain approximately four times more SNPs than occur within genes 23. SNPs may affect the comparative analysis of gene expression levels in qRT-PCR experiments. Therefore, to investigate variation of primer specificity between the indica varieties Cham and DR2, PCR efficiencies were separately computed in a cultivar-specific manner for all gene models. Although individual primer pairs of the TF primer platform can exhibit slight differences for their target genes in different varieties/cultivars (Figure 2), this did not significantly affect the overall applicability of the platform for expression profiling experiments, a finding we have also validated in independent experiments (Caldana et al., manuscript in preparation).

To determine the sensitivity and accuracy of the rice qRT-PCR platform, as reported for other platforms 5, we performed additional experiments. Firstly, we assessed the linearity, sensitivity and accuracy using three weakly expressed genes with preferential expression in either shoots (Os12g38200) or roots (Os03g55610 and Os08g38220). Shoot- and root-derived cDNAs were mixed in different ratios (see Figure 3) and transcript abundance of the three genes was determined by qRT-PCR. Linearised values were calculated as 2(40−CT) MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqaIYaGmdaahaaWcbeqaaiabcIcaOiabisda0iabicdaWiabgkHiTiabboeadnaaBaaameaacqqGubavaeqaaSGaeiykaKcaaaaa@34C2@, where C_T represents the threshold cycle and directly reflects template abundance. A linear relationship was observed in all three cases, suggesting that, as previously reported for Arabidopsis 5, qRT-PCR was sufficiently accurate in rice even for low-abundant transcripts. These relationships were highly significant with R² values (coefficient of determination) of 0.61, 0.94, and 0.90 for Os03g55610, Os08g38220, and Os12g38200, respectively (Figure 3).

We also tested the experimental reproducibility of the rice qRT-PCR platform. Czechowski et al. 5 demonstrated qRT-PCR precision by analysing intra-assay variation using the same pool of cDNAs and inter-assay variation using two different pools of cDNAs synthesised from the same batch of RNA, obtaining R² values of 0.99 and 0.95, respectively. Here, we wanted to determine the variation between different biological replicates which encompasses both technical and biological variation. Using cDNA synthesised from DR2 harvested in three independent experiments we measured the expression of 201 TF genes. The ΔC_T (C_{T_gene of interest} - C_{T_reference gene}) was calculated and the precision of the assay was assessed using the coefficient of variation (CV). Despite the fact that the majority of gene models tested (111 genes) displayed an extremely low expression level (C_T > 35), the obtained mean CV was 14%. This is in good agreement with published expression data for human keratinocyte subclones, in which a CV of 18% was found for genes with a C_T > 30 24. In contrast, CV values are generally higher in microarray-based analyses of genes with such low expression, indicating a lower reproducibility 25. These findings underscore the advantage of qRT-PCR as an alternative and often superior tool for expression profiling studies, especially for the investigation of genes with low expression level.

Generally in qRT-PCR, transcripts of stably expressed genes, also called reference genes, are employed for data normalisation. In rice, previous publications have suggested 18S-rRNA, GADPH, UBI5 and EF-1α as good reference genes 2616. To identify the most suitable reference genes in rice we initially selected nine candidates: 18S-rRNA, ubiquitin (UBQ), actin (ACT), actin1 (ACT1), β-tubulin (TUB), cyclophilin (CYC), elongation factor 1α (EF-1α), which are commonly used house-keeping genes in plants, and expressed protein (EP) and TIP41-like protein (TIP41) found to be good reference genes in Arabidopsis 27. UBQ (Os01g45420), considered as a stable house-keeping gene in various plant species 2716, had unstable expression across three different rice cultivars (data not shown), and was excluded from further studies. Although the abundance of 18S-rRNA remained constant in different rice cultivars and physiological conditions (data not shown), we did not consider it further as a suitable reference for our analyses, primarily because it requires the use of random hexamers instead of oligo(dT) as primers for the reverse transcriptase.

An overview of the remaining seven reference genes is given in Table 1. CYC, EP and TIP41 are expressed at low, TUB, ACT and ACT1 at intermediate, and EF-1α at high levels, respectively. Their expression stability was measured by qRT-PCR in a set of 11 different cDNA samples (Additional file 2), and calculated using the gene expression stability measure (M) implemented in the geNORM software 28. This determines the stability of a reference gene, taking into account the average pair-wise variation (V) of that gene in comparison to all other reference genes tested. Assuming that the difference of gene expression level of two ideal control genes is the same in all experimental conditions (tissues, treatments) compared, the lower a genes M value is, the more stably it is expressed (suggested limit of M<1.5).

To follow the relationship between expression stability and experimental condition, geNORM was run with different input data (Figure 4). The first experimental series used the 11 cDNA samples. Except for CYC, which exhibited an M value of 1.88, all genes displayed values below the threshold of 1.5, proving their stability. EF-1α and EP had the most stable expression and are consequently the best overall reference genes. We also compared expression stability using shoot or root tissue of the different rice cultivars (Figure 4). Although all genes had M values below 1.5, TIP41 was the least stably expressed gene in both tissues. This is in contrast to the stable expression of the orthologous gene from Arabidopsis (At4g34270) across many different tissues 27. Together with our data from UBQ, this highlights the need to test the suitability of a given gene even if its orthologue in another species is an adequate reference gene. We also tested the affect of salt stress on the expression stability of the selected genes. As in the tissue experiment, all genes displayed satisfactory M values and the best ranked genes were ACT1 and CYC, followed by EF-1α. However, the weakly expressed gene CYC was the least stable when more diverse conditions were used. Therefore, considering all the experimental categories, ACT1 and EF-1α were the most stable reference genes. Additionally, EP, similar to its Arabidopsis orthologue (At4g33380) 27, showed good stability in all experiments. Generally, we recommend including more than a single reference gene in each qRT-PCR experiment. Conveniently, the geNORM software also calculates the pair-wise variation (V), which indicates the optimal number of reference genes to be analysed in a given experiment.

The three rice (Oryza sativa L. ssp. indica) cultivars (Cham, DR2 and Lua man) were obtained from the Institute of Biotechnology (Hanoi, Vietnam). Additionally, a Nipponbare (Oryza sativa L. spp. japonica) cultivar provided by the International Rice Research Institute (Manila, Philippines) was used. The plants were grown in hydroponic culture 29 under a day-length of 12 h at 26/22°C (day/night), 70% humidity and 700 μmol m^-2 s^-1 light intensity. Roots and shoots were harvested three weeks after germination. Salt-stressed plants (Cham, DR2, and Lua man) were harvested 30 min or 3 h after the application of 100 mM NaCl, with control samples harvested in parallel.

The roots and shoots of three biological replicates with five plants each were pooled to isolate total RNA using the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. During the extraction, the first on-column DNAse I (Qiagen) digestion was carried out. A second DNAse I (Roche, Mannheim, Germany) digestion was performed on a total of 60 μg of total RNA according to the manufacturer's instructions. Absence of genomic DNA contamination was subsequently confirmed by qRT-PCR using three different primer pairs. The primers were designed to amplify two intergenic regions (AP003727, positions 7366 – 7426: forward 5'-AGAGAAGACCGCCATGTTGG-3' and reverse 5'-CTGGCACCACAAAAACAATGAC-3'; and AP006456, positions 27334624 – 27334684: forward 5'-TATCCACTCGACAGGACGTGC-3' and reverse 5'-CCGGCAGGCAAGCTACTAGAC-3'), and an intron sequence of the gene Os01g01840 (forward 5'-TAGAGAGTTCGATCTTGCGCG-3' and reverse 5'-CGGCCCATTCAATGAAGTCTT-3'). RNA integrity was checked on 1% (w/v) agarose gels and the concentration measured before and after DNAse I digestion. cDNA was synthesized from 5 μg of total RNA using Superscript™ III reverse transcriptase (Invitrogen, Karlsruhe, Germany), according to the manufacturer's instructions. The efficiency of cDNA synthesis was estimated by qRT-PCR using β-tubulin (Os01g59150, forward primer 5'-GGAGTCACATGCTGCCTAAGGTT-3' and reverse primer 5'-TCACTGCCAGCTTACGGAGG-3').

Transcription factor sequences were extracted from version 1 of the Rice Transcription Factor Database 1112 and used to establish the rice TF qRT-PCR platform. The set of 2508 gene models corresponding to 2306 loci of confirmed and putative transcription factors was subsequently used to design primers for ca. 500 genes. The remaining 2000 primer pairs were designed by MWG Biotech AG (Ebersberg, Germany). A standard set of reaction conditions and a set of stringent criteria were used as follows: T_m of 60°C ± 2°C, PCR amplicon length of 60 to 150 bp, primer length of 20 ± 5 bp, and a guanine-cytosine content of 45 to 55%. If gene structure allowed, at least one primer was designed to cover an exon-exon junction. The specificity of the primer pair sequence was checked against Version 2 of the rice transcripts (CDS) from the TIGR Rice Database 15 using the BLAST programme. The EXPECT threshold (statistical significance) was set to 1000, as suggested when searching for short, nearly exact matches 30. The specificity of the amplicons was checked by qRT-PCR dissociation curve analysis and electrophoresis of PCR products on 4% agarose gels. The efficiencies (E) of the polymerase chain reactions were estimated using the LinRegPCR software 21 (Additional file 1).

Potential reference genes were chosen based on published data for rice and other plant species and rice orthologues were identified using the TBLASTN programme 15. The gene models used were: actin (Os03g50890), actin1 (Os05g36290), β-tubulin (Os01g59150), expressed protein (Os06g11070), TIP41-like protein (Os03g55270), cyclophilin (Os08g19610) and elongation factor 1α (Os03g08020). Primer design followed the same criteria as described and details are given in Table 1.

PCR reactions were conducted in an ABI PRISM 7900 HT sequence detection system (Applied Biosystems). A 5 μl reaction containing 0.5 μl of cDNA (1.25 ng/μl), 200 nM of each gene-specific primer and 2.5 μl of SYBR Green master mix (Applied Biosystems Applera, Darmstadt, Germany), was used to monitor double-strand DNA synthesis. The qRT-PCR reactions were carried out following the recommended thermal profile: 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. After 40 cycles, the specificity of the amplifications was tested by heating from 60°C to 95°C with a ramp speed of 1.9°C min^-1, resulting in melting curves. Data analysis was performed using SDS 2.2.1 software (Applied Biosystems). All amplification curves were analysed with a normalized reporter (R_n: the ratio of the fluorescence emission intensity of SYBR Green to the fluorescence signal of the passive reference dye) threshold of 0.2 to obtain the C_T values (threshold cycle). The reference control genes were measured with four replicates in each PCR run, and their average C_T was used for relative expression analyses. TF expression data were normalized by subtracting the mean reference gene C_T value from their C_T value (ΔC_T). The Fold Change value was calculated using the expression 2−ΔΔCT MathType@MTEF@5@5@+=feaafiart1ev1aaatCvAUfKttLearuWrP9MDH5MBPbIqV92AaeXatLxBI9gBaebbnrfifHhDYfgasaacH8akY=wiFfYdH8Gipec8Eeeu0xXdbba9frFj0=OqFfea0dXdd9vqai=hGuQ8kuc9pgc9s8qqaq=dirpe0xb9q8qiLsFr0=vr0=vr0dc8meaabaqaciaacaGaaeqabaqabeGadaaakeaacqaIYaGmdaahaaWcbeqaaiabgkHiTiabfs5aejabfs5aejabboeadnaaBaaameaacqqGubavaeqaaaaaaaa@33ED@, where ΔΔC_T represents ΔC_{Tcondition of interest} - ΔC_{T control}. The obtained results were transformed to log₂ scale.

Mixtures of root and shoot cDNA were prepared as described by Czechowski et al. 5 to give the ratios indicated in Figure 3. Primer pairs for two root-specific genes (Os03g55610, 5'-TACCCCACCCAACACCATCATCTG and 5'-TGAGAAGAAGGCAAAGGCAGTGAG; Os08g38220; 5'-GGGGTTTCCAACTACACCCGATGA and 5'-TCACCATATACCCCACGCGCAA) and one shoot-specific gene (Os12g38200, 5'-TGGTTTTCTCCTCGCTTCCAATCT and 5'-TGCTGCTGCATCTGAGTCCAATT) were used in qRT-PCR experiments.

To analyse the expression stability of the selected reference genes, the geNORM v.3.4 software was used as described by Vandesompele et al. 28. The measured C_T values were transformed so the highest expression of each reference gene is equal to 1 and its expression in all other conditions is relative to this value (as suggested by the geNORM manual). The gene expression stability (M) was calculated and the most stable control genes were determined.