BACKGROUND
the study of gene expression patterns is one of the modern molecular biology cornerstones.
gene expression analyses have provided insight into complex biological processes, increasing our understanding of signalling and metabolic pathways that underlie environmental responses and development.
real-time reverse transcription pcr  is currently the standard method for accurate expression profiling of a moderate number of selected genes, its main advantages being a higher sensitivity and specificity, and a broader quantification range than previous molecular techniques  <cit> .
real-time rt-pcr analysis has become the most common method for verification of microarray expression results  <cit> , reaching a notable level of throughput  <cit> .
regardless of the experimental technique employed, appropriate normalization is essential for obtaining an accurate and reliable quantification of gene expression levels, especially when measuring small expression differences or when working with tissues of different histological origin  <cit> .
the purpose of normalization is to correct for variability associated with the various steps of the experimental procedure, such as differences in initial sample amount, rna recovery, rna integrity, efficiency of cdna synthesis, and differences in the overall transcriptional activity of the tissues or cells analyzed.
among the numerous normalization approaches that have been proposed  <cit>  the use of internal controls or reference genes has become the method of choice  <cit> , because they potentially account for all the above-mentioned sources of variability.
since the internal control and target sequences are naturally present in the biological sample, both will undergo the same type of variation throughout the assay.
the success of this normalization strategy is highly dependent on the choice of the appropriate control gene: its expression level should be relatively constant across the tissues or cells tested, and should not be significantly altered by the experimental pressures introduced  <cit> .
if the expression of the reference gene is affected by an excessive variation the detection of small changes becomes unfeasible or, at worst, erroneous expression patterns could be inferred  <cit> .
there is a general consensus on using housekeeping genes as internal controls in rt-pcr expression analyses.
since housekeeping genes are required for cellular survival, it is assumed that they are stably expressed and are often used without validating their suitability.
however, numerous studies reported that the transcript levels of commonly used housekeeping genes can vary considerably under different experimental conditions  <cit> .
moreover, a reference gene with stable expression in one organism may not be suitable for normalization of gene expression in another  <cit> .
in recent years, it has become clear that it is necessary to validate the expression stability of a candidate control gene in each experimental system prior to its use for normalization.
in this regard, several free software-based applications such as genorm  <cit> , normfinder  <cit>  or qbase  <cit>  permit the statistical identification of the best internal controls from a group of candidate normalization genes in a given set of biological samples.
the combination of these statistical tools with microarray and expressed sequence tags  data sets has been shown to be a valuable source of internal control genes for real-time rt-pcr experiments, providing a new generation of reference genes with very stable expression levels that outperform the classical housekeeping genes  <cit> .
tomato is an important model for genetic and molecular studies, and an international tomato genome project in currently in progress.
however, a systematic study validating internal control genes for expression analyses of different developmental stages has not been accomplished in tomato as has occurred with arabidopsis  <cit> , rice  <cit>  and soybean  <cit> .
searches of the literature reveal a single report in which several classical housekeeping genes are proposed as internal controls based on the relative abundance of tomato est  <cit> .
nevertheless, no genes were identified that showed stable expression across a wide range of developmental conditions and any candidate control gene was further evaluated with a more accurate analytical technique.
in the present report, we tested the performance of  <dig> classical and  <dig> novel housekeeping genes as internal controls for quantitative real-time rt-pcr experiments, in a set of  <dig> samples representing different tissues and organs of tomato plants at different developmental stages.
in addition to  <dig> references genes suitable for transcript normalization in the whole developmental series, we recommended other combinations of internal controls that provide a more accurate normalization in studies focused on less heterogeneous sample panels.
RESULTS
rna quality
a set of  <dig> tissue samples from solanum lycopersicon cv.
ciliegia plants, comprising all tomato organs at different developmental stages, was processed with a commercial kit.
purified total rnas had a mean value of  <dig>   for 260/ <dig> nm ratios and showed, after denaturing electrophoresis, sharp and intense 18s and 25s ribosomal rna bands with a practical absence of smears.
the level of genomic dna  contamination in each rna preparation was estimated by real-time pcr through the amplification of an alpha-tubulin gene sequence .
only rna samples from mature roots and immature green fruit gave a contamination signal, but with threshold cycle  values higher than  <dig>  the cdnas obtained from contaminated rna samples were controlled during the corresponding rt-pcrs by means of reverse transcriptase-minus amplification reactions .
* accession numbers from genbank database or sol genomics network .
primer concentration
* predicted from tomato cdna sequences; ** estimated by agarose gel electrophoresis; *** mean of  <dig> technical replicas; n/a = non-amplification
performance of amplification primers
a total of  <dig> genes were selected as candidates for normalization of gene expression measures during tomato development studies .
these include  <dig> classical  and  <dig> novel  housekeeping genes.
in order to control for gdna contaminations in the cdna samples, pcr primers were designed on different exons  or spanning an exon-exon junction , mainly guided by information about arabidopsis genes.
the performance of the amplification primers was tested by real-time pcr in two ways.
first, aliquots from the  <dig> cdna samples were pooled and used as template in amplification reactions with each primer-pair.
a single band with the expected size  was obtained in each case without signs of primer-dimers formation , as suggested by the previous melting curve analyses.
second, amplification primers were tested using gdna as template .
seven primer-pairs yielded amplicons longer than those obtained with a cdna template , whereas primers for gapdh, tbp, rpl <dig>  and sand genes were unable to amplify genomic sequences.
this result implies that intron position prediction in tomato genes was successful.
as summarized in table  <dig>  six amplicons obtained from gdna have a melting temperature sufficiently different from those of corresponding cdna amplicons to allow detection of gdna interferences in a homogeneous assay.
in the case of efα <dig> primers, real-time rt-pcr should be followed by standard agarose gel electrophoresis.
absolute tm values in table  <dig> should be considered with caution because they depend on the ionic strength of the actual reaction mix and the precision/accuracy of the real-time pcr platform.
finally, in order to optimize pcr conditions, different primer concentrations were tested by real-time rt-pcr with the cdna pool as template.
table  <dig> shows primer concentrations that provided the lowest ct and thus the highest amplification efficiency.
ct data collection
real-time rt-pcr was conducted on the  <dig> cdna samples with the  <dig> primer-pairs.
rt-minus controls were incorporated for mature roots and immature green fruit samples and only with the seven primer-pairs that yielded an amplification signal using gdna as template.
the  <dig> candidate control genes displayed a relatively wide range of expression level with mean ct values between  <dig>   and  <dig>  .
the rt-minus controls for mature roots and immature green fruit reached the fluorescence threshold only with apt, cac and "expressed" primers, but an extra treatment with dnase was not required because the ct values of the mentioned rt-minus reactions were at least  <dig> cycles higher than those in the corresponding rt-pcrs, exceeding the minimum of  <dig> cycles recommended by nolan et al.
<cit> .
amplification specificity was confirmed by melting analysis or, in the case of efα <dig> primers, by agarose gel electrophoresis.
expression stability of housekeeping genes in the whole developmental series
in order to identify the most stably expressed genes during tomato development, the entire ct dataset was analyzed using three different statistical approaches that have been incorporated in free specific vba applets.
the "pairwise comparison strategy"  <cit> , implemented in the genorm software, evaluates the variation of relative quantity  ratios for each gene-pair along the sample series.
the "model-based approach for estimation of expression variation"  <cit> , supported by the normfinder software, estimates intra- and intergroup variation, and thus subdivision of the sample set in at least two coherent groups is required for the correct application of this approach.
in this sense, we initially established the following sample-groups: roots , leaves , inflorescences  and fruits .
since a minimum of  <dig> samples/group is recommended  <cit> , expression data from different organs were also combined into "vegetative"  and "reproductive"  sample-groups.
the third statistical approach determines the expression stability for each control gene as the coefficient of variation  of the relative expression levels after normalization.
this evaluation strategy has been incorporated in the qbase program although limited to the analysis of  <dig> candidate genes  <cit> .
the results of the three evaluation approaches are shown in table  <dig>  it is noteworthy that definition of sample-groups had a notable effect on normfinder output.
only two different normfinder analyses were included in table  <dig> because we believe that sample grouping should not be arbitrary in an effort to adjust group sizes to increment statistical power, but rather that it reflects comparisons that researchers wish to make.
it is remarkable that the normfinder output with  <dig> sample-groups and cv ranking differ only in the relative position of the cac and tip <dig> genes.
the results of the three statistical analyses exhibit several common features: i) cac and tip <dig> always rank as the most stably expressed housekeeping genes; ii), "expressed", tbp and sand also exhibit a remarkable stability of their expression levels and are always included among the  <dig> best performing reference genes; iii) gapdh, efα <dig> and tua show unstable expression patterns and are always classified among the least reliable control genes.
expression data were evaluated with three different statistical approaches, and their outcomes were summarized in a consensus ranking.
expression stability decreases from top to bottom.
details on values of stability parameters are available as additional file  <dig>
since the different statistical analyses applied to the expression data represent complementary strategies, we decided to combine results of the three evaluation approaches in a consensus rank, after averaging the two normfinder outputs.
for this purpose, genes were scored from  <dig>  to  <dig>  based on their relative position in each individual list.
when two candidate genes are co-localized in a particular ranking , both were scored with the average of the two consecutive positions.
from the resulting consensus rank  it can be concluded that the best choice for normalization of expression measures in the entire developmental series are cac and tip <dig> genes, followed by "expressed".
analysis of pairwise variation between two sequential normalization factors  revealed that three genes are sufficient to calculate an accurate sample-specific nf as the geometric mean of their rqs.
that is, the addition of a fourth control gene into the cac/tip41/expressed combination does not significantly change nfs.
the variation value for the pairs nf3/nf <dig>  is lower than the default cut-off value of  <dig>   <cit> .
the mean m and cv values for the cac/tip41/expressed genes in the complete developmental series are m =  <dig>  and cv =  <dig> .
these values are inside the ranges m ≤  <dig> and cv ≤  <dig> , which have been proposed by hellemans et al.
<cit>  as acceptable for heterogeneous sample panels, such as the space-temporal one surveyed in the present study.
unfortunately, reference values for assessing the relevance of normfinder scoring have not been specified by the software's authors  <cit> .
in short, the cac/tip41/expressed gene-triplet is recommended for accurate normalization of gene expression measures encompassing the complete development process in tomato.
to assess the validity of the procedure for the selection of control genes detailed above, the relative expression level of the tofzy gene was estimated in five tomato tissue samples, using the control genes that we recommended for the normalization of gene expression measures in the whole developmental series.
for this purpose, we used tofzy specific primers described previously  <cit>  and applied an efficiency-correction model for relative quantification  <cit> .
our results  were highly concordant with the transcriptional pattern of yuc1/yuc <dig> genes  reported by cheng et al.
<cit>  based on histochemical analysis of gus reporter lines and in situ hybridization.
assessment of normalization in sample subsets
the same evaluation procedure applied to data from the whole developmental series was tested on different sample combinations which, in our understanding would represent plausible experimental contexts.
cotyledons were always included in the leaf sample group because top-ranked genes were not affected by this combination.
the unique exceptions to the analysis routine were the inference of consensus gene-rankings for individual organs , which were constructed without participation of normfinder software because an estimate of the intergroup variation is not possible.
results shown in table  <dig> can be used as a guide for selection of suitable control genes that fulfil specific research needs with regard to the particular developmental series analysed.
the complete consensus rankings are available as additional file  <dig>
for each sample subset, optimal control genes are displayed as arranged in the corresponding consensus ranking .
in brackets are shown optional control genes for individual organs and the resulting stability values .
* pairwise variation of nfn/nfn+ <dig> ratios, n being the number of recommended control genes.
r: roots; l: leaves and cotyledons; i: inflorescences; f: fruits.
the combinations of control genes recommended for the different sample subsets  are basically constructed with those that were ranked among the top five in the analysis of the whole developmental series , with the notable exception of tbp which is now downgraded in most consensus rankings .
it is clear that normalization of expression measures within organs have different requirements from comparisons between organs.
on the one hand, two control genes are sufficient for accurate normalization in individual organs, as indicated by v2/ <dig> values lower than  <dig>  .
the recommended gene-pairs have mean stability values that are acceptable  for relatively homogeneous sample panels  <cit> .
in these cases, a third control gene is included  for those wanting to use a minimum of  <dig> genes for calculating nfs, as suggested by vandesompele et al.
<cit> .
in addition, the sand gene is revealed as appropriate for normalization within organs, but less advisable for between-organs experiments.
on the other hand, when the sample subsets were comprised of  <dig> or  <dig> different organs the evaluation procedure indicated that  <dig> control genes are necessary for a reliable normalization .
the expression levels of the  <dig> proposed triplets of control genes undergo oscillations comparable to those observed in the entire sample set, to judge by the values of the corresponding stability parameters .
moreover, control genes recommended for normalization in the complete developmental series  are also suitable for  <dig> different combinations of plant organs.
the only exception is the subset integrated by inflorescence and fruit samples which can be suitably normalized with the following gene-triplet: cac/sand/rpl <dig>  in this case, if rpl <dig> is substituted by tip <dig>  the next most stable gene in the inflorescence/fruit consensus ranking , the mean values for stability parameters would remain acceptable  and, at the same time, would allow the tool-kit of control genes for normalization during tomato development to be reduced to  <dig> components: cac, tip <dig>  expressed and sand.
discussion
the detection of differentially expressed genes has contributed to understanding how developmental processes are conducted in a biological system such as tomato plant.
in the field of gene-expression analysis, real-time reverse transcription pcr  has become the method of choice for accurate expression profiling of selected genes  <cit> .
correct sample-normalization is an absolute prerequisite for reliable and accurate measurement of gene expression, especially when studying the biological relevance of small differences or when handling samples from different organs or tissues  <cit> .
the actual gold standard for controlling inter-sample variations, both in the amount and quality of cdna inputs, is the use of suitable genes as endogenous controls  <cit> .
however, since there are no universal control genes, a set of potential references must be previously validated in each particular experimental background.
recently, an exhaustive analysis anchored on microarray data about expression profiles in arabidopsis  <cit>  allowed the identification of hundreds of potential reference genes, which show exceptional expression stability throughout development and under a wide range of environmental conditions.
despite its relevance as a model organism, certain biological processes are not tractable in arabidopsis, such as the ripening of fleshy fruits which has received considerable attention in tomato.
in addition, the conclusions derived from studies in arabidopsis cannot be directly extrapolated to any vascular plant species.
for example, ubq <dig> gene shows highly stable expression in arabidopsis  <cit> , whereas it seems unsuitable for normalization in different tissues at different developmental stages in rice and soybean  <cit> .
this emphasizes the importance of preliminary evaluation studies, aimed to identify the most stable housekeeping genes in different organisms.
taking the above-mentioned arguments into account, we accomplished a systematic study of the expression stability of  <dig> housekeeping genes in solanum lycopersicon, along a series composed of  <dig> samples from different tissues/organs at different developmental stages.
in an effort to minimize bias introduced by the validation approach, three different, yet complementary, statistical strategies were used to select the best internal controls for normalization of gene expression studies in tomato.
the pairwise comparison strategy, accessible through the genorm software  <cit> , is a very popular option for verifying the expression stability of candidate genes.
it relies on the principle that variations in the expression ratios of two housekeeping genes reflect the fact that at least one of the two genes is not constantly expressed.
its main advantage is that expression ratios allow a fine control of variations in the amount of cdna inputs, because these oscillations associated to technical variability affect both paired genes equally.
it has been argued that the major weakness of the pairwise comparison approach is its sensitivity to co-regulation, that is, it apparently tends to select those genes with the highest degree of similarity in their expression profile  <cit> .
however, it should be noted that the stability measure provided by genorm  is the mean pairwise variation between a gene and all other tested candidates, and thus a pair of highly co-regulated genes could soon be eliminated during the selection process if they show high inter-sample variability.
in addition, the advantage of two co-regulated genes is inversely proportional to the number of candidate genes being validated.
an obvious prediction about behaviour of two co-regulated genes in the pairwise variation approach is that they will be scored with a similar m value.
indeed, there are numerous examples in the literature of genes belonging to the same functional class  that are not top-ranked by the genorm software, but which occupy closed positions in the ranking.
whatever that means, and since it is very difficult to foresee common expression patterns, the threshold cycle data were analyzed with two other statistical strategies that are less sensitive towards co-regulation of the candidate genes.
on the one hand, the "model-based approach" implemented in the normfinder software examines variation within and between sample groups that must be defined by the user.
on the other hand, overall expression variation of each candidate gene was measured as the coefficient of variation  of the normalized relative quantities .
the normfinder approach stands out because it makes a balance of two sources of variation, but it does not account for systematic errors during sample preparation.
nevertheless, the cv strategy overcomes this drawback through the handling of normalized quantities, and may be a good alternative when the sample set cannot be appropriately subdivided.
although other valid statistical strategies have been successfully applied to control gene selection  <cit> , the above-mentioned approaches are usually preferred because they are supported by user-friendly software.
since the  <dig> statistical approaches complement one another their outcomes were equally weighted and combined into a consensus ranking.
as the main result of this analysis, based on real-time pcr data, we proposed a tool-kit of control genes suitable for normalization of gene expression measures in a wide variety of samples in tomato.
this tool-kit is composed of  <dig> housekeeping genes , which are recommended in different combinations depending on the sample origin .
our analysis suggests that studies involving different tomato organs require at least  <dig> control genes for reliable and accurate normalization, while two control genes are sufficient for experiments within particular organs.
the method of calculating a sample-specific normalization factor as the geometric mean of multiple carefully selected housekeeping genes  <cit>  is currently the golden standard  <cit> .
this approach has been adopted by many researchers and has been empirically and statistically validated  <cit> .
although the minimal use of three control genes has been proposed for the correct normalization of rt-pcr data  <cit>  the actual optimal number of control genes should arise from a balance between economic considerations and accuracy, keeping in mind that normalization with multiple genes is less error-prone than single gene normalization  <cit> .
among the housekeeping genes evaluated in the present study, dnaj, gapdh and tua genes have been previously described as "candidate controls" in tomato plants  <cit> .
these genes were selected after the expression analysis of  <dig> transcripts in  <dig> expressed sequence tag libraries, but none of them was described as a suitable control gene for all tissues.
our results, based on data obtained with a more accurate and precise technique, lead to the conclusion that dnaj gene may be useful for normalization in inflorescence samples  and, to a lesser extent, in leaves, fruits or a leaf/inflorescence developmental series .
this is in accord with the results of cocker and davis  <cit> .
however, we suggest that gapdh and, especially, tua should be avoided as control genes because their expression stability is far from acceptable.
for instance, the nrqs of tua gene showed cvs higher than 180% in leaf and fruit samples.
as another contribution of the present report, our results indicate that reliable normalization of the whole tomato developmental series is possible with the cac, tip <dig> and expressed genes.
finally, the results reported herein are in good agreement with those described in arabidopsis by czechowski et al.
guided by microarray expression data  <cit> .
in fact, the  <dig> control genes that we recommended for normalization in tomato are among the  <dig> top-ranked genes in arabidopsis, although with a different relative position in the respective rankings.
these novel control genes, as in arabidopsis, are superior to traditional ones in terms of expression stability.
CONCLUSIONS
this work constitutes the first in-depth study aimed to validate the optimal control genes for the quantification of transcript levels during tomato development using real-time rt-pcr technology.
we have tested the expression stabilities of  <dig> candidate genes in a set of  <dig> tissue samples from tomato plants.
as a result of this evaluation, we recommend  <dig> non-classical housekeeping genes as superior references for normalization of gene expression measures in different tomato developmental stages, and provide primer sequences whose performance in real-time pcr experiments is demonstrated.
finally, we have provided useful background information about the procedure of control gene selection in quantitative rt-pcr studies of gene expression.

 the study of gene expression patterns is one of the modern molecular biology cornerstones. gene expression analyses have provided insight into complex biological processes, increasing our understanding of signalling and metabolic pathways that underlie environmental responses and development. real-time reverse transcription pcr is currently the standard method for accurate expression profiling of a moderate number of selected genes, its main advantages being a higher sensitivity and specificity, and a broader quantification range than previous molecular techniques. real-time rt-pcr analysis has become the most common method for verification of microarray expression results, reaching a notable level of throughput. regardless of the experimental technique employed, appropriate normalization is essential for obtaining an accurate and reliable quantification of gene expression levels, especially when measuring small expression differences or when working with tissues of different histological origin. the purpose of normalization is to correct for variability associated with the various steps of the experimental procedure, such as differences in initial sample amount, rna recovery, rna integrity, efficiency of cdna synthesis, and differences in the overall transcriptional activity of the tissues or cells analyzed. among the numerous normalization approaches that have been proposed the use of internal controls or reference genes has become the method of choice, because they potentially account for all the above-mentioned sources of variability. since the internal control and target sequences are naturally present in the biological sample, both will undergo the same type of variation throughout the assay. the success of this normalization strategy is highly dependent on the choice of the appropriate control gene: its expression level should be relatively constant across the tissues or cells tested, and should not be significantly altered by the experimental pressures introduced. if the expression of the reference gene is affected by an excessive variation the detection of small changes becomes unfeasible or, at worst, erroneous expression patterns could be inferred. there is a general consensus on using housekeeping genes as internal controls in rt-pcr expression analyses. since housekeeping genes are required for cellular survival, it is assumed that they are stably expressed and are often used without validating their suitability. however, numerous studies reported that the transcript levels of commonly used housekeeping genes can vary considerably under different experimental conditions. moreover, a reference gene with stable expression in one organism may not be suitable for normalization of gene expression in another. in recent years, it has become clear that it is necessary to validate the expression stability of a candidate control gene in each experimental system prior to its use for normalization. in this regard, several free software-based applications such as genorm, normfinder or qbase permit the statistical identification of the best internal controls from a group of candidate normalization genes in a given set of biological samples. the combination of these statistical tools with microarray and expressed sequence tags data sets has been shown to be a valuable source of internal control genes for real-time rt-pcr experiments, providing a new generation of reference genes with very stable expression levels that outperform the classical housekeeping genes. tomato is an important model for genetic and molecular studies, and an international tomato genome project in currently in progress. however, a systematic study validating internal control genes for expression analyses of different developmental stages has not been accomplished in tomato as has occurred with arabidopsis, rice and soybean. searches of the literature reveal a single report in which several classical housekeeping genes are proposed as internal controls based on the relative abundance of tomato est. nevertheless, no genes were identified that showed stable expression across a wide range of developmental conditions and any candidate control gene was further evaluated with a more accurate analytical technique. in the present report, we tested the performance of classical and novel housekeeping genes as internal controls for quantitative real-time rt-pcr experiments, in a set of samples representing different tissues and organs of tomato plants at different developmental stages. in addition to references genes suitable for transcript normalization in the whole developmental series, we recommended other combinations of internal controls that provide a more accurate normalization in studies focused on less heterogeneous sample panels.  rna quality a set of tissue samples from solanum lycopersicon cv. ciliegia plants, comprising all tomato organs at different developmental stages, was processed with a commercial kit. purified total rnas had a mean value of for 260/ nm ratios and showed, after denaturing electrophoresis, sharp and intense 18s and 25s ribosomal rna bands with a practical absence of smears. the level of genomic dna contamination in each rna preparation was estimated by real-time pcr through the amplification of an alpha-tubulin gene sequence. only rna samples from mature roots and immature green fruit gave a contamination signal, but with threshold cycle values higher than the cdnas obtained from contaminated rna samples were controlled during the corresponding rt-pcrs by means of reverse transcriptase-minus amplification reactions. * accession numbers from genbank database or sol genomics network. primer concentration * predicted from tomato cdna sequences; ** estimated by agarose gel electrophoresis; *** mean of technical replicas; n/a = non-amplification performance of amplification primers a total of genes were selected as candidates for normalization of gene expression measures during tomato development studies. these include classical and novel housekeeping genes. in order to control for gdna contaminations in the cdna samples, pcr primers were designed on different exons or spanning an exon-exon junction, mainly guided by information about arabidopsis genes. the performance of the amplification primers was tested by real-time pcr in two ways. first, aliquots from the cdna samples were pooled and used as template in amplification reactions with each primer-pair. a single band with the expected size was obtained in each case without signs of primer-dimers formation, as suggested by the previous melting curve analyses. second, amplification primers were tested using gdna as template. seven primer-pairs yielded amplicons longer than those obtained with a cdna template, whereas primers for gapdh, tbp, rpl and sand genes were unable to amplify genomic sequences. this result implies that intron position prediction in tomato genes was successful. as summarized in table six amplicons obtained from gdna have a melting temperature sufficiently different from those of corresponding cdna amplicons to allow detection of gdna interferences in a homogeneous assay. in the case of efα primers, real-time rt-pcr should be followed by standard agarose gel electrophoresis. absolute tm values in table should be considered with caution because they depend on the ionic strength of the actual reaction mix and the precision/accuracy of the real-time pcr platform. finally, in order to optimize pcr conditions, different primer concentrations were tested by real-time rt-pcr with the cdna pool as template. table shows primer concentrations that provided the lowest ct and thus the highest amplification efficiency. ct data collection real-time rt-pcr was conducted on the cdna samples with the primer-pairs. rt-minus controls were incorporated for mature roots and immature green fruit samples and only with the seven primer-pairs that yielded an amplification signal using gdna as template. the candidate control genes displayed a relatively wide range of expression level with mean ct values between and. the rt-minus controls for mature roots and immature green fruit reached the fluorescence threshold only with apt, cac and "expressed" primers, but an extra treatment with dnase was not required because the ct values of the mentioned rt-minus reactions were at least cycles higher than those in the corresponding rt-pcrs, exceeding the minimum of cycles recommended by nolan et al.. amplification specificity was confirmed by melting analysis or, in the case of efα primers, by agarose gel electrophoresis. expression stability of housekeeping genes in the whole developmental series in order to identify the most stably expressed genes during tomato development, the entire ct dataset was analyzed using three different statistical approaches that have been incorporated in free specific vba applets. the "pairwise comparison strategy", implemented in the genorm software, evaluates the variation of relative quantity ratios for each gene-pair along the sample series. the "model-based approach for estimation of expression variation", supported by the normfinder software, estimates intra- and intergroup variation, and thus subdivision of the sample set in at least two coherent groups is required for the correct application of this approach. in this sense, we initially established the following sample-groups: roots, leaves, inflorescences and fruits. since a minimum of samples/group is recommended, expression data from different organs were also combined into "vegetative" and "reproductive" sample-groups. the third statistical approach determines the expression stability for each control gene as the coefficient of variation of the relative expression levels after normalization. this evaluation strategy has been incorporated in the qbase program although limited to the analysis of candidate genes. the results of the three evaluation approaches are shown in table it is noteworthy that definition of sample-groups had a notable effect on normfinder output. only two different normfinder analyses were included in table because we believe that sample grouping should not be arbitrary in an effort to adjust group sizes to increment statistical power, but rather that it reflects comparisons that researchers wish to make. it is remarkable that the normfinder output with sample-groups and cv ranking differ only in the relative position of the cac and tip genes. the results of the three statistical analyses exhibit several common features: i) cac and tip always rank as the most stably expressed housekeeping genes; ii), "expressed", tbp and sand also exhibit a remarkable stability of their expression levels and are always included among the best performing reference genes; iii) gapdh, efα and tua show unstable expression patterns and are always classified among the least reliable control genes. expression data were evaluated with three different statistical approaches, and their outcomes were summarized in a consensus ranking. expression stability decreases from top to bottom. details on values of stability parameters are available as additional file since the different statistical analyses applied to the expression data represent complementary strategies, we decided to combine results of the three evaluation approaches in a consensus rank, after averaging the two normfinder outputs. for this purpose, genes were scored from to based on their relative position in each individual list. when two candidate genes are co-localized in a particular ranking, both were scored with the average of the two consecutive positions. from the resulting consensus rank it can be concluded that the best choice for normalization of expression measures in the entire developmental series are cac and tip genes, followed by "expressed". analysis of pairwise variation between two sequential normalization factors revealed that three genes are sufficient to calculate an accurate sample-specific nf as the geometric mean of their rqs. that is, the addition of a fourth control gene into the cac/tip41/expressed combination does not significantly change nfs. the variation value for the pairs nf3/nf is lower than the default cut-off value of. the mean m and cv values for the cac/tip41/expressed genes in the complete developmental series are m = and cv =. these values are inside the ranges m ≤ and cv ≤, which have been proposed by hellemans et al. as acceptable for heterogeneous sample panels, such as the space-temporal one surveyed in the present study. unfortunately, reference values for assessing the relevance of normfinder scoring have not been specified by the software's authors. in short, the cac/tip41/expressed gene-triplet is recommended for accurate normalization of gene expression measures encompassing the complete development process in tomato. to assess the validity of the procedure for the selection of control genes detailed above, the relative expression level of the tofzy gene was estimated in five tomato tissue samples, using the control genes that we recommended for the normalization of gene expression measures in the whole developmental series. for this purpose, we used tofzy specific primers described previously and applied an efficiency-correction model for relative quantification. our results were highly concordant with the transcriptional pattern of yuc1/yuc genes reported by cheng et al. based on histochemical analysis of gus reporter lines and in situ hybridization. assessment of normalization in sample subsets the same evaluation procedure applied to data from the whole developmental series was tested on different sample combinations which, in our understanding would represent plausible experimental contexts. cotyledons were always included in the leaf sample group because top-ranked genes were not affected by this combination. the unique exceptions to the analysis routine were the inference of consensus gene-rankings for individual organs, which were constructed without participation of normfinder software because an estimate of the intergroup variation is not possible. results shown in table can be used as a guide for selection of suitable control genes that fulfil specific research needs with regard to the particular developmental series analysed. the complete consensus rankings are available as additional file for each sample subset, optimal control genes are displayed as arranged in the corresponding consensus ranking. in brackets are shown optional control genes for individual organs and the resulting stability values. * pairwise variation of nfn/nfn+ ratios, n being the number of recommended control genes. r: roots; l: leaves and cotyledons; i: inflorescences; f: fruits. the combinations of control genes recommended for the different sample subsets are basically constructed with those that were ranked among the top five in the analysis of the whole developmental series, with the notable exception of tbp which is now downgraded in most consensus rankings. it is clear that normalization of expression measures within organs have different requirements from comparisons between organs. on the one hand, two control genes are sufficient for accurate normalization in individual organs, as indicated by v2/ values lower than. the recommended gene-pairs have mean stability values that are acceptable for relatively homogeneous sample panels. in these cases, a third control gene is included for those wanting to use a minimum of genes for calculating nfs, as suggested by vandesompele et al.. in addition, the sand gene is revealed as appropriate for normalization within organs, but less advisable for between-organs experiments. on the other hand, when the sample subsets were comprised of or different organs the evaluation procedure indicated that control genes are necessary for a reliable normalization. the expression levels of the proposed triplets of control genes undergo oscillations comparable to those observed in the entire sample set, to judge by the values of the corresponding stability parameters. moreover, control genes recommended for normalization in the complete developmental series are also suitable for different combinations of plant organs. the only exception is the subset integrated by inflorescence and fruit samples which can be suitably normalized with the following gene-triplet: cac/sand/rpl in this case, if rpl is substituted by tip the next most stable gene in the inflorescence/fruit consensus ranking, the mean values for stability parameters would remain acceptable and, at the same time, would allow the tool-kit of control genes for normalization during tomato development to be reduced to components: cac, tip expressed and sand. discussion the detection of differentially expressed genes has contributed to understanding how developmental processes are conducted in a biological system such as tomato plant. in the field of gene-expression analysis, real-time reverse transcription pcr has become the method of choice for accurate expression profiling of selected genes. correct sample-normalization is an absolute prerequisite for reliable and accurate measurement of gene expression, especially when studying the biological relevance of small differences or when handling samples from different organs or tissues. the actual gold standard for controlling inter-sample variations, both in the amount and quality of cdna inputs, is the use of suitable genes as endogenous controls. however, since there are no universal control genes, a set of potential references must be previously validated in each particular experimental background. recently, an exhaustive analysis anchored on microarray data about expression profiles in arabidopsis allowed the identification of hundreds of potential reference genes, which show exceptional expression stability throughout development and under a wide range of environmental conditions. despite its relevance as a model organism, certain biological processes are not tractable in arabidopsis, such as the ripening of fleshy fruits which has received considerable attention in tomato. in addition, the conclusions derived from studies in arabidopsis cannot be directly extrapolated to any vascular plant species. for example, ubq gene shows highly stable expression in arabidopsis, whereas it seems unsuitable for normalization in different tissues at different developmental stages in rice and soybean. this emphasizes the importance of preliminary evaluation studies, aimed to identify the most stable housekeeping genes in different organisms. taking the above-mentioned arguments into account, we accomplished a systematic study of the expression stability of housekeeping genes in solanum lycopersicon, along a series composed of samples from different tissues/organs at different developmental stages. in an effort to minimize bias introduced by the validation approach, three different, yet complementary, statistical strategies were used to select the best internal controls for normalization of gene expression studies in tomato. the pairwise comparison strategy, accessible through the genorm software, is a very popular option for verifying the expression stability of candidate genes. it relies on the principle that variations in the expression ratios of two housekeeping genes reflect the fact that at least one of the two genes is not constantly expressed. its main advantage is that expression ratios allow a fine control of variations in the amount of cdna inputs, because these oscillations associated to technical variability affect both paired genes equally. it has been argued that the major weakness of the pairwise comparison approach is its sensitivity to co-regulation, that is, it apparently tends to select those genes with the highest degree of similarity in their expression profile. however, it should be noted that the stability measure provided by genorm is the mean pairwise variation between a gene and all other tested candidates, and thus a pair of highly co-regulated genes could soon be eliminated during the selection process if they show high inter-sample variability. in addition, the advantage of two co-regulated genes is inversely proportional to the number of candidate genes being validated. an obvious prediction about behaviour of two co-regulated genes in the pairwise variation approach is that they will be scored with a similar m value. indeed, there are numerous examples in the literature of genes belonging to the same functional class that are not top-ranked by the genorm software, but which occupy closed positions in the ranking. whatever that means, and since it is very difficult to foresee common expression patterns, the threshold cycle data were analyzed with two other statistical strategies that are less sensitive towards co-regulation of the candidate genes. on the one hand, the "model-based approach" implemented in the normfinder software examines variation within and between sample groups that must be defined by the user. on the other hand, overall expression variation of each candidate gene was measured as the coefficient of variation of the normalized relative quantities. the normfinder approach stands out because it makes a balance of two sources of variation, but it does not account for systematic errors during sample preparation. nevertheless, the cv strategy overcomes this drawback through the handling of normalized quantities, and may be a good alternative when the sample set cannot be appropriately subdivided. although other valid statistical strategies have been successfully applied to control gene selection, the above-mentioned approaches are usually preferred because they are supported by user-friendly software. since the statistical approaches complement one another their outcomes were equally weighted and combined into a consensus ranking. as the main result of this analysis, based on real-time pcr data, we proposed a tool-kit of control genes suitable for normalization of gene expression measures in a wide variety of samples in tomato. this tool-kit is composed of housekeeping genes, which are recommended in different combinations depending on the sample origin. our analysis suggests that studies involving different tomato organs require at least control genes for reliable and accurate normalization, while two control genes are sufficient for experiments within particular organs. the method of calculating a sample-specific normalization factor as the geometric mean of multiple carefully selected housekeeping genes is currently the golden standard. this approach has been adopted by many researchers and has been empirically and statistically validated. although the minimal use of three control genes has been proposed for the correct normalization of rt-pcr data the actual optimal number of control genes should arise from a balance between economic considerations and accuracy, keeping in mind that normalization with multiple genes is less error-prone than single gene normalization. among the housekeeping genes evaluated in the present study, dnaj, gapdh and tua genes have been previously described as "candidate controls" in tomato plants. these genes were selected after the expression analysis of transcripts in expressed sequence tag libraries, but none of them was described as a suitable control gene for all tissues. our results, based on data obtained with a more accurate and precise technique, lead to the conclusion that dnaj gene may be useful for normalization in inflorescence samples and, to a lesser extent, in leaves, fruits or a leaf/inflorescence developmental series. this is in accord with the results of cocker and davis. however, we suggest that gapdh and, especially, tua should be avoided as control genes because their expression stability is far from acceptable. for instance, the nrqs of tua gene showed cvs higher than 180% in leaf and fruit samples. as another contribution of the present report, our results indicate that reliable normalization of the whole tomato developmental series is possible with the cac, tip and expressed genes. finally, the results reported herein are in good agreement with those described in arabidopsis by czechowski et al. guided by microarray expression data. in fact, the control genes that we recommended for normalization in tomato are among the top-ranked genes in arabidopsis, although with a different relative position in the respective rankings. these novel control genes, as in arabidopsis, are superior to traditional ones in terms of expression stability.  this work constitutes the first in-depth study aimed to validate the optimal control genes for the quantification of transcript levels during tomato development using real-time rt-pcr technology. we have tested the expression stabilities of candidate genes in a set of tissue samples from tomato plants. as a result of this evaluation, we recommend non-classical housekeeping genes as superior references for normalization of gene expression measures in different tomato developmental stages, and provide primer sequences whose performance in real-time pcr experiments is demonstrated. finally, we have provided useful background information about the procedure of control gene selection in quantitative rt-pcr studies of gene expression. 
in this study, we assess the expression stability of traditional and novel housekeeping genes in a set of samples representing different tissues and organs of tomato plants at different developmental stages. then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. first, we designed, tested and optimized amplification primers for real-time rt-pcr. the elucidation of gene expression patterns leads to a better understanding of biological processes. real-time quantitative rt-pcr has become the standard method for in-depth studies of gene expression. this work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time rt-pcr studies of gene expression during tomato development process. from our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability. the purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from rna to cdna, or differences between tissues or cells in overall transcriptional activity. a biologically meaningful reporting of target mrna quantities requires accurate and reliable normalization in order to identify real gene-specific variation. the validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. in the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time rt-pcr. our analysis suggests that sgn-u, sgn-u, sgn-u and sgn-u genes provide superior transcript normalization in tomato development studies. we recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process.
 the elucidation of gene expression patterns leads to a better understanding of biological processes. real-time quantitative rt-pcr has become the standard method for in-depth studies of gene expression. a biologically meaningful reporting of target mrna quantities requires accurate and reliable normalization in order to identify real gene-specific variation. the purpose of normalization is to control several variables such as different amounts and quality of starting material, variable enzymatic efficiencies of retrotranscription from rna to cdna, or differences between tissues or cells in overall transcriptional activity. the validity of a housekeeping gene as endogenous control relies on the stability of its expression level across the sample panel being analysed. in the present report we describe the first systematic evaluation of potential internal controls during tomato development process to identify which are the most reliable for transcript quantification by real-time rt-pcr.  in this study, we assess the expression stability of traditional and novel housekeeping genes in a set of samples representing different tissues and organs of tomato plants at different developmental stages. first, we designed, tested and optimized amplification primers for real-time rt-pcr. then, expression data from each candidate gene were evaluated with three complementary approaches based on different statistical procedures. our analysis suggests that sgn-u, sgn-u, sgn-u and sgn-u genes provide superior transcript normalization in tomato development studies. we recommend different combinations of these exceptionally stable housekeeping genes for suited normalization of different developmental series, including the complete tomato development process.  this work constitutes the first effort for the selection of optimal endogenous controls for quantitative real-time rt-pcr studies of gene expression during tomato development process. from our study a tool-kit of control genes emerges that outperform the traditional genes in terms of expression stability. 