BACKGROUND
the study of biological regulations is very often correlated to quantification assays.
in order to detect differential expression of a gene in distinct biological samples, such as tissue types or under different experimental conditions, the invention of quantitative pcr  has transformed the field of gene expression analysis in living organisms  <cit> .
in comparison to classical reverse transcription-polymerase chain reaction , the main advantages of qpcr are higher sensitivity, specificity and broad quantification range of up to seven orders of magnitude  <cit> .
regardless of being an extremely powerful technique, qpcr has its pitfalls, the most important one being the need of appropriate data normalization with a reference gene  <cit> .
according to andersen et al.
<cit> , accurate data normalization is an absolute requirement for correct measurement of gene expression.
expression of the reference gene used to normalize qpcr analyses should be unaffected throughout many biological contexts; otherwise, it may lead to erroneous results  <cit> .
until recently, several such genes  have been used as internal controls for gene expression analyses under the assumption of stable expression  <cit> .
however, several reports have demonstrated that the expression levels of these so-called reference genes differ among different tissue/organ types  <cit> .
consequently, these genes are unsuitable as transcriptional inner controls, and their use to normalize qpcr data in different tissues may induce significant experimental errors that could result in inappropriate biological data interpretation  <cit> .
recognizing the importance of reference gene in normalization of qpcr data, various housekeeping genes have been evaluated for stable expression under specific conditions in various organisms.
in plants, only a few of them have been investigated in some detail in rice  <cit> , poplar  <cit> , potato  <cit> , soybean  <cit>  and arabidopsis thaliana  <cit> .
so far, suitable internal controls for gene expression studies have not been defined for coffea arabica.
coffee is an agricultural crop of significant economic importance.
coffea arabica l.  is typical of the highland growing regions and is responsible for almost 75% of world production  <cit> .
in this study, we report the validation of housekeeping genes to identify the most suitable internal reference gene for normalization of qpcr data obtained among five different tissues/organs  of c. arabica.
to further validate our results, we evaluated the expression levels of our best reference genes at different developmental stages of flowers and cherries and under a specific biotic stress.
following the current literature, five candidate reference genes, namely alcohol dehydrogenase , polyubiquitin , 14-3- <dig>  β-actin  and glyceraldehyde-3-phosphate dehydrogenase , were selected.
in addition to these commonly used internal controls, three other genes coding for a cysteine proteinase , a caffeine synthase  and the 60s ribosomal protein l <dig> , respectively, were included in this analysis.
these potential reference genes were ranked according to their expression profiles and stability.
RESULTS
the expression profile of eight candidate reference genes  was firstly assessed by qpcr over a panel of five coffee tissue/organ samples .
descriptive analysis of the reference candidate genes
descriptive statistics of the derived crossing points , based on bestkeeper program  <cit> , were calculated to investigate the variation level of each candidate gene following pfaffl et al.
<cit> .
according to this analysis , the gene with lowest expression level was actin, for which cp values were obtained around cycles 31–34; while the highest was gapdh, whose cp values were obtained around cycles 21– <dig>  the expression levels of 14-3- <dig>  ccs, gapdh, and rpl <dig> presented fluctuations of approximately ±  <dig>  x-fold , whereas poly expression showed higher ranges of cp variation  as well as up- down-regulation .
the coefficient of variation  of the assay was  <dig> % , which is within the range  of previously reported values for qpcr  <cit> .
abbreviations: n: number of samples; cp: crossing-point; gm : geometric cp mean; am : arithmetic cp mean; min  and max : cp threshold values; sd : cp standard deviation; cv : variance coefficient expressed as percentage of cp level; min  and max : threshold expression levels expressed as absolute x-fold over- or under-regulation coefficient; sd : standard deviation of absolute regulation coefficient.
descriptive statistics for the expression analyses of each reference candidate gene in the five distinct coffee plant tissue/organ types was also obtained .
according to this analysis, the gene that exhibit the minor gene expression variation among the analyzed tissues was gapdh , while the gene with major variation was ccs .
in addition, ccs also presented the highest expression variation in flowers and fruits tissue samples, and therefore it cannot be used as a reference gene.
numerous studies have shown that the expression of housekeeping genes can vary under given situations  <cit> .
this may partly be explained by the fact that housekeeping genes are not only implicated in the basal cell metabolism but also participate in other cellular functions  <cit> .
a model-based approach for estimation of expression variation
the model-based variance estimation approach, a visual basic application for microsoft excel   <cit> , was used to evaluate expression stability of reference candidate genes.
this analysis allowed the ranking of candidate genes since the estimated variation directly indicates the introduced error associated with their use.
according to this method, the gene with minimal estimated intra- and intertissue variation was gapdh  while the gene with the maximal variation was poly  , thus corroborating the results obtained in descriptive analysis.
ranking the candidate reference genes
the relationship between the stability value and the intra- and intertissue expression variations is present in figure  <dig>  this figure clearly demonstrates the distinct specificities of the investigated genes.
according to this analysis, the best candidate gene should present the minimal combined inter- and intra-tissue expression variation.
consistent with the descriptive analysis  and the model-based variance estimation approach , gapdh showed not only the highest expression levels but was also the most stable gene studied.
in figure  <dig>  it can be observed that almost all genes presented average of log expression levels near  <dig> .
log difference > <dig>  or < <dig>  implies that variability in expression levels is significant, so the gene could be incorrectly used as reference gene for normalization.
in this context, the poly gene presents the highest intertissue expression variation .
thus, among the tested genes, gapdh, followed by rpl <dig> and 14-3- <dig>  showed the most stable expression over the investigated panel of five distinct coffee tissue/organ types.
the candidate genes were also ranked according to their m values using the genorm program.
the average expression stabilities  of all tested genes were lower than  <dig> , with 14-3- <dig> and actin showing the most stable expression .
although actin gene has shown highest stability following genorm analysis, this gene presented the lowest expression profile according the bestkeeper analysis.
corroborating the previous analysis, poly remained the least stable gene.
as a whole, our analysis indicates that housekeeping genes are differently regulated in different tissues/organs of coffee plants and may exhibit variable expression patterns.
the observed differences in gene expression ratios along a comprehensive panel of tissues/organs are consistent with the data presented by barber et al.
<cit> , iskandar et al.
<cit>  and jain et al.
<cit> .
our results also provide evidences that normalizations to the expression level of a single gene in samples from distinct tissue types may induce to errors, thus corroborating previous studies  <cit> .
comparison of the identified best reference genes to published data
in order to validate our potential candidate reference genes , the expression stability of these genes under the influence of a specific biotic stress was investigated.
in this case, the obtained results were compared to those dealing with similar coffee gene expression analysis but using ubiquitin as a reference gene for normalization  <cit> .
the comparison was conduced by linear regressions analyzes of the cp difference  obtained from the assayed expression levels of the tested genes in leaves of c. arabica inoculated, or not , with hemileia vastatrix.
the average cp  was calculated for each gene and the Δcp  was determined for each time-point .
as it can be observed in figure  <dig>  the regression lines for 14-3- <dig> and gapdh have slopes close to zero , indicating similar expression levels in inoculated and non-inoculated leaves, and reinforcing their use as effective normalization genes  <cit> .
in contrast, the slope value for rpl <dig> was significantly different from zero and higher than the one obtained for ubiquitin , thus limiting its use as a normalization factor under biotic stress condition.
validation of data results in different developmental stages of flowers and coffee cherries
an additional validation step of the expression levels of gapdh, 14-3- <dig>  rpl <dig> and ubiquitin was performed using unpooled tissue samples from flower and cherry developmental series.
the employed sample set is given in the additional file  <dig>
in this assay, the highly expressed gene was gapdh  followed by ubiquitin , while 14-3- <dig> and rpl <dig> presented the same mean cp .
the comparison of gene contributions is present in figure  <dig>  as already observed, gapdh showed the greatest stability in expression among all coffee tissue/organ samples analyzed, while expression of rpl <dig> and 14-3- <dig> varied the most, especially in flowers at stage  <dig> of development .
as mentioned earlier, ubiquitin was used as a standard reference gene for comparison.
general remarks about the selected reference genes
according to these results, the gene encoding glyceraldeyde-3-phosphate dehydrogenase , an enzyme of glycolysis  <cit> , outperformed all other reference genes tested and should therefore be considered a suitable reference gene for expression studies in arabica coffee plants.
this observation corroborates the quantification of gapdh expression in different tissues of sugarcane  <cit>  and eucalyptus  <cit> .
in contrast, several reports in human and animal systems have suggested that this reference gene has limitations for its use as internal control due to its marked variability of expression among tissue types  <cit> .
the assayed 14-3- <dig> gene also showed a stable expression  that was supported by the descriptive analysis  and confirmed in the biotic stress assay .
however, its expression presented some variation among different stages of coffee flower development .
papini-terzi et al.
<cit>  recommended 14-3- <dig> as a suitable reference gene for expression normalization in a wide range of tissue samples of sugarcane.
the gene encoding the transcription regulator and structural constituent of the 60s subunit of the cytosolic ribosome  could also be used as an internal control in gene expression studies in c. arabica, due to its stability  and acceptable variation among tissue/organ types .
our results are in agreement with previous published data for this gene since small variation among tissue types was detected by descriptive analysis .
nevertheless, in leaves of c. arabica inoculated with h. vastatrix, it was observed that the expression ratio of rpl <dig>  was not constant and the absolute value of rpl <dig> linear regression slope was superior to that observed for a commonly used coffee normalization gene  .
in addition, this gene, like 14-3- <dig>  presented a variable expression level among different stages of coffee flower development .
in sugarcane, the relative expression of rpl35- <dig>  a gene coding for the ribosomal l35- <dig> 60s protein, was also reported to be stable  <cit> .
these authors estimated the sugarcane leaf transcriptome using serial analysis of gene expression  and reported that tag associated with the rpl35- <dig> transcript presented minimum variation among the analyzed sage libraries.
the remaining tested genes showed to be unsuitable as internal controls for normalization purposes in c. arabica.
CONCLUSIONS
this study provides the most extensive collection of arabica coffee tissue/organ mrna expression data for eight reference candidate genes.
our analysis evidenced stable levels of gapdh, 14-3- <dig> and rpl <dig> mrna in different coffea arabica tissue/organ types.
consequently, these genes can be used for accurate and reliable normalization in future gene expression studies in coffee .
in this respect, we suggest gapdh as the most relevant reference gene for accurate normalization purposes in c. arabica, showing almost constant expression levels in the investigated experimental set-up.
moreover, we have shown that depending on the reference inner control gene, the within-tissue variation of mrna expression levels is generally small, whereas among tissues/organs the variation can be substantial.
this indicates that normalizations to a single gene across different tissue types are unwise.
since the variation observed between normal tissues of different types may in part be due to the different metabolic demands of those tissues, comparisons within a tissue type between normal and diseased states are similarly unwise.

 the study of biological regulations is very often correlated to quantification assays. in order to detect differential expression of a gene in distinct biological samples, such as tissue types or under different experimental conditions, the invention of quantitative pcr has transformed the field of gene expression analysis in living organisms. in comparison to classical reverse transcription-polymerase chain reaction, the main advantages of qpcr are higher sensitivity, specificity and broad quantification range of up to seven orders of magnitude. regardless of being an extremely powerful technique, qpcr has its pitfalls, the most important one being the need of appropriate data normalization with a reference gene. according to andersen et al., accurate data normalization is an absolute requirement for correct measurement of gene expression. expression of the reference gene used to normalize qpcr analyses should be unaffected throughout many biological contexts; otherwise, it may lead to erroneous results. until recently, several such genes have been used as internal controls for gene expression analyses under the assumption of stable expression. however, several reports have demonstrated that the expression levels of these so-called reference genes differ among different tissue/organ types. consequently, these genes are unsuitable as transcriptional inner controls, and their use to normalize qpcr data in different tissues may induce significant experimental errors that could result in inappropriate biological data interpretation. recognizing the importance of reference gene in normalization of qpcr data, various housekeeping genes have been evaluated for stable expression under specific conditions in various organisms. in plants, only a few of them have been investigated in some detail in rice, poplar, potato, soybean and arabidopsis thaliana. so far, suitable internal controls for gene expression studies have not been defined for coffea arabica. coffee is an agricultural crop of significant economic importance. coffea arabica l. is typical of the highland growing regions and is responsible for almost 75% of world production. in this study, we report the validation of housekeeping genes to identify the most suitable internal reference gene for normalization of qpcr data obtained among five different tissues/organs of c. arabica. to further validate our results, we evaluated the expression levels of our best reference genes at different developmental stages of flowers and cherries and under a specific biotic stress. following the current literature, five candidate reference genes, namely alcohol dehydrogenase, polyubiquitin, 14-3- β-actin and glyceraldehyde-3-phosphate dehydrogenase, were selected. in addition to these commonly used internal controls, three other genes coding for a cysteine proteinase, a caffeine synthase and the 60s ribosomal protein l, respectively, were included in this analysis. these potential reference genes were ranked according to their expression profiles and stability.  the expression profile of eight candidate reference genes was firstly assessed by qpcr over a panel of five coffee tissue/organ samples. descriptive analysis of the reference candidate genes descriptive statistics of the derived crossing points, based on bestkeeper program, were calculated to investigate the variation level of each candidate gene following pfaffl et al.. according to this analysis, the gene with lowest expression level was actin, for which cp values were obtained around cycles 31–34; while the highest was gapdh, whose cp values were obtained around cycles 21– the expression levels of 14-3- ccs, gapdh, and rpl presented fluctuations of approximately ± x-fold, whereas poly expression showed higher ranges of cp variation as well as up- down-regulation. the coefficient of variation of the assay was %, which is within the range of previously reported values for qpcr. abbreviations: n: number of samples; cp: crossing-point; gm: geometric cp mean; am: arithmetic cp mean; min and max: cp threshold values; sd: cp standard deviation; cv: variance coefficient expressed as percentage of cp level; min and max: threshold expression levels expressed as absolute x-fold over- or under-regulation coefficient; sd: standard deviation of absolute regulation coefficient. descriptive statistics for the expression analyses of each reference candidate gene in the five distinct coffee plant tissue/organ types was also obtained. according to this analysis, the gene that exhibit the minor gene expression variation among the analyzed tissues was gapdh, while the gene with major variation was ccs. in addition, ccs also presented the highest expression variation in flowers and fruits tissue samples, and therefore it cannot be used as a reference gene. numerous studies have shown that the expression of housekeeping genes can vary under given situations. this may partly be explained by the fact that housekeeping genes are not only implicated in the basal cell metabolism but also participate in other cellular functions. a model-based approach for estimation of expression variation the model-based variance estimation approach, a visual basic application for microsoft excel, was used to evaluate expression stability of reference candidate genes. this analysis allowed the ranking of candidate genes since the estimated variation directly indicates the introduced error associated with their use. according to this method, the gene with minimal estimated intra- and intertissue variation was gapdh while the gene with the maximal variation was poly, thus corroborating the results obtained in descriptive analysis. ranking the candidate reference genes the relationship between the stability value and the intra- and intertissue expression variations is present in figure this figure clearly demonstrates the distinct specificities of the investigated genes. according to this analysis, the best candidate gene should present the minimal combined inter- and intra-tissue expression variation. consistent with the descriptive analysis and the model-based variance estimation approach, gapdh showed not only the highest expression levels but was also the most stable gene studied. in figure it can be observed that almost all genes presented average of log expression levels near. log difference > or < implies that variability in expression levels is significant, so the gene could be incorrectly used as reference gene for normalization. in this context, the poly gene presents the highest intertissue expression variation. thus, among the tested genes, gapdh, followed by rpl and 14-3- showed the most stable expression over the investigated panel of five distinct coffee tissue/organ types. the candidate genes were also ranked according to their m values using the genorm program. the average expression stabilities of all tested genes were lower than, with 14-3- and actin showing the most stable expression. although actin gene has shown highest stability following genorm analysis, this gene presented the lowest expression profile according the bestkeeper analysis. corroborating the previous analysis, poly remained the least stable gene. as a whole, our analysis indicates that housekeeping genes are differently regulated in different tissues/organs of coffee plants and may exhibit variable expression patterns. the observed differences in gene expression ratios along a comprehensive panel of tissues/organs are consistent with the data presented by barber et al., iskandar et al. and jain et al.. our results also provide evidences that normalizations to the expression level of a single gene in samples from distinct tissue types may induce to errors, thus corroborating previous studies. comparison of the identified best reference genes to published data in order to validate our potential candidate reference genes, the expression stability of these genes under the influence of a specific biotic stress was investigated. in this case, the obtained results were compared to those dealing with similar coffee gene expression analysis but using ubiquitin as a reference gene for normalization. the comparison was conduced by linear regressions analyzes of the cp difference obtained from the assayed expression levels of the tested genes in leaves of c. arabica inoculated, or not, with hemileia vastatrix. the average cp was calculated for each gene and the Δcp was determined for each time-point. as it can be observed in figure the regression lines for 14-3- and gapdh have slopes close to zero, indicating similar expression levels in inoculated and non-inoculated leaves, and reinforcing their use as effective normalization genes. in contrast, the slope value for rpl was significantly different from zero and higher than the one obtained for ubiquitin, thus limiting its use as a normalization factor under biotic stress condition. validation of data results in different developmental stages of flowers and coffee cherries an additional validation step of the expression levels of gapdh, 14-3- rpl and ubiquitin was performed using unpooled tissue samples from flower and cherry developmental series. the employed sample set is given in the additional file in this assay, the highly expressed gene was gapdh followed by ubiquitin, while 14-3- and rpl presented the same mean cp. the comparison of gene contributions is present in figure as already observed, gapdh showed the greatest stability in expression among all coffee tissue/organ samples analyzed, while expression of rpl and 14-3- varied the most, especially in flowers at stage of development. as mentioned earlier, ubiquitin was used as a standard reference gene for comparison. general remarks about the selected reference genes according to these results, the gene encoding glyceraldeyde-3-phosphate dehydrogenase, an enzyme of glycolysis, outperformed all other reference genes tested and should therefore be considered a suitable reference gene for expression studies in arabica coffee plants. this observation corroborates the quantification of gapdh expression in different tissues of sugarcane and eucalyptus. in contrast, several reports in human and animal systems have suggested that this reference gene has limitations for its use as internal control due to its marked variability of expression among tissue types. the assayed 14-3- gene also showed a stable expression that was supported by the descriptive analysis and confirmed in the biotic stress assay. however, its expression presented some variation among different stages of coffee flower development. papini-terzi et al. recommended 14-3- as a suitable reference gene for expression normalization in a wide range of tissue samples of sugarcane. the gene encoding the transcription regulator and structural constituent of the 60s subunit of the cytosolic ribosome could also be used as an internal control in gene expression studies in c. arabica, due to its stability and acceptable variation among tissue/organ types. our results are in agreement with previous published data for this gene since small variation among tissue types was detected by descriptive analysis. nevertheless, in leaves of c. arabica inoculated with h. vastatrix, it was observed that the expression ratio of rpl was not constant and the absolute value of rpl linear regression slope was superior to that observed for a commonly used coffee normalization gene. in addition, this gene, like 14-3- presented a variable expression level among different stages of coffee flower development. in sugarcane, the relative expression of rpl35- a gene coding for the ribosomal l35- 60s protein, was also reported to be stable. these authors estimated the sugarcane leaf transcriptome using serial analysis of gene expression and reported that tag associated with the rpl35- transcript presented minimum variation among the analyzed sage libraries. the remaining tested genes showed to be unsuitable as internal controls for normalization purposes in c. arabica.  this study provides the most extensive collection of arabica coffee tissue/organ mrna expression data for eight reference candidate genes. our analysis evidenced stable levels of gapdh, 14-3- and rpl mrna in different coffea arabica tissue/organ types. consequently, these genes can be used for accurate and reliable normalization in future gene expression studies in coffee. in this respect, we suggest gapdh as the most relevant reference gene for accurate normalization purposes in c. arabica, showing almost constant expression levels in the investigated experimental set-up. moreover, we have shown that depending on the reference inner control gene, the within-tissue variation of mrna expression levels is generally small, whereas among tissues/organs the variation can be substantial. this indicates that normalizations to a single gene across different tissue types are unwise. since the variation observed between normal tissues of different types may in part be due to the different metabolic demands of those tissues, comparisons within a tissue type between normal and diseased states are similarly unwise. 
the aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. in addition to these commonly used internal controls, three other genes encoding a cysteine proteinase, a caffeine synthase and the 60s ribosomal protein l were also tested. the obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions. based on our results, gapdh, followed by 14-3- and rpl were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. the expression levels of five frequently used housekeeping genes, namely alcohol dehydrogenase, 14-3- polyubiquitin, β-actin and glyceraldehyde-3-phosphate dehydrogenase was assessed by quantitative real-time rt-pcr over a set of five tissue/organ samples of coffea arabica plants. to date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including coffea arabica. gapdh is therefore the recommended reference gene for measuring gene expression in coffea arabica. quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant. we also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress. their stability and suitability as reference genes were validated by genorm, normfinder and bestkeeper programs.
 quantitative data from gene expression experiments are often normalized by transcription levels of reference or housekeeping genes. an inherent assumption for their use is that the expression of these genes is highly uniform in living organisms during various phases of development, in different cell types and under diverse environmental conditions. to date, the validation of reference genes in plants has received very little attention and suitable reference genes have not been defined for a great number of crop species including coffea arabica. the aim of the research reported herein was to compare the relative expression of a set of potential reference genes across different types of tissue/organ samples of coffee. we also validated the expression profiles of the selected reference genes at various stages of development and under a specific biotic stress.  the expression levels of five frequently used housekeeping genes, namely alcohol dehydrogenase, 14-3- polyubiquitin, β-actin and glyceraldehyde-3-phosphate dehydrogenase was assessed by quantitative real-time rt-pcr over a set of five tissue/organ samples of coffea arabica plants. in addition to these commonly used internal controls, three other genes encoding a cysteine proteinase, a caffeine synthase and the 60s ribosomal protein l were also tested. their stability and suitability as reference genes were validated by genorm, normfinder and bestkeeper programs. the obtained results revealed significantly variable expression levels of all reference genes analyzed, with the exception of gapdh, which showed no significant changes in expression among the investigated experimental conditions.  our data suggests that the expression of housekeeping genes is not completely stable in coffee. based on our results, gapdh, followed by 14-3- and rpl were found to be homogeneously expressed and are therefore adequate for normalization purposes, showing equivalent transcript levels in different tissue/organ samples. gapdh is therefore the recommended reference gene for measuring gene expression in coffea arabica. its use will enable more accurate and reliable normalization of tissue/organ-specific gene expression studies in this important cherry crop plant. 