BACKGROUND
recent research has shown that genetic components in gene transcription as well as genetic variability in coding sequences are of evolutionary importance  <cit> .
quantitative reverse transcription polymerase chain reaction  is a technique for the quantification of gene expression at the mrna level that combines the advantages of specificity, sensitivity, speed, throughput and reproducibility.
therefore it is a powerful tool in experimental research  <cit> .
the mrna level itself is not only influenced by regulation of gene expression.
many other conditions, e.g.
nutrition, differences in size and components of the tissue, can influence the mrna level of the target gene.
therefore it is a straightforward solution to measure the mrna of the target gene relative to a housekeeping gene  that is supposed to reflect the status of the fish or tissue and not to be regulated in the selected treatments  <cit> .
in the majority of recent studies either the genes coding for beta-actin , 18s rrna  or glyceraldehyd-3-phosphate dehydrogenase  have been used as reference genes  <cit> .
in the case of the three-spined stickleback, gasterosteus aculeatus, actb was used as the reference gene of choice  <cit> , and until now no other genes have been tested for expression stability and compared with actb in this species.
recent studies in teleost fish and other taxa  <cit>  suggest that genes cannot be used as a reference gene per se without testing their expression stability under the conditions of the desired experiment.
furthermore it has been found that the most stably expressed gene is not necessarily the same in different organs  <cit> .
therefore the main purpose of this study was to test diverse genes for their suitability as reference genes in different tissues and under different immunological challenges  <cit> .
the choice of a suitable reference gene can be a circular problem, because the expression data of this target gene itself also needs to be standardised.
one possible solution is to determine the most stable candidate in a group of several genes  <cit> .
several procedures for this have been suggested, but have rarely been compared.
here we compared  <dig> different algorithms, implemented in the programs genorm  <cit> , bestkeeper  <cit>  and normfinder  <cit> .
we discuss differences in results, taking into account the different approaches to identify the most stably expressed reference gene.
aim of the present study is to identify reference genes that can be used for expression analysis of immune-relevant genes during an immunological challenge.
therefore our study focused on three immunologically active organs of g. aculeatus.
first, the head kidney, a major primary and secondary lymphoid organ in teleosts, functioning as a hematopoetic tissue as well as a site for antigen presentation and cell differentiation  <cit> .
second, the spleen, a major secondary lymphoid organ of teleosts, with essential functions in blood filtering, antigen trapping and processing  <cit> .
third, the gills were chosen.
the gill tissue is in the first line of defence against suspended pathogens in the aquatic environment and therefore is infiltrated with immune cells  <cit> .
for example, wegner et al.
<cit>  found very high rates of mhc  expression in the gills.
we analysed also the gene expression of leukocytes isolated from head kidney and spleen from both a control group and parasite-infected sticklebacks.
for this, we used an in vitro challenge with parasite antigens and pokeweed mitogen  as a positive control  <cit> .
most of the candidate reference genes had not been sequenced before in g. aculeatus.
thus, homologous genes in other teleostei were used to find the sequences of the genes in the three-spined stickleback.
the homologous genes were aligned with an expressed sequence tag  library and a whole genome shotgun  library of the three-spined stickleback.
then est and wgs sequences were aligned to determine exon-intron boundaries for the design of primers that span these regions in the transcripts.
in this study we present a method for the reliable measurement of gene expression in g. aculeatus.
RESULTS
evaluation of pcr conditions and primers
all primer pairs were tested in pcrs with cdna and gdna.
they were supposed to amplify a single product with a cdna template, but not to amplify a gdna template.
all primers tested did not amplify gdna except the primers that target 18s rrna .
the reason for this is that no exon-intron boundaries are present in this target sequence.
consequently, the 18s rrna primers amplify both cdna and gdna.
the application of 18s rrna primers to lysates from no-reverse-transcription assays  showed that the dnase digestion during mrna extraction was not always satisfactory .
therefore some of the samples contained cdna and gdna during pcrs.
furthermore the ppia and the g6pd gene  were excluded from further analysis, because no product was amplified with primers for these genes during pcr .
in order to optimise specificity and efficiency, pcr was tested with various concentrations of our new primer sets, and also a variety of annealing temperatures .
after optimisation of pcr conditions, amplification products from cdna were sequenced and checked for their identity in genbank.
this showed that the  <dig> primer sets that led to amplicon accumulation were stickleback genes.
a dissociation analysis was performed after each pcr run with cdna as a template, in order to show that each primer set amplified the expected single product .
selection of housekeeping genes
there is no reason to expect a single gene to be the most stably expressed hkg in all tissues  <cit> .
therefore, data for every organ and every experiment were tested separately, while the data of the different treatments were combined to determine an appropriate reference gene for our study.
even though the three programmes we used presented different orders of the four most stably expressed genes  in cultures of spleen cells , these four selected genes are recognised as stably expressed by all three algorithms used .
the results suggest that all the other genes are less suitable as reference genes.
therefore one of the four selected genes or a combination of them should preferably be chosen as reference genes in cell cultures of spleens.
in cultures of head kidney cells the same four genes were selected to be the most stably expressed genes in these experiments .
there was, however, more disagreement between the programmes concerning the ranking of the reference gene candidates.
in whole organs, the differences between the three approaches were more pronounced than in cell cultures.
nevertheless some general observations were made.
two genes, ubc and rpl13a, were ranked first by at least one of the programmes in each of the three organs.
if the experiment is restricted to one of the organs, other candidate genes can be considered as well.
in spleens  eef1a seems to be a reliable candidate gene, while b2m might be a good alternative in head kidneys .
furthermore, hprt <dig> was ranked second by the programme normfinder and third by genorm in gills .
we encountered failure of single pcr assay series , and we assume that this was related to the accidental omission of primers and/or pipetting errors.
hence, we decided not to include the genes gapd and actb in the analysis of whole head kidney organs, and actb in the analysis of the gills.
this was necessary to run the algorithms with sufficient sample numbers, because normfinder and genorm require complete data sets, i.e.
data from all genes and from the same pcr run.
the exclusion of single genes seemed to be justified, because none of them was one of the top candidate genes when taking into consideration the ranking after using the reduced data set: head kidneys: normfinder: actb, rank 9; gapd, 3; genorm: actb, rank 9; gapd, 3; bestkeeper: actb rank 8; gapd  <dig>  results from gills: normfinder: actb, rank 8; genorm: actb rank8; bestkeeper rank  <dig>  .
nf = normfinder; gn = genorm; bk = bestkeeper
nf = normfinder; gn = genorm; bk = bestkeeper
nf = normfinder; gn = genorm; bk = bestkeeper
nf = normfinder; gn = genorm; bk = bestkeeper
nf = normfinder; gn = genorm; bk = bestkeeper
discussion
primer design and pcr
the pcr primer design for the housekeeping genes  evaluated in this study faced a general problem.
we had to design primers using sequences from north american sticklebacks in order to amplify the cdna of sticklebacks from our local populations in germany.
expected sequence differences between the north american and the local stickleback may lead to two problems due to mismatches between primer and target sequence.
first, the pcr efficiencies in our assays may differ between target genes.
this, however, is monitored with the algorithms used in this study, and thus taken into consideration in all calculations presented.
second, total failure of pcrs may occur.
this may explain why it was not possible to amplify all candidate genes of the local stickleback population.
additional problems may arise from the contamination of the cdna template with genomic dna.
the minus rt controls of all samples evaluated in this study showed indeed that the dnase treatment of total rna did not eliminate the gdna in every sample.
hence, in some minus rt control qpcr assays we observed a cp value, which means that a significant pcr signal arose from gdna contamination, despite the fact that our primers had a high melting temperature and were designed to span exon-intron boundaries in the transcripts.
due to the comparison of the cp values between minus rt control qpcr assays and those assays with cdna as a template , we can estimate that the bias introduced by gdna is a maximum of 3% .
therefore primers that are not designed across intron/exon boundaries, and thus amplify gdna, cannot be used to measure gene expression in our pcr assays.
hence, the 18s rrna without introns was not a suitable reference gene in our study per se.
the primer concentration in the pcr runs was very high .
this was supposed to outweigh the high annealing/extension temperature of the pcr, because the assay was designed towards specificity rather than efficiency .
the sensitivity of our pcr assays was demonstrated by the fact that cdna from  <dig> cells was sufficient to measure the expression of  <dig> genes in parallel pcr runs.
the additional purification of mrna from total rna was not necessary before reverse-transcription.
hence, the possible loss of mrna during this additional step did not outweigh the increased efficiency of the following pcrs .
it has already been shown that pcr can be inhibited by the enzyme reverse transcriptase, if unpurified cdna is used as a template  <cit> .
therefore we used a precipitation step to purify the reverse-transcribed cdna.
this also led to improved pcr efficiency .
after completion of the thermal programme, all rt-pcrs in this study were subjected to a dissociation analysis to check the identity  of the pcr products.
an example for the ten different target gene pcr products from a single cdna sample is shown in figure  <dig>  assays that were not dominated by a single pcr product were excluded from further algorithm analysis.
initially, during optimisation pcr conditions for this study, the pcr products of the new primer pairs were sequenced and checked in genbank to verify the identity of the target genes.
even though we cannot exclude the amplification of isotypic variants of single genes, our method of gene expression quantification is reliable, because the dissociation analysis would detect high amounts of by-products.
housekeeping genes
in this study, we evaluated candidate genes for quantitative real-time pcr assays for immune response gene expression studies in g. aculeatus.
unfortunately, there is no universal reference gene that is stably expressed in all tissues and under all biological conditions  <cit> .
it is therefore necessary to ensure that no significant regulation occurs during an immunological challenge before a gene can be chosen as standard for relative expression analysis.
the potential reference gene should also be stably expressed in tissues with different proportions of lymphocytes and granulocytes as this usually is the case in spleens of natural populations.
therefore we did not separate the different cell types.
additionally, the chosen gene itself has to be standardised, hence there is a circular problem that needs to be solved.
one possible solution is to take more than one gene as the target gene.
pfaffl's solution  <cit>  is to calculate a bestkeeper's index, which is the geometric mean of those genes, that are expressed with a standard deviation  lower than  <dig>  this condition, however, may be too strict, because hardly any of the genes in our cell culture experiments were expressed with a sd lower than  <dig>  in the infection experiments, the sd of the candidate genes was by far too high to include any of them in the calculation of the bestkeeper's index.
all other sources of variation, e.g.
amount of template, would have to be highly controlled.
in the infection experiments, the expression was measured with total rna from whole organs.
this led to a sd too high to calculate a bestkeeper's index according to pfaffl's conditions.
bestkeeper's prerequisites seem to require a very limited experimental setup.
therefore, this algorithm can only be used under limited conditions, whereas a suitable reference gene should be stably expressed under a variety of conditions.
furthermore the quality of each reference candidate gene is determined only by the standard deviation of its expression in different samples.
hence, bestkeeper cannot solve the circular problem outlined above.
the software genorm  <cit>  suffers from a different problem.
in this approach the stability of a candidate gene is determined by pair-wise comparison of variation of expression ratios.
therefore the quality of a reference gene depends on the set of candidate genes that are included in the analysis.
the genorm calculation is only reliable if either most of the other candidate genes are already known to be stably expressed or the candidate genes are not co-regulated.
instead of the most stably expressed gene, genorm tends to select the gene with the highest degree of similarity to the expression pattern of candidate genes in the whole data set.
it has been shown that the elimination of genes from the analysis changed the ranking of the candidate genes  <cit> .
in this study we made similar observations .
the normfinder software  <cit>  combines the advantages of the two other approaches by an estimation of both the intra- and the inter-group expression variation.
normfinder directly and robustly evaluates gene expression stability  <cit> .
it should therefore be preferred to the other two approaches evaluated in this study.
in cell cultures, rpl13a and gapd seem to be the best reference genes .
the other two approaches did not give any reason to exclude rpl <dig> and gapd as the best reference gene under these circumstances.
actb and ubc were also good candidates that should be taken into account when more than one reference gene is desired or required.
in the infection experiments, results were less straightforward.
in spleens  normfinder suggested ubc as the most stable gene.
taking into account the results of all approaches, rpl13a is as stable as ubc, therefore these two genes should be equally suitable as reference genes.
bestkeeper and genorm preferred ubc and rpl13a as the gene of choice for head kidneys , but normfinder suggested that ubc should be preferred.
in the gills  ubc and rpl13a were the best pair of reference genes.
the hprt <dig> may be used as a reference gene in gills and head kidneys as well.
CONCLUSIONS
the study highlights the necessity of pilot studies for selecting the best reference gene in gene expression analysis.
furthermore we can confirm recent studies that question the status of the gene for beta-actin  as a general reference gene.
obviously there is no single best gene for all experiments and tissues, even though there seem to be some candidate genes that are stably expressed in a variety of assays and tissues.
our results showed that in most of the tissues and treatments hkg, which could not be used so far due to unknown sequences, proved to be more stably expressed than the gene for beta-actin.
as they were the most stably expressed genes in all tissues examined, we suggest using the genes of the l13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analyses in g. aculeatus.

 recent research has shown that genetic components in gene transcription as well as genetic variability in coding sequences are of evolutionary importance. quantitative reverse transcription polymerase chain reaction is a technique for the quantification of gene expression at the mrna level that combines the advantages of specificity, sensitivity, speed, throughput and reproducibility. therefore it is a powerful tool in experimental research. the mrna level itself is not only influenced by regulation of gene expression. many other conditions, e.g. nutrition, differences in size and components of the tissue, can influence the mrna level of the target gene. therefore it is a straightforward solution to measure the mrna of the target gene relative to a housekeeping gene that is supposed to reflect the status of the fish or tissue and not to be regulated in the selected treatments. in the majority of recent studies either the genes coding for beta-actin, 18s rrna or glyceraldehyd-3-phosphate dehydrogenase have been used as reference genes. in the case of the three-spined stickleback, gasterosteus aculeatus, actb was used as the reference gene of choice, and until now no other genes have been tested for expression stability and compared with actb in this species. recent studies in teleost fish and other taxa suggest that genes cannot be used as a reference gene per se without testing their expression stability under the conditions of the desired experiment. furthermore it has been found that the most stably expressed gene is not necessarily the same in different organs. therefore the main purpose of this study was to test diverse genes for their suitability as reference genes in different tissues and under different immunological challenges. the choice of a suitable reference gene can be a circular problem, because the expression data of this target gene itself also needs to be standardised. one possible solution is to determine the most stable candidate in a group of several genes. several procedures for this have been suggested, but have rarely been compared. here we compared different algorithms, implemented in the programs genorm, bestkeeper and normfinder. we discuss differences in results, taking into account the different approaches to identify the most stably expressed reference gene. aim of the present study is to identify reference genes that can be used for expression analysis of immune-relevant genes during an immunological challenge. therefore our study focused on three immunologically active organs of g. aculeatus. first, the head kidney, a major primary and secondary lymphoid organ in teleosts, functioning as a hematopoetic tissue as well as a site for antigen presentation and cell differentiation. second, the spleen, a major secondary lymphoid organ of teleosts, with essential functions in blood filtering, antigen trapping and processing. third, the gills were chosen. the gill tissue is in the first line of defence against suspended pathogens in the aquatic environment and therefore is infiltrated with immune cells. for example, wegner et al. found very high rates of mhc expression in the gills. we analysed also the gene expression of leukocytes isolated from head kidney and spleen from both a control group and parasite-infected sticklebacks. for this, we used an in vitro challenge with parasite antigens and pokeweed mitogen as a positive control. most of the candidate reference genes had not been sequenced before in g. aculeatus. thus, homologous genes in other teleostei were used to find the sequences of the genes in the three-spined stickleback. the homologous genes were aligned with an expressed sequence tag library and a whole genome shotgun library of the three-spined stickleback. then est and wgs sequences were aligned to determine exon-intron boundaries for the design of primers that span these regions in the transcripts. in this study we present a method for the reliable measurement of gene expression in g. aculeatus.  evaluation of pcr conditions and primers all primer pairs were tested in pcrs with cdna and gdna. they were supposed to amplify a single product with a cdna template, but not to amplify a gdna template. all primers tested did not amplify gdna except the primers that target 18s rrna. the reason for this is that no exon-intron boundaries are present in this target sequence. consequently, the 18s rrna primers amplify both cdna and gdna. the application of 18s rrna primers to lysates from no-reverse-transcription assays showed that the dnase digestion during mrna extraction was not always satisfactory. therefore some of the samples contained cdna and gdna during pcrs. furthermore the ppia and the g6pd gene were excluded from further analysis, because no product was amplified with primers for these genes during pcr. in order to optimise specificity and efficiency, pcr was tested with various concentrations of our new primer sets, and also a variety of annealing temperatures. after optimisation of pcr conditions, amplification products from cdna were sequenced and checked for their identity in genbank. this showed that the primer sets that led to amplicon accumulation were stickleback genes. a dissociation analysis was performed after each pcr run with cdna as a template, in order to show that each primer set amplified the expected single product. selection of housekeeping genes there is no reason to expect a single gene to be the most stably expressed hkg in all tissues. therefore, data for every organ and every experiment were tested separately, while the data of the different treatments were combined to determine an appropriate reference gene for our study. even though the three programmes we used presented different orders of the four most stably expressed genes in cultures of spleen cells, these four selected genes are recognised as stably expressed by all three algorithms used. the results suggest that all the other genes are less suitable as reference genes. therefore one of the four selected genes or a combination of them should preferably be chosen as reference genes in cell cultures of spleens. in cultures of head kidney cells the same four genes were selected to be the most stably expressed genes in these experiments. there was, however, more disagreement between the programmes concerning the ranking of the reference gene candidates. in whole organs, the differences between the three approaches were more pronounced than in cell cultures. nevertheless some general observations were made. two genes, ubc and rpl13a, were ranked first by at least one of the programmes in each of the three organs. if the experiment is restricted to one of the organs, other candidate genes can be considered as well. in spleens eef1a seems to be a reliable candidate gene, while b2m might be a good alternative in head kidneys. furthermore, hprt was ranked second by the programme normfinder and third by genorm in gills. we encountered failure of single pcr assay series, and we assume that this was related to the accidental omission of primers and/or pipetting errors. hence, we decided not to include the genes gapd and actb in the analysis of whole head kidney organs, and actb in the analysis of the gills. this was necessary to run the algorithms with sufficient sample numbers, because normfinder and genorm require complete data sets, i.e. data from all genes and from the same pcr run. the exclusion of single genes seemed to be justified, because none of them was one of the top candidate genes when taking into consideration the ranking after using the reduced data set: head kidneys: normfinder: actb, rank 9; gapd, 3; genorm: actb, rank 9; gapd, 3; bestkeeper: actb rank 8; gapd results from gills: normfinder: actb, rank 8; genorm: actb rank8; bestkeeper rank. nf = normfinder; gn = genorm; bk = bestkeeper nf = normfinder; gn = genorm; bk = bestkeeper nf = normfinder; gn = genorm; bk = bestkeeper nf = normfinder; gn = genorm; bk = bestkeeper nf = normfinder; gn = genorm; bk = bestkeeper discussion primer design and pcr the pcr primer design for the housekeeping genes evaluated in this study faced a general problem. we had to design primers using sequences from north american sticklebacks in order to amplify the cdna of sticklebacks from our local populations in germany. expected sequence differences between the north american and the local stickleback may lead to two problems due to mismatches between primer and target sequence. first, the pcr efficiencies in our assays may differ between target genes. this, however, is monitored with the algorithms used in this study, and thus taken into consideration in all calculations presented. second, total failure of pcrs may occur. this may explain why it was not possible to amplify all candidate genes of the local stickleback population. additional problems may arise from the contamination of the cdna template with genomic dna. the minus rt controls of all samples evaluated in this study showed indeed that the dnase treatment of total rna did not eliminate the gdna in every sample. hence, in some minus rt control qpcr assays we observed a cp value, which means that a significant pcr signal arose from gdna contamination, despite the fact that our primers had a high melting temperature and were designed to span exon-intron boundaries in the transcripts. due to the comparison of the cp values between minus rt control qpcr assays and those assays with cdna as a template, we can estimate that the bias introduced by gdna is a maximum of 3%. therefore primers that are not designed across intron/exon boundaries, and thus amplify gdna, cannot be used to measure gene expression in our pcr assays. hence, the 18s rrna without introns was not a suitable reference gene in our study per se. the primer concentration in the pcr runs was very high. this was supposed to outweigh the high annealing/extension temperature of the pcr, because the assay was designed towards specificity rather than efficiency. the sensitivity of our pcr assays was demonstrated by the fact that cdna from cells was sufficient to measure the expression of genes in parallel pcr runs. the additional purification of mrna from total rna was not necessary before reverse-transcription. hence, the possible loss of mrna during this additional step did not outweigh the increased efficiency of the following pcrs. it has already been shown that pcr can be inhibited by the enzyme reverse transcriptase, if unpurified cdna is used as a template. therefore we used a precipitation step to purify the reverse-transcribed cdna. this also led to improved pcr efficiency. after completion of the thermal programme, all rt-pcrs in this study were subjected to a dissociation analysis to check the identity of the pcr products. an example for the ten different target gene pcr products from a single cdna sample is shown in figure assays that were not dominated by a single pcr product were excluded from further algorithm analysis. initially, during optimisation pcr conditions for this study, the pcr products of the new primer pairs were sequenced and checked in genbank to verify the identity of the target genes. even though we cannot exclude the amplification of isotypic variants of single genes, our method of gene expression quantification is reliable, because the dissociation analysis would detect high amounts of by-products. housekeeping genes in this study, we evaluated candidate genes for quantitative real-time pcr assays for immune response gene expression studies in g. aculeatus. unfortunately, there is no universal reference gene that is stably expressed in all tissues and under all biological conditions. it is therefore necessary to ensure that no significant regulation occurs during an immunological challenge before a gene can be chosen as standard for relative expression analysis. the potential reference gene should also be stably expressed in tissues with different proportions of lymphocytes and granulocytes as this usually is the case in spleens of natural populations. therefore we did not separate the different cell types. additionally, the chosen gene itself has to be standardised, hence there is a circular problem that needs to be solved. one possible solution is to take more than one gene as the target gene. pfaffl's solution is to calculate a bestkeeper's index, which is the geometric mean of those genes, that are expressed with a standard deviation lower than this condition, however, may be too strict, because hardly any of the genes in our cell culture experiments were expressed with a sd lower than in the infection experiments, the sd of the candidate genes was by far too high to include any of them in the calculation of the bestkeeper's index. all other sources of variation, e.g. amount of template, would have to be highly controlled. in the infection experiments, the expression was measured with total rna from whole organs. this led to a sd too high to calculate a bestkeeper's index according to pfaffl's conditions. bestkeeper's prerequisites seem to require a very limited experimental setup. therefore, this algorithm can only be used under limited conditions, whereas a suitable reference gene should be stably expressed under a variety of conditions. furthermore the quality of each reference candidate gene is determined only by the standard deviation of its expression in different samples. hence, bestkeeper cannot solve the circular problem outlined above. the software genorm suffers from a different problem. in this approach the stability of a candidate gene is determined by pair-wise comparison of variation of expression ratios. therefore the quality of a reference gene depends on the set of candidate genes that are included in the analysis. the genorm calculation is only reliable if either most of the other candidate genes are already known to be stably expressed or the candidate genes are not co-regulated. instead of the most stably expressed gene, genorm tends to select the gene with the highest degree of similarity to the expression pattern of candidate genes in the whole data set. it has been shown that the elimination of genes from the analysis changed the ranking of the candidate genes. in this study we made similar observations. the normfinder software combines the advantages of the two other approaches by an estimation of both the intra- and the inter-group expression variation. normfinder directly and robustly evaluates gene expression stability. it should therefore be preferred to the other two approaches evaluated in this study. in cell cultures, rpl13a and gapd seem to be the best reference genes. the other two approaches did not give any reason to exclude rpl and gapd as the best reference gene under these circumstances. actb and ubc were also good candidates that should be taken into account when more than one reference gene is desired or required. in the infection experiments, results were less straightforward. in spleens normfinder suggested ubc as the most stable gene. taking into account the results of all approaches, rpl13a is as stable as ubc, therefore these two genes should be equally suitable as reference genes. bestkeeper and genorm preferred ubc and rpl13a as the gene of choice for head kidneys, but normfinder suggested that ubc should be preferred. in the gills ubc and rpl13a were the best pair of reference genes. the hprt may be used as a reference gene in gills and head kidneys as well.  the study highlights the necessity of pilot studies for selecting the best reference gene in gene expression analysis. furthermore we can confirm recent studies that question the status of the gene for beta-actin as a general reference gene. obviously there is no single best gene for all experiments and tissues, even though there seem to be some candidate genes that are stably expressed in a variety of assays and tissues. our results showed that in most of the tissues and treatments hkg, which could not be used so far due to unknown sequences, proved to be more stably expressed than the gene for beta-actin. as they were the most stably expressed genes in all tissues examined, we suggest using the genes of the l13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analyses in g. aculeatus. 
the expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms bestkeeper, genorm and normfinder. our results showed that in most of the tissues and treatments hkg that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin. in this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene, depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. as they were the most stably expressed genes in all tissues examined, we suggest using the genes for the l13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in gasterosteus aculeatus. in the three-spined stickleback, gasterosteus aculeatus, other hkg than the one for beta-actin have not been established so far. to establish a reliable method for the measurement of immune gene expression in gasterosteus aculeatus, sequences from the now available genome database and an est library of the same species were used to select oligonucleotide primers for hkg, in order to perform quantitative reverse-transcription pcr.
 during the last years the quantification of immune response under immunological challenges, e.g. parasitation, has been a major focus of research. in this context, the expression of immune response genes in teleost fish has been surveyed for scientific and commercial purposes. despite the fact that it was shown in teleostei and other taxa that the gene for beta-actin is not the most stably expressed housekeeping gene, depending on the tissue and experimental treatment, the gene has been used as a reference gene in such studies. in the three-spined stickleback, gasterosteus aculeatus, other hkg than the one for beta-actin have not been established so far.  to establish a reliable method for the measurement of immune gene expression in gasterosteus aculeatus, sequences from the now available genome database and an est library of the same species were used to select oligonucleotide primers for hkg, in order to perform quantitative reverse-transcription pcr. the expression stability of ten candidate reference genes was evaluated in three different tissues, and in five parasite treatment groups, using the three algorithms bestkeeper, genorm and normfinder. our results showed that in most of the tissues and treatments hkg that could not be used so far due to unknown sequences, proved to be more stably expressed than the one for beta-actin.  as they were the most stably expressed genes in all tissues examined, we suggest using the genes for the l13a ribosomal binding protein and ubiquitin as alternative or additional reference genes in expression analysis in gasterosteus aculeatus. 