BACKGROUND
quantitative real-time reverse transcription pcr  is an extremely sensitive technique that allows the precise measurement of gene expression across more than seven orders of magnitude <cit> .
rt-pcr is often considered the gold standard for quantifying gene expression, and is commonly used to validate techniques with greater throughput but less overall sensitivity, such as microarray analysis  <cit> .
rt-pcr relies on the use of fluorescent dyes to quantify transcript amplification, with the amplification cycle number at which these dyes/transcripts are detected  giving an indication as to the relative abundance of the target molecules.
the sensitivity of rt-pcr makes it a powerful tool for gene expression measurement, especially when sample quantities are limited or a transcript is expressed at a low level.
however, this sensitivity also means that a great deal of care must be taken with regards to experimental design and implementation of the procedure.
when designing an experiment to evaluate gene expression in a group of samples, a number of critical factors must be kept constant.
these include rna extraction, dnase treatments, and cdna synthesis.
normalisation of rt-pcr results is required to control inter-sample differences that may arise as a result of these sample processing steps, and ensure the gene expression of target transcripts are robustly quantifiable  <cit> .
the most common method for normalising rt-pcr data involves the use of one or more endogenous control genes.
an ideal endogenous control gene is one that is stably expressed within the samples to be compared, regardless of tissue differences, experimental conditions, or treatments.
choosing an endogenous control gene to normalise gene expression data is one of the most crucial steps in the experimental design.
genes used as endogenous controls in rt-pcr experiments are often chosen with little prior knowledge of their expression over the experimental conditions examined, and are often selected arbitrarily from a pool of commonly used endogenous control genes such as gapdh, and β-actin  <cit> .
the most widely used endogenous control gene in studies of endometrial gene expression is gapdh  <cit> .
however, the suitability of gapdh as an endogenous control gene has recently come into question, especially due to its potential regulation in a wide variety of physiological states  <cit> , making it a questionable choice for rt-pcr normalisation  <cit> .
over the past three decades, genetic selection for milk production has resulted in a significant decline in dairy cattle fertility  <cit> .
the fertilisation rate in dairy cattle is around 90% and does not differ between low-moderate and high-producing animals.
however, the calving rate in lower producing animals is approximately 55%, whereas in high-producing animals this rate is approximately 35%.
pregnancy losses are thought to occur primarily during the pregnancy recognition/pre-implantation period  <cit> , making studies of endometrial gene expression critical to further understanding of pregnancy establishment, recognition and maintenance within the bovine reproductive cycle.
the primary aim of this study was to identify suitable endogenous control genes for analysis of endometrial tissues from pregnant and cycling bovines.
this study also aimed to investigate the potential use of microarray data analysis for identification of novel endogenous control genes, and the effect of endogenous control gene selection on the calculated expression of a target gene.
a total of  <dig> candidate endogenous control genes were analysed in  <dig> samples representing two different tissues  from  <dig> animals.
these animals were either pregnant or cycling at day  <dig> of the reproductive cycle, and represented holstein-friesian cows from two divergent genetic backgrounds , and new zealand ).
two strategies were employed to identify the candidates.
five genes were selected on the basis that they had been previously used as housekeeping genes  <cit> , and an additional  <dig> novel genes were derived from a microarray experiment based around the same  <dig> samples used in the current analysis.
genespring gx software was used to generate a list through filtering on expression stability across the  <dig> samples.
this list was subjected to genorm  <cit>  and normfinder  <cit>  analysis to identify the  <dig> most suitable genes.
the suitability of all  <dig> genes was then tested through statistical analyses, including a comparison of expression stability as determined by genorm and normfinder algorithms.
the effect of using these endogenous control genes was then evaluated using relative quantification of a gene known to be differentially expressed in the study.
RESULTS
microarray analysis of the  <dig> endometrial samples revealed  <dig> transcripts with a high degree of expression stability .
genorm and normfinder were utilised to identify the  <dig> most stably expressed transcripts for further analysis.
for rt-pcr design, full length transcripts were identified by querying microarray probe sequences against the bovine genome  using ncbi blast .
gene expression levels of the candidate endogenous control genes  are displayed in table  <dig> and figure  <dig>  cq values for sample replicates had very low variability with a mean intra-assay coefficient of variation  of  <dig> %.
all genes had low overall variability, with the cq range between  <dig>  and  <dig>  cycles, standard deviations ranging from  <dig>  to  <dig>  cycles, and cv values ranging from  <dig>  to  <dig> % table  <dig>
significance calculations between gene expression data for pregnant and cycling animals were performed on cq and relative concentration values, as estimated through absolute quantification using the roche lc <dig> software.
no significant differences  in means or variances  between pregnant and cycling endometrial tissues were apparent for any candidate genes  for variance between pregnant and cycling animals).
the means and variances between the two different strains of holstein-friesians also lacked significance .
cqs and relative concentration variances between the two different tissue types showed no significant differences, however means were significantly different for all genes, except rps15a , p =  <dig>  ).
the differences in means calculated between tissue subtypes likely reflect the distinct morphological and functional differences between caruncular and intercaruncular endometrium, which relate to their respective roles in reproduction.
the 'caruncules' of the endometrium are specialised projections that are the site of embryo attachment.
caruncules become highly vascularised, and are the major site for small molecule and gaseous exchange.
in comparison, intercaruncular tissue is highly glandular and responsible for early nourishment of the embryo through secretions of large molecules into the uterus  <cit> .
the intercaruncular tissue is often thought to be more important in early pregnancy and, therefore, the majority of expression studies in pre-implantation bovine endometrium focus solely on intercaruncular tissue gene expression <cit> .
there is very little reported expression analysis of caruncular endometrium and no known studies comparing expression profiles of the two tissues.
expression stability testing of candidate genes
to further analyse the suitability of the candidate genes for use as endogenous controls for bovine endometrial tissues, expression stability was assessed using the genorm  <cit>  and normfinder  <cit>  algorithms.
genorm rankings for the  <dig> genes tested are presented in table  <dig> and figure 2a.
genorm identified suz <dig> and znf <dig> as the most stably expressed genes of the  <dig> candidates.
four of the five most stable genes were those derived from microarray data; the other was rps15a, which was selected from the literature.
by contrast, four of the five least stable genes were chosen from the literature, including gapdh.
the most stably expressed gene identified by normfinder was suz <dig> , which was also one of the two most stable genes identified in the genorm analysis.
the best combination of genes identified by normfinder was suz <dig> and c2orf <dig>  the five most stably expressed genes according to normfinder consisted entirely of microarray-derived genes, while the least stable five contained three out of five genes selected from the literature, again including gapdh.
the comparative ranking of all genes for both genorm and normfinder algorithm analyses is displayed in table  <dig>
normfinder rankings of genes through analysis of inter- and intra group variability .
genorm rankings of genes based on pairwise analysis of expression stability.
genorm calculates an expression stability value  for each candidate gene based on pairwise comparisons of variability.
each gene is compared to every other gene to determine the two genes that contain the least variation.
the stability value calculated for each gene is used to rank genes from least to most stable.
the authors of the method give an m-value of  <dig>  as a cut-off for suitability as an endogenous control gene.
the principal behind the pairwise stability measure ranking is that two ideal candidate normalisation genes should have an equal expression ratio in all samples  <cit> .
in the present study, all genes are well below the stipulated  <dig>  m-value.
the program then calculates a normalisation factor in each sample for the most stable genes .
genorm also calculates the optimal number of endogenous control genes to be used in the analysis of gene expression .
this value is determined by locating the point where addition of the next most stable gene does not significantly affect the normalisation factor, using a cut-off value of  <dig> .
in this study, the value for  <dig> genes was  <dig> , suggesting that  <dig> genes should be sufficient to normalise the experimental data.
genorm calculates the minimum number of genes required for accurate normalisation.
in this case, two genes were sufficient .
the number of genes necessary for accurate normalisation is defined as the point at which the addition of a gene has a non-significant effect on the calculation of the normalisation factor, the threshold for a non-significant difference being  <dig> .
normfinder is another freely available tool for the identification of stable endogenous control genes.
the main point of difference between the two methods is that normfinder takes into account both inter- and intra-group variability.
the program not only identifies the most stable pair of endogenous control genes but also identifies the best overall endogenous control gene.
the calculation of variability between groups is especially important in the present study considering the significant expression differences between the two tissue subtypes.
the use of the two most stably expressed genes, in this case suz <dig> and c2orf <dig> , should provide sufficient normalisation for tissue comparisons as normfinder selects the best combination of genes whilst taking into account any grouping effects such as tissue type.
the differences in rankings of gene stability using the two algorithms could be due to the fact that they use very different methods to assess gene stability.
genorm selects genes based on the pairwise variation between genes.
the two most stably expressed genes are therefore those genes that share an expression profile.
in contrast, normfinder uses a model based algorithm that takes into account overall stability as well as any groups that may be present in the sample set.
for example, if there are any grouping effects on gene expression a gene would be ranked lower than one that demonstrated variability not associated with any particular group.
effect of endogenous control gene on target gene relative quantification
temporal down-regulation of endometrial oxytocin receptor  expression is a hallmark of early pregnancy, with embryonic interferon tau  thought to elicit this response  <cit> .
given the expectation of differential oxtr expression between pregnant and cycling animals, the effect of control gene stability on gene expression values of oxtr in the  <dig> endometrial samples was tested.
oxtr expression was significantly greater in cycling than in pregnant cows regardless of the endogenous control gene used .
notably, oxtr expression in pregnant animals was greater on average and more variable in na animals  than for nz animals , and was also related to embryo size .
the use of different endogenous control genes had no effect on oxtr expression differences in these group comparisons.
however, there were differences in the normalised oxtr expression values that were inconsistent across the  <dig> samples, depending on which endogenous control genes were employed.
for example, in sample pregnant nz-5a the calculated relative oxtr expression value was  <dig>  when normalised to suz <dig>   <dig>  when normalised to gapdh,  <dig>  when normalised to uxt, and  <dig>  when normalised to both suz <dig> and znf <dig>  another sample , the values were  <dig> ,  <dig> ,  <dig> , and  <dig>  when normalised to suz <dig>  gapdh, uxt and both suz <dig> and znf <dig>  respectively.
the average calculated fold change difference between pregnant and cycling animals was not affected by the choice of normalisation gene, possibly due to the large difference in expression level for this comparison .
however, the choice of reference gene could be important when normalising genes that exhibit more subtle variation between experimental groups, given the considerable variation in expression shown between individual samples.
CONCLUSIONS
this study provides the first reported assessment of endogenous control genes for use in expression studies in bovine endometrium.
normalisation is a critical factor in reporting rt-pcr expression data, providing a necessary control for error associated with sample preparation.
normalisation using endogenous control genes provides a means of controlling this error, provided the gene used are stably expressed across all samples under investigation.
the study described here tested  <dig> candidate reference genes across  <dig> bovine endometrial samples representing a range of physiological states and tissue subtypes.
this study evaluated the suitability of both commonly used and novel experimentally derived reference genes for use in normalisation of rt-pcr data.
candidates derived via microarray analysis were superior to existing, commonly used endogenous control genes, demonstrating the suitability of using microarray data for deriving novel endogenous control genes.
this study also highlighted the importance of accurate normalisation with a stable endogenous control gene, by demonstrating relative expression of a differentially expressed gene when normalised using control genes of varying stability.
suz <dig> was ranked first for stability across samples as determined by the statistical algorithms used in genorm and normfinder, and is therefore proposed as the best gene for normalisation of rt-pcr data in the current study.

 quantitative real-time reverse transcription pcr is an extremely sensitive technique that allows the precise measurement of gene expression across more than seven orders of magnitude. rt-pcr is often considered the gold standard for quantifying gene expression, and is commonly used to validate techniques with greater throughput but less overall sensitivity, such as microarray analysis. rt-pcr relies on the use of fluorescent dyes to quantify transcript amplification, with the amplification cycle number at which these dyes/transcripts are detected giving an indication as to the relative abundance of the target molecules. the sensitivity of rt-pcr makes it a powerful tool for gene expression measurement, especially when sample quantities are limited or a transcript is expressed at a low level. however, this sensitivity also means that a great deal of care must be taken with regards to experimental design and implementation of the procedure. when designing an experiment to evaluate gene expression in a group of samples, a number of critical factors must be kept constant. these include rna extraction, dnase treatments, and cdna synthesis. normalisation of rt-pcr results is required to control inter-sample differences that may arise as a result of these sample processing steps, and ensure the gene expression of target transcripts are robustly quantifiable. the most common method for normalising rt-pcr data involves the use of one or more endogenous control genes. an ideal endogenous control gene is one that is stably expressed within the samples to be compared, regardless of tissue differences, experimental conditions, or treatments. choosing an endogenous control gene to normalise gene expression data is one of the most crucial steps in the experimental design. genes used as endogenous controls in rt-pcr experiments are often chosen with little prior knowledge of their expression over the experimental conditions examined, and are often selected arbitrarily from a pool of commonly used endogenous control genes such as gapdh, and β-actin. the most widely used endogenous control gene in studies of endometrial gene expression is gapdh. however, the suitability of gapdh as an endogenous control gene has recently come into question, especially due to its potential regulation in a wide variety of physiological states, making it a questionable choice for rt-pcr normalisation. over the past three decades, genetic selection for milk production has resulted in a significant decline in dairy cattle fertility. the fertilisation rate in dairy cattle is around 90% and does not differ between low-moderate and high-producing animals. however, the calving rate in lower producing animals is approximately 55%, whereas in high-producing animals this rate is approximately 35%. pregnancy losses are thought to occur primarily during the pregnancy recognition/pre-implantation period, making studies of endometrial gene expression critical to further understanding of pregnancy establishment, recognition and maintenance within the bovine reproductive cycle. the primary aim of this study was to identify suitable endogenous control genes for analysis of endometrial tissues from pregnant and cycling bovines. this study also aimed to investigate the potential use of microarray data analysis for identification of novel endogenous control genes, and the effect of endogenous control gene selection on the calculated expression of a target gene. a total of candidate endogenous control genes were analysed in samples representing two different tissues from animals. these animals were either pregnant or cycling at day of the reproductive cycle, and represented holstein-friesian cows from two divergent genetic backgrounds, and new zealand ). two strategies were employed to identify the candidates. five genes were selected on the basis that they had been previously used as housekeeping genes, and an additional novel genes were derived from a microarray experiment based around the same samples used in the current analysis. genespring gx software was used to generate a list through filtering on expression stability across the samples. this list was subjected to genorm and normfinder analysis to identify the most suitable genes. the suitability of all genes was then tested through statistical analyses, including a comparison of expression stability as determined by genorm and normfinder algorithms. the effect of using these endogenous control genes was then evaluated using relative quantification of a gene known to be differentially expressed in the study.  microarray analysis of the endometrial samples revealed transcripts with a high degree of expression stability. genorm and normfinder were utilised to identify the most stably expressed transcripts for further analysis. for rt-pcr design, full length transcripts were identified by querying microarray probe sequences against the bovine genome using ncbi blast. gene expression levels of the candidate endogenous control genes are displayed in table and figure cq values for sample replicates had very low variability with a mean intra-assay coefficient of variation of %. all genes had low overall variability, with the cq range between and cycles, standard deviations ranging from to cycles, and cv values ranging from to % table significance calculations between gene expression data for pregnant and cycling animals were performed on cq and relative concentration values, as estimated through absolute quantification using the roche lc software. no significant differences in means or variances between pregnant and cycling endometrial tissues were apparent for any candidate genes for variance between pregnant and cycling animals). the means and variances between the two different strains of holstein-friesians also lacked significance. cqs and relative concentration variances between the two different tissue types showed no significant differences, however means were significantly different for all genes, except rps15a, p = ). the differences in means calculated between tissue subtypes likely reflect the distinct morphological and functional differences between caruncular and intercaruncular endometrium, which relate to their respective roles in reproduction. the'caruncules' of the endometrium are specialised projections that are the site of embryo attachment. caruncules become highly vascularised, and are the major site for small molecule and gaseous exchange. in comparison, intercaruncular tissue is highly glandular and responsible for early nourishment of the embryo through secretions of large molecules into the uterus. the intercaruncular tissue is often thought to be more important in early pregnancy and, therefore, the majority of expression studies in pre-implantation bovine endometrium focus solely on intercaruncular tissue gene expression. there is very little reported expression analysis of caruncular endometrium and no known studies comparing expression profiles of the two tissues. expression stability testing of candidate genes to further analyse the suitability of the candidate genes for use as endogenous controls for bovine endometrial tissues, expression stability was assessed using the genorm and normfinder algorithms. genorm rankings for the genes tested are presented in table and figure 2a. genorm identified suz and znf as the most stably expressed genes of the candidates. four of the five most stable genes were those derived from microarray data; the other was rps15a, which was selected from the literature. by contrast, four of the five least stable genes were chosen from the literature, including gapdh. the most stably expressed gene identified by normfinder was suz, which was also one of the two most stable genes identified in the genorm analysis. the best combination of genes identified by normfinder was suz and c2orf the five most stably expressed genes according to normfinder consisted entirely of microarray-derived genes, while the least stable five contained three out of five genes selected from the literature, again including gapdh. the comparative ranking of all genes for both genorm and normfinder algorithm analyses is displayed in table normfinder rankings of genes through analysis of inter- and intra group variability. genorm rankings of genes based on pairwise analysis of expression stability. genorm calculates an expression stability value for each candidate gene based on pairwise comparisons of variability. each gene is compared to every other gene to determine the two genes that contain the least variation. the stability value calculated for each gene is used to rank genes from least to most stable. the authors of the method give an m-value of as a cut-off for suitability as an endogenous control gene. the principal behind the pairwise stability measure ranking is that two ideal candidate normalisation genes should have an equal expression ratio in all samples. in the present study, all genes are well below the stipulated m-value. the program then calculates a normalisation factor in each sample for the most stable genes. genorm also calculates the optimal number of endogenous control genes to be used in the analysis of gene expression. this value is determined by locating the point where addition of the next most stable gene does not significantly affect the normalisation factor, using a cut-off value of. in this study, the value for genes was, suggesting that genes should be sufficient to normalise the experimental data. genorm calculates the minimum number of genes required for accurate normalisation. in this case, two genes were sufficient. the number of genes necessary for accurate normalisation is defined as the point at which the addition of a gene has a non-significant effect on the calculation of the normalisation factor, the threshold for a non-significant difference being. normfinder is another freely available tool for the identification of stable endogenous control genes. the main point of difference between the two methods is that normfinder takes into account both inter- and intra-group variability. the program not only identifies the most stable pair of endogenous control genes but also identifies the best overall endogenous control gene. the calculation of variability between groups is especially important in the present study considering the significant expression differences between the two tissue subtypes. the use of the two most stably expressed genes, in this case suz and c2orf, should provide sufficient normalisation for tissue comparisons as normfinder selects the best combination of genes whilst taking into account any grouping effects such as tissue type. the differences in rankings of gene stability using the two algorithms could be due to the fact that they use very different methods to assess gene stability. genorm selects genes based on the pairwise variation between genes. the two most stably expressed genes are therefore those genes that share an expression profile. in contrast, normfinder uses a model based algorithm that takes into account overall stability as well as any groups that may be present in the sample set. for example, if there are any grouping effects on gene expression a gene would be ranked lower than one that demonstrated variability not associated with any particular group. effect of endogenous control gene on target gene relative quantification temporal down-regulation of endometrial oxytocin receptor expression is a hallmark of early pregnancy, with embryonic interferon tau thought to elicit this response. given the expectation of differential oxtr expression between pregnant and cycling animals, the effect of control gene stability on gene expression values of oxtr in the endometrial samples was tested. oxtr expression was significantly greater in cycling than in pregnant cows regardless of the endogenous control gene used. notably, oxtr expression in pregnant animals was greater on average and more variable in na animals than for nz animals, and was also related to embryo size. the use of different endogenous control genes had no effect on oxtr expression differences in these group comparisons. however, there were differences in the normalised oxtr expression values that were inconsistent across the samples, depending on which endogenous control genes were employed. for example, in sample pregnant nz-5a the calculated relative oxtr expression value was when normalised to suz when normalised to gapdh, when normalised to uxt, and when normalised to both suz and znf another sample, the values were,,, and when normalised to suz gapdh, uxt and both suz and znf respectively. the average calculated fold change difference between pregnant and cycling animals was not affected by the choice of normalisation gene, possibly due to the large difference in expression level for this comparison. however, the choice of reference gene could be important when normalising genes that exhibit more subtle variation between experimental groups, given the considerable variation in expression shown between individual samples.  this study provides the first reported assessment of endogenous control genes for use in expression studies in bovine endometrium. normalisation is a critical factor in reporting rt-pcr expression data, providing a necessary control for error associated with sample preparation. normalisation using endogenous control genes provides a means of controlling this error, provided the gene used are stably expressed across all samples under investigation. the study described here tested candidate reference genes across bovine endometrial samples representing a range of physiological states and tissue subtypes. this study evaluated the suitability of both commonly used and novel experimentally derived reference genes for use in normalisation of rt-pcr data. candidates derived via microarray analysis were superior to existing, commonly used endogenous control genes, demonstrating the suitability of using microarray data for deriving novel endogenous control genes. this study also highlighted the importance of accurate normalisation with a stable endogenous control gene, by demonstrating relative expression of a differentially expressed gene when normalised using control genes of varying stability. suz was ranked first for stability across samples as determined by the statistical algorithms used in genorm and normfinder, and is therefore proposed as the best gene for normalisation of rt-pcr data in the current study. 
gene stability was assessed using the statistical algorithms genorm and normfinder. fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling holstein-friesian dairy cows from two divergent genetic backgrounds. the expression profiles of five commonly used endogenous control genes and experimentally derived candidate endogenous control genes were analysed across samples to determine the most stably expressed gene. based on the results of this analysis, suz is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle. quantitative real-time pcr gene expression results are generally normalised using endogenous control genes. these reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. the suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. this study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates. there has been no systematic investigation of endogenous control genes for bovine endometrium to date.
 quantitative real-time pcr gene expression results are generally normalised using endogenous control genes. these reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. there has been no systematic investigation of endogenous control genes for bovine endometrium to date. the suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling holstein-friesian dairy cows from two divergent genetic backgrounds.  the expression profiles of five commonly used endogenous control genes and experimentally derived candidate endogenous control genes were analysed across samples to determine the most stably expressed gene. gene stability was assessed using the statistical algorithms genorm and normfinder. all genes presented with low overall variability. however, when used to normalise a differentially expressed gene in the samples, the reported relative gene expression levels were significantly affected by the control gene chosen. based on the results of this analysis, suz is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle.  this study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates. 