BACKGROUND
oxygen-glucose deprivation  and middle cerebral artery occlusion  are accepted as in-vitro and in-vivo cerebral ischaemia models  <cit> .
the permanent mcao  focal ischaemia model is widely used in stroke research and is helpful in elucidating physiopathological causes and identifying prognostic markers and potential therapeutic targets  <cit> .
real-time polymerase chain reaction  is a powerful technique for gene expression studies due to its high sensitivity, specificity and broad quantification range for high throughput and accurate expression profiling of selected genes  <cit> .
the data obtained by quantitative rt-pcr  is typically normalised with an internal control, often referred to as a housekeeping gene.
up to now, several mathematical methods, such as normfinder  <cit> , genorm  <cit>  and 2-Δc't method  <cit> , have been developed to analyse the variability of the expression of candidate housekeeping genes.
the ideal housekeeping gene for qrt-pcr would be one whose mrna is consistently expressed at the same level in all samples under investigation, regardless of tissue type, disease state, medication or experimental conditions, and would have expression levels comparable to that of the target  <cit> .
however, no ideal reference gene has yet been discovered and it is reasonable to suppose that such a gene may not exist.
although different genes have been used, several previous studies have shown that the suitability of a particular reference gene depends on the insult, lesion and experimental conditions applied to the cell culture or experimental animal  <cit> .
therefore, careful evaluation and validation of control genes is required prior to using them in each individual case to avoid possible inaccuracies stemming from the use of an unsuitable reference gene  <cit> .
in addition, no validated housekeeping genes have been reported for the relative quantification of the mrna expression profile activated in in-vitro ischaemic conditions.
with regards to the in-vivo model , glyceraldehyde-3-phosphate dehydrogenase   <cit> , peptidyl-prolyl cis-trans isomerasa   <cit> , ribosomal protein l13a  <cit>  and hypoxanthine phosphoribosyltransferase   <cit>  have previously been accepted as control genes.
the aim of this study was to confirm the validity of these reference genes in our pmcao samples and to test their usefulness as endogenous controls in the in-vitro model using primary cultures of rat astrocytes.
furthermore, we aimed to analyse the expression stability of other reference genes that have been used, beta- <dig> microglobulin  and 18s rrna  <cit> , glucoronidase b   <cit> , succinate dehydrogenase complex, subunit a   <cit> , tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide   <cit>  and a new candidate gene for the in-vivo model, ornithine decarboxylase antizyme  <dig> , which has been hypothesised as being suitable in experimental conditions involving high protein turn-over  <cit> .
RESULTS
evaluation and validation of selected candidate housekeeping genes in an in-vitro ischaemia model
the results of gene expression of gapdh, hprt, ppia, 18s rrna, β2m, oaz <dig>  gusb, ywhaz, sdha and rpl13a from astrocytes exposed to ogd during  <dig> hours and collected at  <dig> minutes,  <dig> minutes,  <dig>   <dig>   <dig> and  <dig> hours after reperfusion are shown in figure  <dig>
the expression of gapdh was found to have significant fluctuations in the levels of mrna  increasing significantly at  <dig> h after reperfusion  whereas no significant differences were observed at other points .
the expression of hprt, in comparison to the control group, was found to fluctuate significantly in all the groups as a whole .
this fluctuation was a significant increase at  <dig> min ,  <dig> min  and  <dig> h after reperfusion  .
significant differences in the expression of ppia were found between time groups .
levels increased at  <dig> and  <dig> min  and at  <dig> h and  <dig> h  after reperfusion in comparison with the control group .
the 18s rrna mrna showed significant level fluctuations .
levels decreased at  <dig> h  and  <dig> h  after reperfusion .
changes in β2m mrna levels were found to be significant between time groups .
β2m expression decreased at  <dig> min ,  <dig> min and  <dig> h  and  <dig> h  in comparison with the control group .
oaz <dig> also showed significant differences in expression between groups .
the expression was stable at the first four analysis times but decreased significantly at  <dig> h  and  <dig> h  .
significant fluctuations in the mrna expression of gusb were found  decreasing at  <dig> h after reperfusion  .
the expression of ywhaz was found to have significant fluctuations in mrna levels  .
analysis of the same samples with a specific gene expression assay for sdha and rpl13a did not reveal any significant differences in expressions at the different time points when analysis was made  .
evaluation and validation of selected candidate housekeeping genes in an in-vivo cerebral ischaemia model
the gene expression of candidate housekeeping genes, β2m, rpl13a, oaz <dig>  gapdh, ppia, gusb, 18srrna, sdha, ywhaz and hprt, in cerebral cortex samples collected at  <dig> minutes and  <dig>   <dig>   <dig>   <dig> and  <dig> hours after pmcao is shown in figure  <dig>
β2m expression presented a significant increase at  <dig> and  <dig> h  and was significantly different between groups  .
the expression of rpl13a increased  <dig> h after pmcao  with a significant fluctuation between groups  .
the level of mrna variation of oaz <dig> was found to be significant for all the time groups taken as a whole  .
significant differences were found between groups for the expression of ppia  with mrna decreasing significantly at  <dig> and  <dig> h  after pmcao .
the expression of gusb was found to fluctuate significantly between the different test points .
at  <dig> h and  <dig> h, levels of gusb mrna had increased  .
the level of the mrna of 18s rrna showed significant fluctuation  decreasing significantly at  <dig> h  and  <dig> h  .
changes in gapdh mrna levels were found to be significant between time groups .
gapdh expression decreased at  <dig> min ,  <dig> h  and  <dig> h  in comparison with the control group .
mrna expression was invariable under in-vivo ischaemic brain conditions for the sdha , hprt  and ywhaz  genes .
the stability of these three genes was evaluated using genorm and normfinder algorithms.
genorm identified ywhaz and sdha as the most stable pairwise combination of reference genes for the in-vivo ischaemia model .
figure 3b shows the pairwise variation value  between two sequential normalization factors containing an increasing number of genes.
as we only analysed three genes, the figure shows the pairwise variation value of v2/ <dig> .
according to vandesompele et al.
<cit> , the ideal pairwise variation value is less than  <dig>  and therefore two genes, in our case ywhaz and sdha, are enough for normalization.
normfinder identified ywhaz as the most stable gene with a stability value of  <dig>  and, as with genorm, the best two genes for normalization were ywhaz and sdha with a stability value of  <dig>  .
discussion
the selection of the housekeeping gene is crucial to the normalization of quantitative gene expression results.
this is even more important in those settings in which gene expression is severely affected, as after cerebral ischaemia.
no validated housekeeping genes have been reported for the relative quantification of the mrna expression profile activated in in-vitro ischaemic conditions.
our present study discards the use of gapdh, ppia, β2m, 18s rrna, hprt, oaz <dig>  gusb and ywhaz as reference genes and finds rpl13a and sdha to be the best control genes for the in-vitro model in the conditions we have tested.
with regards to the pmcao model, four studies have reported the suitability of several reference genes for this ischaemia model.
in table  <dig> we summarise the methodologies employed and results obtained both for previously published and the present studies.
earlier investigations only tested from two to four candidate control genes finding invariable expressions for rpl13a, hprt, ppia and gapdh genes.
in the case of gapdh, results were contradictory: three studies validated the gene  <cit>  whereas another rejected it  <cit> .
our results show that gapdh, rpl13a and ppia together with other candidate genes, namely β2m, gusb, 18s rrna and oaz <dig>  are not appropriate as housekeeping genes for the pmcao model.
as previously reported by meldgaard et al.
when using the pmcao model in mice  <cit> , we have found hprt to be a suitable housekeeping gene for the in-vivo ischaemia model.
moreover we have identified two new housekeeping genes for this model, sdha and ywhaz, which have not been previously analysed.
according to the results obtained with the normfinder and genorm algorithms, these two genes are more stable and so the best suited for normalization of relative quantification gene expression studies.
several factors have an influence on the variability of control genes and may lead to differences between studies.
in the same way that gene control expression may vary depending on whether the model is permanent  or transient   <cit> , different methodologies employed to perform the pmcao, electrocautery or intraluminal filament, may have an influence on the gene expression.
sample collection and the control sample used are other factors which must be taken into consideration.
three different cerebral regions have been collected in each previous study: the whole of the infarcted cortex  <cit> , the whole of the infarcted hemisphere  <cit>  and the ischaemic penumbra cortex  <cit> .
in our study we have collected the infarcted and peri-infarcted cortical area to analyse changes in gene expression in the affected region.
with regards to the control sample there are also different possibilities: brain samples from sham or naive rats, or healthy contralateral hemispheres.
we consider sham rats to be the best control because this control minimises bias due to surgery.
in our opinion, the use of the healthy contralateral hemisphere may lead to erroneuos conclusions because studies have reported structural changes as well as changes in gene expression in the contralateral hemisphere after ischaemia  <cit> .
finally, the time course studied is also a relevant factor.
we have validated sdha, ywhaz and hprt as a suitable housekeeping genes for the pmcao model conducting an analysis of the control situation and after ischaemia at frequent time intervals up to  <dig> hours, which vary from those used in earlier studies  <cit> .
considering all the variables that may influence gene expression, the validation of candidate reference genes in each individual experimental condition is crucial.
the genorm  <cit>  and normfinder  <cit>  algorithms are now widely used to determine the most stable reference genes from a set of candidates genes with invariable expression  <cit> .
we have only used algorithms on genes identified as stable housekeeping genes in the in-vivo model in our analysis as the software requires a minimum of three genes for the analysis.
in order to check the value of the validation of endogenous controls, we have used different housekeeping genes analysed in this study to normalise the expression of cyclooxygenase  <dig>  and bcl <dig>  genes which are known to be involved in cerebral ischaemia .
cox <dig> is an inflammatory mediator gene which undergoes a significant increase due to ischaemia  <cit> .
bcl <dig> is a protooncogene that codifies an antiapoptotic protein, which has been described as participating in neuroprotective mechanisms in ischaemic stroke although changes in the expression are limited to an increase in the neurons within the peri-infarct region  <cit> .
in the two cases we have observed differences in the results obtained when suitable and unsuitable control genes are used.
these are even more critical when the differences in expression of the analysed gene are minor, as is the case with bcl <dig>  which has a different expression pattern depending on the housekeeping gene that is used.
CONCLUSIONS
in designing any study into relative gene expression quantification it is necessary to perform a validation of the housekeeping genes used which takes into account the specific experimental conditions to be encountered and the time course to be employed.
we report a detailed investigation of the effects of experimental ischaemia on the expression of potential housekeeping genes including genes typically used and new candidates as internal controls in qrt-pcr assays.
this analysis was facilitated by the sensitivity and efficiency of real-time quantitative pcr.
we determined that gapdh, rpl13a and ppia, which have previously been validated as housekeeping genes in quantitative expression studies with the pmcao model, had significant differences in their expression in our samples collected at different time points.
our study confirms the suitability of hprt as a housekeeping gene for this in-vivo model and identifies two new housekeeping genes, sdha and ywhaz, which are more stable than hprt.
we have also identified, to the best of our knowledge for the first time, reference genes  for the ogd in-vitro model using primary astrocyte cultures.
the study also highlights the importance of validating control genes for each individual study as gene expression is influenced by many different factors which can lead to mistaken conclusions.

 oxygen-glucose deprivation and middle cerebral artery occlusion are accepted as in-vitro and in-vivo cerebral ischaemia models. the permanent mcao focal ischaemia model is widely used in stroke research and is helpful in elucidating physiopathological causes and identifying prognostic markers and potential therapeutic targets. real-time polymerase chain reaction is a powerful technique for gene expression studies due to its high sensitivity, specificity and broad quantification range for high throughput and accurate expression profiling of selected genes. the data obtained by quantitative rt-pcr is typically normalised with an internal control, often referred to as a housekeeping gene. up to now, several mathematical methods, such as normfinder, genorm and 2-Δc't method, have been developed to analyse the variability of the expression of candidate housekeeping genes. the ideal housekeeping gene for qrt-pcr would be one whose mrna is consistently expressed at the same level in all samples under investigation, regardless of tissue type, disease state, medication or experimental conditions, and would have expression levels comparable to that of the target. however, no ideal reference gene has yet been discovered and it is reasonable to suppose that such a gene may not exist. although different genes have been used, several previous studies have shown that the suitability of a particular reference gene depends on the insult, lesion and experimental conditions applied to the cell culture or experimental animal. therefore, careful evaluation and validation of control genes is required prior to using them in each individual case to avoid possible inaccuracies stemming from the use of an unsuitable reference gene. in addition, no validated housekeeping genes have been reported for the relative quantification of the mrna expression profile activated in in-vitro ischaemic conditions. with regards to the in-vivo model, glyceraldehyde-3-phosphate dehydrogenase, peptidyl-prolyl cis-trans isomerasa, ribosomal protein l13a and hypoxanthine phosphoribosyltransferase have previously been accepted as control genes. the aim of this study was to confirm the validity of these reference genes in our pmcao samples and to test their usefulness as endogenous controls in the in-vitro model using primary cultures of rat astrocytes. furthermore, we aimed to analyse the expression stability of other reference genes that have been used, beta- microglobulin and 18s rrna, glucoronidase b, succinate dehydrogenase complex, subunit a, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide and a new candidate gene for the in-vivo model, ornithine decarboxylase antizyme, which has been hypothesised as being suitable in experimental conditions involving high protein turn-over.  evaluation and validation of selected candidate housekeeping genes in an in-vitro ischaemia model the results of gene expression of gapdh, hprt, ppia, 18s rrna, β2m, oaz gusb, ywhaz, sdha and rpl13a from astrocytes exposed to ogd during hours and collected at minutes, minutes, and hours after reperfusion are shown in figure the expression of gapdh was found to have significant fluctuations in the levels of mrna increasing significantly at h after reperfusion whereas no significant differences were observed at other points. the expression of hprt, in comparison to the control group, was found to fluctuate significantly in all the groups as a whole. this fluctuation was a significant increase at min, min and h after reperfusion. significant differences in the expression of ppia were found between time groups. levels increased at and min and at h and h after reperfusion in comparison with the control group. the 18s rrna mrna showed significant level fluctuations. levels decreased at h and h after reperfusion. changes in β2m mrna levels were found to be significant between time groups. β2m expression decreased at min, min and h and h in comparison with the control group. oaz also showed significant differences in expression between groups. the expression was stable at the first four analysis times but decreased significantly at h and h. significant fluctuations in the mrna expression of gusb were found decreasing at h after reperfusion. the expression of ywhaz was found to have significant fluctuations in mrna levels. analysis of the same samples with a specific gene expression assay for sdha and rpl13a did not reveal any significant differences in expressions at the different time points when analysis was made. evaluation and validation of selected candidate housekeeping genes in an in-vivo cerebral ischaemia model the gene expression of candidate housekeeping genes, β2m, rpl13a, oaz gapdh, ppia, gusb, 18srrna, sdha, ywhaz and hprt, in cerebral cortex samples collected at minutes and and hours after pmcao is shown in figure β2m expression presented a significant increase at and h and was significantly different between groups. the expression of rpl13a increased h after pmcao with a significant fluctuation between groups. the level of mrna variation of oaz was found to be significant for all the time groups taken as a whole. significant differences were found between groups for the expression of ppia with mrna decreasing significantly at and h after pmcao. the expression of gusb was found to fluctuate significantly between the different test points. at h and h, levels of gusb mrna had increased. the level of the mrna of 18s rrna showed significant fluctuation decreasing significantly at h and h. changes in gapdh mrna levels were found to be significant between time groups. gapdh expression decreased at min, h and h in comparison with the control group. mrna expression was invariable under in-vivo ischaemic brain conditions for the sdha, hprt and ywhaz genes. the stability of these three genes was evaluated using genorm and normfinder algorithms. genorm identified ywhaz and sdha as the most stable pairwise combination of reference genes for the in-vivo ischaemia model. figure 3b shows the pairwise variation value between two sequential normalization factors containing an increasing number of genes. as we only analysed three genes, the figure shows the pairwise variation value of v2/. according to vandesompele et al., the ideal pairwise variation value is less than and therefore two genes, in our case ywhaz and sdha, are enough for normalization. normfinder identified ywhaz as the most stable gene with a stability value of and, as with genorm, the best two genes for normalization were ywhaz and sdha with a stability value of. discussion the selection of the housekeeping gene is crucial to the normalization of quantitative gene expression results. this is even more important in those settings in which gene expression is severely affected, as after cerebral ischaemia. no validated housekeeping genes have been reported for the relative quantification of the mrna expression profile activated in in-vitro ischaemic conditions. our present study discards the use of gapdh, ppia, β2m, 18s rrna, hprt, oaz gusb and ywhaz as reference genes and finds rpl13a and sdha to be the best control genes for the in-vitro model in the conditions we have tested. with regards to the pmcao model, four studies have reported the suitability of several reference genes for this ischaemia model. in table we summarise the methodologies employed and results obtained both for previously published and the present studies. earlier investigations only tested from two to four candidate control genes finding invariable expressions for rpl13a, hprt, ppia and gapdh genes. in the case of gapdh, results were contradictory: three studies validated the gene whereas another rejected it. our results show that gapdh, rpl13a and ppia together with other candidate genes, namely β2m, gusb, 18s rrna and oaz are not appropriate as housekeeping genes for the pmcao model. as previously reported by meldgaard et al. when using the pmcao model in mice, we have found hprt to be a suitable housekeeping gene for the in-vivo ischaemia model. moreover we have identified two new housekeeping genes for this model, sdha and ywhaz, which have not been previously analysed. according to the results obtained with the normfinder and genorm algorithms, these two genes are more stable and so the best suited for normalization of relative quantification gene expression studies. several factors have an influence on the variability of control genes and may lead to differences between studies. in the same way that gene control expression may vary depending on whether the model is permanent or transient, different methodologies employed to perform the pmcao, electrocautery or intraluminal filament, may have an influence on the gene expression. sample collection and the control sample used are other factors which must be taken into consideration. three different cerebral regions have been collected in each previous study: the whole of the infarcted cortex, the whole of the infarcted hemisphere and the ischaemic penumbra cortex. in our study we have collected the infarcted and peri-infarcted cortical area to analyse changes in gene expression in the affected region. with regards to the control sample there are also different possibilities: brain samples from sham or naive rats, or healthy contralateral hemispheres. we consider sham rats to be the best control because this control minimises bias due to surgery. in our opinion, the use of the healthy contralateral hemisphere may lead to erroneuos conclusions because studies have reported structural changes as well as changes in gene expression in the contralateral hemisphere after ischaemia. finally, the time course studied is also a relevant factor. we have validated sdha, ywhaz and hprt as a suitable housekeeping genes for the pmcao model conducting an analysis of the control situation and after ischaemia at frequent time intervals up to hours, which vary from those used in earlier studies. considering all the variables that may influence gene expression, the validation of candidate reference genes in each individual experimental condition is crucial. the genorm and normfinder algorithms are now widely used to determine the most stable reference genes from a set of candidates genes with invariable expression. we have only used algorithms on genes identified as stable housekeeping genes in the in-vivo model in our analysis as the software requires a minimum of three genes for the analysis. in order to check the value of the validation of endogenous controls, we have used different housekeeping genes analysed in this study to normalise the expression of cyclooxygenase and bcl genes which are known to be involved in cerebral ischaemia. cox is an inflammatory mediator gene which undergoes a significant increase due to ischaemia. bcl is a protooncogene that codifies an antiapoptotic protein, which has been described as participating in neuroprotective mechanisms in ischaemic stroke although changes in the expression are limited to an increase in the neurons within the peri-infarct region. in the two cases we have observed differences in the results obtained when suitable and unsuitable control genes are used. these are even more critical when the differences in expression of the analysed gene are minor, as is the case with bcl which has a different expression pattern depending on the housekeeping gene that is used.  in designing any study into relative gene expression quantification it is necessary to perform a validation of the housekeeping genes used which takes into account the specific experimental conditions to be encountered and the time course to be employed. we report a detailed investigation of the effects of experimental ischaemia on the expression of potential housekeeping genes including genes typically used and new candidates as internal controls in qrt-pcr assays. this analysis was facilitated by the sensitivity and efficiency of real-time quantitative pcr. we determined that gapdh, rpl13a and ppia, which have previously been validated as housekeeping genes in quantitative expression studies with the pmcao model, had significant differences in their expression in our samples collected at different time points. our study confirms the suitability of hprt as a housekeeping gene for this in-vivo model and identifies two new housekeeping genes, sdha and ywhaz, which are more stable than hprt. we have also identified, to the best of our knowledge for the first time, reference genes for the ogd in-vitro model using primary astrocyte cultures. the study also highlights the importance of validating control genes for each individual study as gene expression is influenced by many different factors which can lead to mistaken conclusions. 
genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup. no validated housekeeping genes have been reported for the relative quantification of the mrna expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. the present study aims to determine the expression stability of ten housekeeping genes to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models. the expression stability of the candidate reference genes was evaluated using the 2-Δc't method and anova followed by dunnett's test. for the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for rpl13a and sdha were found to have significantly different levels of mrna expression. we have validated endogenous control genes for qrt-pcr analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. the normalization of relative qrt-pcr data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. for normalization purposes, rpl13a and sdha are found to be the most suitable genes for the in-vitro model and sdha and ywhaz for the in-vivo model. studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. these different levels were also found in the case of the in-vivo model of pmcao in rats except for hprt, sdha and ywhaz mrna, where the expression did not vary.
 studies of gene expression in experimental cerebral ischaemia models can contribute to understanding the pathophysiology of brain ischaemia and to identifying prognostic markers and potential therapeutic targets. the normalization of relative qrt-pcr data using a suitable reference gene is a crucial prerequisite for obtaining reliable conclusions. no validated housekeeping genes have been reported for the relative quantification of the mrna expression profile activated in in-vitro ischaemic conditions, whereas for the in-vivo model different reference genes have been used. the present study aims to determine the expression stability of ten housekeeping genes to establish their suitability as control genes for in-vitro and in-vivo cerebral ischaemia models.  the expression stability of the candidate reference genes was evaluated using the 2-Δc't method and anova followed by dunnett's test. for the in-vitro model using primary cultures of rat astrocytes, all genes analysed except for rpl13a and sdha were found to have significantly different levels of mrna expression. these different levels were also found in the case of the in-vivo model of pmcao in rats except for hprt, sdha and ywhaz mrna, where the expression did not vary. sdha and ywhaz were identified by genorm and normfinder as the two most stable genes.  we have validated endogenous control genes for qrt-pcr analysis of gene expression in in-vitro and in-vivo cerebral ischaemia models. for normalization purposes, rpl13a and sdha are found to be the most suitable genes for the in-vitro model and sdha and ywhaz for the in-vivo model. genes previously used as housekeeping genes for the in-vivo model in the literature were not validated as good control genes in the present study, showing the need for careful evaluation for each new experimental setup. 