T lymphocytes may have an important role in the pathogenesis of smoking-related emphysema, according to a new study by researchers from Houston, Texas, United States. “We now know that T cells are not only present in chronic obstructive pulmonary disease [COPD], but are harmful,” comments Steven Shapiro from Brigham and Women's Hospital ,  Harvard Medical School, who was not involved in the study. “We also now have a pathway that could be interrupted to prevent lung destruction in COPD.”,Farrah Kheradmand and colleagues took lung samples from 28 ex-smokers who had been admitted to hospital for lung resection: 18 patients had moderate to severe COPD as well as evidence of emphysema, and ten patients had none. The researchers isolated lung lymphocytes from the samples and used two-color flow cytometry to phenotypically characterize the cells. They found that lymphocytes taken from patients with emphysema expressed more CCR5 and CXCR3 receptors, which are associated with a particular type of T cell called T helper 1 (Th1), than did those from control individuals. By contrast, expression of CCR4 receptors, which are found on T helper 2 (Th2) cells, was very low in both control and emphysema groups.In a separate experiment, Kheradmand's team showed that lung lymphocytes taken from patients with emphysema secreted more of three other proteins—interferon gamma, monokine induced by interferon (MIG), and interferon-inducible protein 10 (IP-10)—than control patients. MIG and IP-10 are known to be produced by injured epithelial cells and are ligands for CXCR3 receptors, which are expressed by Th1 cells. Importantly, the researchers were also able to show that isolated peripheral lung macrophages secreted matrix metalloproteinase-12 (MMP12), an enzyme that degrades elastin—a protein important for lung elasticity—in the lungs, in response to IP-10 and MIG. Together these findings, say the authors, indicate that Th1 cells, but not Th2 cells, are required for producing the elastin-destroying lung environment of emphysema.The researchers now intend to investigate the antigens that drive the Th1-based inflammation that underlies emphysema. “Ultimately, we seek to understand the biochemistry of tobacco smoke that triggers inflammation in the first place, and whether such insight might explain other environmentally triggered lung diseases,” explains Kheradmand. “To understand such detailed immune mechanisms, we really need an improved experimental model of disease, and this we are currently working on.”


Background The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon.
Background
The neuroprotective effect of xenon has been demonstrated for glutamatergic neurons. In the present study it is investigated if dopaminergic neurons, i.e. nerve-growth-factor differentiated PC-12 cells, are protected as well against hypoxia-induced cell damage in the presence of xenon.Results Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a N 2 -saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N 2 -treated cells. Replacement of N 2  by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering of Ca 2+  using the Ca-chelator 1, 2- bis (2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid  tetrakis (acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the essential participation of intracellular Ca 2+ -ions in the process of xenon-induced neuroprotection.
Results
Pheochromocytoma cells differentiated by addition of nerve growth factor were placed in a N 2 -saturated atmosphere, a treatment that induced release of dopamine, reaching a maximum after 30 min. By determining extracellular lactate dehydrogenase concentration as marker for concomitant cellular damage, a substantial increase of enzymatic activity was found for N 2 -treated cells. Replacement of N 2  by xenon in such a hypoxic atmosphere resulted in complete protection against cellular damage and prevention of hypoxia-induced dopamine release. Intracellular buffering of Ca 2+  using the Ca-chelator 1, 2- bis (2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid  tetrakis (acetoxymethyl) ester (BAPTA) reduced the neuroprotective effect of xenon indicating the essential participation of intracellular Ca 2+ -ions in the process of xenon-induced neuroprotection.bistetrakisConclusions The results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation.
Conclusions
The results presented demonstrate the outstanding property of xenon to protect neuron-like cells in a hypoxic situation.

Background Originally, hypoxia/ischemia-induced alterations in neuronal function have been attributed to be an over-release of neurotransmitters, including dopamine and glutamate. Many studies have been performed on the mechanisms of glutamate-induced neuronal damage [ 1 , 2 ] but relatively few have investigated the hypoxia-induced damage in dopaminergic neurons [ 3 - 6 ]. In recent years several lines of evidence have suggested that effects other than excitotoxic mechanisms may also participate in hypoxia-induced cell damage such as cortical spreading depression [ 7 , 8 ]. Rat pheochromocytoma (PC-12) cells are catecholaminergic, excitable cells that have been widely used as an  in vitro  model for neuronal cells [ 9 ] possessing both D1- and D2-dopamine receptors [ 10 ]. In these cells hypoxia causes a transient release of dopamine resulting from a complex cellular response consisting of increased dopamine release and reduced uptake rate. Such increased dopamine concentration has been shown to be associated with cellular damage indicated by an elevated release of lactate dehydrogenase (LDH) from the cells [ 6 , 11 ]. Numerous approaches have been undertaken to reduce hypoxia-induced neurotoxicity [ 2 , 12 ]. The pathological increase of extracellular neurotransmitter concentration presents probably one of the first indicators for such damage although it is not clear to what extent it contributes directly. Thus, a reduction or even complete suppression of such an increase of neurotransmitter concentration after the primary neuronal damage would suggest a high probability for protection from the hypoxic insult. Recently, we have shown that the noble gas xenon prevents in hypoxic cortical neurons hypoxia-induced cell damage and glutamate release [ 13 , 14 ]. Such neuroprotective potential has been confirmed by Ma et al. [ 15 ] and Wilhelm et al.[ 16 ], and related to its property of being an NMDA-receptor antagonist. In the present paper, however, we show that also in the dopaminergic PC-12-system xenon exhibits profound neuroprotective properties for hypoxic cells thus underlining its usefulness as a general neuroprotectant.
Background
Originally, hypoxia/ischemia-induced alterations in neuronal function have been attributed to be an over-release of neurotransmitters, including dopamine and glutamate. Many studies have been performed on the mechanisms of glutamate-induced neuronal damage [ 1 , 2 ] but relatively few have investigated the hypoxia-induced damage in dopaminergic neurons [ 3 - 6 ]. In recent years several lines of evidence have suggested that effects other than excitotoxic mechanisms may also participate in hypoxia-induced cell damage such as cortical spreading depression [ 7 , 8 ]. Rat pheochromocytoma (PC-12) cells are catecholaminergic, excitable cells that have been widely used as an  in vitro  model for neuronal cells [ 9 ] possessing both D1- and D2-dopamine receptors [ 10 ]. In these cells hypoxia causes a transient release of dopamine resulting from a complex cellular response consisting of increased dopamine release and reduced uptake rate. Such increased dopamine concentration has been shown to be associated with cellular damage indicated by an elevated release of lactate dehydrogenase (LDH) from the cells [ 6 , 11 ].in vitroNumerous approaches have been undertaken to reduce hypoxia-induced neurotoxicity [ 2 , 12 ]. The pathological increase of extracellular neurotransmitter concentration presents probably one of the first indicators for such damage although it is not clear to what extent it contributes directly. Thus, a reduction or even complete suppression of such an increase of neurotransmitter concentration after the primary neuronal damage would suggest a high probability for protection from the hypoxic insult. Recently, we have shown that the noble gas xenon prevents in hypoxic cortical neurons hypoxia-induced cell damage and glutamate release [ 13 , 14 ]. Such neuroprotective potential has been confirmed by Ma et al. [ 15 ] and Wilhelm et al.[ 16 ], and related to its property of being an NMDA-receptor antagonist. In the present paper, however, we show that also in the dopaminergic PC-12-system xenon exhibits profound neuroprotective properties for hypoxic cells thus underlining its usefulness as a general neuroprotectant.Results Release of dopamine under hypoxic conditions Cells kept under normoxic conditions did not release dopamine during the time period studied. If, however, they were kept in an atmosphere consisting of 100% nitrogene, considerable amounts of dopamine were found in the extracellular space reaching a maximum at 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by xenon, no such increase in dopamine concentration was found (Fig.  1a ). The level of extracellular dopamine remained as low as in cells kept under normoxic conditions. Hypoxia-induced cellular damage In order to test if such hypoxia damaged the cells, extracellular LDH was determined after a two-hour period of treatment. A low level of LDH was found in cells kept under normoxic conditions whereas cells kept under nitrogen showed a significant release of LDH indicating severe cellular damage (Fig.  1b ). If instead of nitrogen xenon was used to create such hypoxic condition, the LDH level remained at the same low level as in controls. Effect of the dopamine reuptake inhibitor GBR 1209 Hypoxia-induced extracellular increase of dopamine could be caused either by elevated release of dopamine or by a reduced, or even inhibited, dopamine uptake. If hypoxia caused faster release but did not interfere with uptake, uptake-inhibitors would cause a higher concentration of dopamine in the extracellular space. On the other hand, if the release was constant but the re-uptake inhibited by hypoxia, additional inhibition of uptake by inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the extracellular dopamine concentration did not change in a normoxic or xenon environment. However, in nitrogen the extracellular dopamine concentration did not reach exactly the same value as in pure nitrogen, the dopamine level was slightly but significantly reduced, thus supporting the view that hypoxia-induced extracellular dopamine increase was caused by an enhanced release of dopamine and – to a lesser extent – an interference with the uptake mechanism (Fig.  2 ). Effects of the dopamine receptor antagonists SCH 23390 and sulpiride To test if indeed the hypoxia-induced increase of extracellular dopamine itself caused the cell damage measured by the increase in extracellular LDH, dopamine receptor antagonists were used. Since they prevent dopamine binding they should provide protection of dopamine-induced damage. If the D1 receptor antagonist SCH 23390 was used during the incubation period, then at the highest dose of 10 nM, a reduction of nitrogen-induced external LDH-increase could be seen. However, even at this highest applied dose of SCH 23390, there was still only a less than 50% reduction in extracellular LDH (Fig.  3a ). If, on the other hand, the D2 receptor antagonist sulpiride was used, no reduction in the nitrogen-induced LDH-release was found (Fig.  3b ). Both compounds did not change the xenon-induced suppression of cellular damage. Cellular damage induced by external addition of dopamine To analyze if indeed the increased external dopamine was detrimental to cells, they were incubated in the presence of 100 nM dopamine, either for 30 min followed by 120 min in normal medium, or continuously for 150 min. As shown in fig.  4 , column (c), even the 30 min incubation with 100 nm dopamine (the lesser of the two dopamine challenges) was sufficient to cause considerable cell damage. Such damage was further increased if dopamine was present for the whole period of time of 150 min (column (e). We asked then if xenon not only prevented the release of dopamine in a hypoxic situation but could even reduce the damage caused by external dopamine. Cells were incubated for 30 min in normal buffer containing 100 nM dopamine followed by 120 min in xenon-saturated buffer, without dopamine. As shown in column (d), the dopamine-induced damage to the cells as seen in column (c) was significantly reduced. If the cells were incubated for 150 min in xenon-saturated buffer containing dopamine, even under those conditions the damage was low compared to cells exposed to dopamine in normal buffer (column (f)). Buffering of intracellular Ca 2+ -ions using BAPTA In order to test if changes in intracellular Ca 2+  were required for the neuroprotective effect of xenon, cells were incubated with the cell-permeant Ca-chelator BAPTA-AM. As shown in fig.  5 , chelating intracellular Ca 2+  does not damage the cells  per se  (control + 10 μM BAPTA). Surprisingly, such chelation reduces significantly the neuroprotective effect of xenon, indicating an essential role for intracellular Ca 2+  for this effect to occur. A slight but significant reduction in cellular damage is observed when BAPTA-treated cells are incubated in nitrogen-saturated buffer. Comparison with another dopaminergic cell system To exclude that the results obtained were limited to the PC-12-system itself, hypoxia-induced dopamine release and cell damage was investigated in rat embryonic primary mesencephalic cell cultures that are known to contain 0.5 – 1.5% of dopaminergic cells [ 26 ]. As shown in fig.  6 , in an hypoxic atmosphere a very similar pattern of dopamine and LDH release is obtained compared to PC-12 cells. Xenon prevents also in these primary cells the hypoxia-induced neurotransmitter and LDH release.
Results
Release of dopamine under hypoxic conditions Cells kept under normoxic conditions did not release dopamine during the time period studied. If, however, they were kept in an atmosphere consisting of 100% nitrogene, considerable amounts of dopamine were found in the extracellular space reaching a maximum at 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by xenon, no such increase in dopamine concentration was found (Fig.  1a ). The level of extracellular dopamine remained as low as in cells kept under normoxic conditions.
Release of dopamine under hypoxic conditions
Cells kept under normoxic conditions did not release dopamine during the time period studied. If, however, they were kept in an atmosphere consisting of 100% nitrogene, considerable amounts of dopamine were found in the extracellular space reaching a maximum at 30 min of incubation, followed by a subsequent decrease. If under the same conditions nitrogen was replaced by xenon, no such increase in dopamine concentration was found (Fig.  1a ). The level of extracellular dopamine remained as low as in cells kept under normoxic conditions.Hypoxia-induced cellular damage In order to test if such hypoxia damaged the cells, extracellular LDH was determined after a two-hour period of treatment. A low level of LDH was found in cells kept under normoxic conditions whereas cells kept under nitrogen showed a significant release of LDH indicating severe cellular damage (Fig.  1b ). If instead of nitrogen xenon was used to create such hypoxic condition, the LDH level remained at the same low level as in controls.
Hypoxia-induced cellular damage
In order to test if such hypoxia damaged the cells, extracellular LDH was determined after a two-hour period of treatment. A low level of LDH was found in cells kept under normoxic conditions whereas cells kept under nitrogen showed a significant release of LDH indicating severe cellular damage (Fig.  1b ). If instead of nitrogen xenon was used to create such hypoxic condition, the LDH level remained at the same low level as in controls.Effect of the dopamine reuptake inhibitor GBR 1209 Hypoxia-induced extracellular increase of dopamine could be caused either by elevated release of dopamine or by a reduced, or even inhibited, dopamine uptake. If hypoxia caused faster release but did not interfere with uptake, uptake-inhibitors would cause a higher concentration of dopamine in the extracellular space. On the other hand, if the release was constant but the re-uptake inhibited by hypoxia, additional inhibition of uptake by inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the extracellular dopamine concentration did not change in a normoxic or xenon environment. However, in nitrogen the extracellular dopamine concentration did not reach exactly the same value as in pure nitrogen, the dopamine level was slightly but significantly reduced, thus supporting the view that hypoxia-induced extracellular dopamine increase was caused by an enhanced release of dopamine and – to a lesser extent – an interference with the uptake mechanism (Fig.  2 ).
Effect of the dopamine reuptake inhibitor GBR 1209
Hypoxia-induced extracellular increase of dopamine could be caused either by elevated release of dopamine or by a reduced, or even inhibited, dopamine uptake. If hypoxia caused faster release but did not interfere with uptake, uptake-inhibitors would cause a higher concentration of dopamine in the extracellular space. On the other hand, if the release was constant but the re-uptake inhibited by hypoxia, additional inhibition of uptake by inhibitors would have no or little effect. In the presence of 5 nM of the dopamine reuptake inhibitor GBR 1209 the extracellular dopamine concentration did not change in a normoxic or xenon environment. However, in nitrogen the extracellular dopamine concentration did not reach exactly the same value as in pure nitrogen, the dopamine level was slightly but significantly reduced, thus supporting the view that hypoxia-induced extracellular dopamine increase was caused by an enhanced release of dopamine and – to a lesser extent – an interference with the uptake mechanism (Fig.  2 ).Effects of the dopamine receptor antagonists SCH 23390 and sulpiride To test if indeed the hypoxia-induced increase of extracellular dopamine itself caused the cell damage measured by the increase in extracellular LDH, dopamine receptor antagonists were used. Since they prevent dopamine binding they should provide protection of dopamine-induced damage. If the D1 receptor antagonist SCH 23390 was used during the incubation period, then at the highest dose of 10 nM, a reduction of nitrogen-induced external LDH-increase could be seen. However, even at this highest applied dose of SCH 23390, there was still only a less than 50% reduction in extracellular LDH (Fig.  3a ). If, on the other hand, the D2 receptor antagonist sulpiride was used, no reduction in the nitrogen-induced LDH-release was found (Fig.  3b ). Both compounds did not change the xenon-induced suppression of cellular damage.
Effects of the dopamine receptor antagonists SCH 23390 and sulpiride
To test if indeed the hypoxia-induced increase of extracellular dopamine itself caused the cell damage measured by the increase in extracellular LDH, dopamine receptor antagonists were used. Since they prevent dopamine binding they should provide protection of dopamine-induced damage. If the D1 receptor antagonist SCH 23390 was used during the incubation period, then at the highest dose of 10 nM, a reduction of nitrogen-induced external LDH-increase could be seen. However, even at this highest applied dose of SCH 23390, there was still only a less than 50% reduction in extracellular LDH (Fig.  3a ). If, on the other hand, the D2 receptor antagonist sulpiride was used, no reduction in the nitrogen-induced LDH-release was found (Fig.  3b ). Both compounds did not change the xenon-induced suppression of cellular damage.Cellular damage induced by external addition of dopamine To analyze if indeed the increased external dopamine was detrimental to cells, they were incubated in the presence of 100 nM dopamine, either for 30 min followed by 120 min in normal medium, or continuously for 150 min. As shown in fig.  4 , column (c), even the 30 min incubation with 100 nm dopamine (the lesser of the two dopamine challenges) was sufficient to cause considerable cell damage. Such damage was further increased if dopamine was present for the whole period of time of 150 min (column (e). We asked then if xenon not only prevented the release of dopamine in a hypoxic situation but could even reduce the damage caused by external dopamine. Cells were incubated for 30 min in normal buffer containing 100 nM dopamine followed by 120 min in xenon-saturated buffer, without dopamine. As shown in column (d), the dopamine-induced damage to the cells as seen in column (c) was significantly reduced. If the cells were incubated for 150 min in xenon-saturated buffer containing dopamine, even under those conditions the damage was low compared to cells exposed to dopamine in normal buffer (column (f)).
Cellular damage induced by external addition of dopamine
To analyze if indeed the increased external dopamine was detrimental to cells, they were incubated in the presence of 100 nM dopamine, either for 30 min followed by 120 min in normal medium, or continuously for 150 min. As shown in fig.  4 , column (c), even the 30 min incubation with 100 nm dopamine (the lesser of the two dopamine challenges) was sufficient to cause considerable cell damage. Such damage was further increased if dopamine was present for the whole period of time of 150 min (column (e). We asked then if xenon not only prevented the release of dopamine in a hypoxic situation but could even reduce the damage caused by external dopamine. Cells were incubated for 30 min in normal buffer containing 100 nM dopamine followed by 120 min in xenon-saturated buffer, without dopamine. As shown in column (d), the dopamine-induced damage to the cells as seen in column (c) was significantly reduced. If the cells were incubated for 150 min in xenon-saturated buffer containing dopamine, even under those conditions the damage was low compared to cells exposed to dopamine in normal buffer (column (f)).Buffering of intracellular Ca 2+ -ions using BAPTA In order to test if changes in intracellular Ca 2+  were required for the neuroprotective effect of xenon, cells were incubated with the cell-permeant Ca-chelator BAPTA-AM. As shown in fig.  5 , chelating intracellular Ca 2+  does not damage the cells  per se  (control + 10 μM BAPTA). Surprisingly, such chelation reduces significantly the neuroprotective effect of xenon, indicating an essential role for intracellular Ca 2+  for this effect to occur. A slight but significant reduction in cellular damage is observed when BAPTA-treated cells are incubated in nitrogen-saturated buffer.
Buffering of intracellular Ca 2+ -ions using BAPTA
In order to test if changes in intracellular Ca 2+  were required for the neuroprotective effect of xenon, cells were incubated with the cell-permeant Ca-chelator BAPTA-AM. As shown in fig.  5 , chelating intracellular Ca 2+  does not damage the cells  per se  (control + 10 μM BAPTA). Surprisingly, such chelation reduces significantly the neuroprotective effect of xenon, indicating an essential role for intracellular Ca 2+  for this effect to occur. A slight but significant reduction in cellular damage is observed when BAPTA-treated cells are incubated in nitrogen-saturated buffer.per seComparison with another dopaminergic cell system To exclude that the results obtained were limited to the PC-12-system itself, hypoxia-induced dopamine release and cell damage was investigated in rat embryonic primary mesencephalic cell cultures that are known to contain 0.5 – 1.5% of dopaminergic cells [ 26 ]. As shown in fig.  6 , in an hypoxic atmosphere a very similar pattern of dopamine and LDH release is obtained compared to PC-12 cells. Xenon prevents also in these primary cells the hypoxia-induced neurotransmitter and LDH release.
Comparison with another dopaminergic cell system
To exclude that the results obtained were limited to the PC-12-system itself, hypoxia-induced dopamine release and cell damage was investigated in rat embryonic primary mesencephalic cell cultures that are known to contain 0.5 – 1.5% of dopaminergic cells [ 26 ]. As shown in fig.  6 , in an hypoxic atmosphere a very similar pattern of dopamine and LDH release is obtained compared to PC-12 cells. Xenon prevents also in these primary cells the hypoxia-induced neurotransmitter and LDH release.Discussion In hypoxia/ischemia a key feature of secondary damage after the primary neuron-damaging event is the over-release of neurotransmitters [ 17 ]. Consequently, an interference with the hypoxia-induced release mechanism with respect to its control systems may be extremely useful to reduce cellular damage. The results presented here show that xenon has such properties, namely to prevent cellular damage and neurotransmitter release in a hypoxic situation thus qualifying it as an almost ideal early neuroprotectant. Concerning possible cellular targets for xenon, a first indication for the participation of Ca 2+ -regulated events was obtained when it was shown that xenon blocked cells in metaphase and that the block could be lifted by artificial small intracellular Ca 2+ - increases [ 18 ]. Since the CaM KII complex is known to play a decisive role in the metaphase/anaphase transition, it was tested if the CaMKII-inhibitor KN-93 had likewise metaphase-blocking properties. Such effects were obtained [ 19 ]. It is well known that in dopaminergic differentiated PC-12 cells, the CaMKII complex is involved in the regulation of neurotransmitter release [ 20 - 22 ] as well as its participation in a multitude of other Ca 2+ -dependent regulatory events [ 23 ]. Thus, it appears to be plausible that one of the targets for xenon might be the CaMKII complex, either directly or via interference with other Ca 2+ -dependent systems. One of those may be the Ca 2+ /calmodulin-activated calcineurin system that has been implicated in the regulation of monoamine release [ 24 ]. Alternatively, xenon might interact upstream of these regulatory systems with other Ca 2+ -dependent events required to occur in hypoxia-induced cell damage. Such a scenario is suggested by our demonstration that the neuroprotective effect of xenon is strongly reduced if PC-12 cells are loaded with BAPTA. Thus, at present all evidence obtained by us [ 13 , 14 , 18 , 19 ] and others [ 15 , 16 ] establish a complex and composite picture of targets susceptible to xenon including NMDA receptors, Ca 2+ -regulating and -regulated systems up to the activaton of transcription factors whereby such targets are probably not essentially and sequentially linked to each other. To summarize briefly our main findings: (1) The presence of xenon blocks hypoxia-induced dopamine release in dopaminergic cells. (2) Hypoxia-induced dopamine increase is caused by an enhanced release of dopamine rather than a reduced uptake of dopamine. (3) When measuring LDH release as a marker of cellular damage, xenon was found to block such release, which suggests that xenon reduces hypoxia-induced cellular damage. (4) Increased extracellular dopamine can damage dopaminergic cells directly. This is mainly mediated by D1 receptor agonism rather than D2. (5) Such direct extracellular dopamine-induced damage can be reduced by the presence of xenon, even when the increase in extracellular dopamine has not been caused by an episode of cellular hypoxia. (6) The above described protective effects of xenon depend on the presence of calcium ions. Further studies will show if indeed in the hypoxic cell multiple intracellular targets exist for xenon and how they are orchestrated together to result in cellular protection.
Discussion
In hypoxia/ischemia a key feature of secondary damage after the primary neuron-damaging event is the over-release of neurotransmitters [ 17 ]. Consequently, an interference with the hypoxia-induced release mechanism with respect to its control systems may be extremely useful to reduce cellular damage. The results presented here show that xenon has such properties, namely to prevent cellular damage and neurotransmitter release in a hypoxic situation thus qualifying it as an almost ideal early neuroprotectant. Concerning possible cellular targets for xenon, a first indication for the participation of Ca 2+ -regulated events was obtained when it was shown that xenon blocked cells in metaphase and that the block could be lifted by artificial small intracellular Ca 2+ - increases [ 18 ]. Since the CaM KII complex is known to play a decisive role in the metaphase/anaphase transition, it was tested if the CaMKII-inhibitor KN-93 had likewise metaphase-blocking properties. Such effects were obtained [ 19 ]. It is well known that in dopaminergic differentiated PC-12 cells, the CaMKII complex is involved in the regulation of neurotransmitter release [ 20 - 22 ] as well as its participation in a multitude of other Ca 2+ -dependent regulatory events [ 23 ]. Thus, it appears to be plausible that one of the targets for xenon might be the CaMKII complex, either directly or via interference with other Ca 2+ -dependent systems. One of those may be the Ca 2+ /calmodulin-activated calcineurin system that has been implicated in the regulation of monoamine release [ 24 ]. Alternatively, xenon might interact upstream of these regulatory systems with other Ca 2+ -dependent events required to occur in hypoxia-induced cell damage. Such a scenario is suggested by our demonstration that the neuroprotective effect of xenon is strongly reduced if PC-12 cells are loaded with BAPTA. Thus, at present all evidence obtained by us [ 13 , 14 , 18 , 19 ] and others [ 15 , 16 ] establish a complex and composite picture of targets susceptible to xenon including NMDA receptors, Ca 2+ -regulating and -regulated systems up to the activaton of transcription factors whereby such targets are probably not essentially and sequentially linked to each other.To summarize briefly our main findings: (1) The presence of xenon blocks hypoxia-induced dopamine release in dopaminergic cells. (2) Hypoxia-induced dopamine increase is caused by an enhanced release of dopamine rather than a reduced uptake of dopamine. (3) When measuring LDH release as a marker of cellular damage, xenon was found to block such release, which suggests that xenon reduces hypoxia-induced cellular damage. (4) Increased extracellular dopamine can damage dopaminergic cells directly. This is mainly mediated by D1 receptor agonism rather than D2. (5) Such direct extracellular dopamine-induced damage can be reduced by the presence of xenon, even when the increase in extracellular dopamine has not been caused by an episode of cellular hypoxia. (6) The above described protective effects of xenon depend on the presence of calcium ions.Further studies will show if indeed in the hypoxic cell multiple intracellular targets exist for xenon and how they are orchestrated together to result in cellular protection.Conclusions Based on the present results obtained with NGF-differentiated PC-12 cells and on the literature cited in this paper, xenon appears to be a neuroprotectant for a broad spectrum of neuronal cells; given its proven non-toxicity based on its long clinical use, it may come close to fulfilling the requirements for an ideal or "gold standard" neuroprotectant.
Conclusions
Based on the present results obtained with NGF-differentiated PC-12 cells and on the literature cited in this paper, xenon appears to be a neuroprotectant for a broad spectrum of neuronal cells; given its proven non-toxicity based on its long clinical use, it may come close to fulfilling the requirements for an ideal or "gold standard" neuroprotectant.Methods Cells Rat pheochromocytoma cells (PC-12) were maintained in RPMI 1640 medium containing 5% fetal calf serum, 10% horse serum, at 37°C, 5% CO 2 . For experiments, cells were seeded in 24-well plates at a density of 1 × 10 5  cells/well and nerve-growth-factor (Promega, Heidelberg, Germany) was added (0.4 μg/ml) whereupon cells entered differentiation. They were used on day five after the addition of growth factor [ 25 ]. Primary dopaminergic cells from rat embryonic brain were prepared as described (26) and used on day 14. Determination of dopamine and LDH Hypoxia-treatment was performed as described [ 13 ]. Samples from individual wells were taken at the intervals indicated and deproteinated using 5% perchloric acid (1:1 = vol/vol). Supernatants were transferred to Eppendorff tubes and the same volume (0.5 ml) of 0.4 M perchloric acid was added, mixed on vortex and centrifuged (6000 rpm, 3 min) to remove cell debris. Dopamine concentration was determined by high-pressure liquid chromatography (Bio-Tek, Neufahrn, Germany) using an electrochemical detector (Biometra, Göttingen, Germany). Cellular damage after the experiment was assessed by measuring spectrophotometrically the concentration of LDH in the original supernatant, before the addition of perchloric acid (Roche Diagnostics, Mannheim, Germany). Chemicals Gases were supplied by AGA-Linde (Berlin, Germany). 1,2- bis (2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid  tetrakis (acetoxymethyl) ester (BAPTA-AM) was purchased from Molecular Probes, (Leiden, The Netherlands), and all standard chemical products were obtained from Merck (Berlin, Germany). Statistical analysis All experiments were repeated at least five times, i.e. in five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups were analyzed using one-way ANOVA with Dunnett's multiple comparison post test or two-way ANOVA with Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with  p  values less than 0.05 were considered statistically significant.
Methods
Cells Rat pheochromocytoma cells (PC-12) were maintained in RPMI 1640 medium containing 5% fetal calf serum, 10% horse serum, at 37°C, 5% CO 2 . For experiments, cells were seeded in 24-well plates at a density of 1 × 10 5  cells/well and nerve-growth-factor (Promega, Heidelberg, Germany) was added (0.4 μg/ml) whereupon cells entered differentiation. They were used on day five after the addition of growth factor [ 25 ]. Primary dopaminergic cells from rat embryonic brain were prepared as described (26) and used on day 14.
Cells
Rat pheochromocytoma cells (PC-12) were maintained in RPMI 1640 medium containing 5% fetal calf serum, 10% horse serum, at 37°C, 5% CO 2 . For experiments, cells were seeded in 24-well plates at a density of 1 × 10 5  cells/well and nerve-growth-factor (Promega, Heidelberg, Germany) was added (0.4 μg/ml) whereupon cells entered differentiation. They were used on day five after the addition of growth factor [ 25 ]. Primary dopaminergic cells from rat embryonic brain were prepared as described (26) and used on day 14.Determination of dopamine and LDH Hypoxia-treatment was performed as described [ 13 ]. Samples from individual wells were taken at the intervals indicated and deproteinated using 5% perchloric acid (1:1 = vol/vol). Supernatants were transferred to Eppendorff tubes and the same volume (0.5 ml) of 0.4 M perchloric acid was added, mixed on vortex and centrifuged (6000 rpm, 3 min) to remove cell debris. Dopamine concentration was determined by high-pressure liquid chromatography (Bio-Tek, Neufahrn, Germany) using an electrochemical detector (Biometra, Göttingen, Germany). Cellular damage after the experiment was assessed by measuring spectrophotometrically the concentration of LDH in the original supernatant, before the addition of perchloric acid (Roche Diagnostics, Mannheim, Germany).
Determination of dopamine and LDH
Hypoxia-treatment was performed as described [ 13 ]. Samples from individual wells were taken at the intervals indicated and deproteinated using 5% perchloric acid (1:1 = vol/vol). Supernatants were transferred to Eppendorff tubes and the same volume (0.5 ml) of 0.4 M perchloric acid was added, mixed on vortex and centrifuged (6000 rpm, 3 min) to remove cell debris. Dopamine concentration was determined by high-pressure liquid chromatography (Bio-Tek, Neufahrn, Germany) using an electrochemical detector (Biometra, Göttingen, Germany). Cellular damage after the experiment was assessed by measuring spectrophotometrically the concentration of LDH in the original supernatant, before the addition of perchloric acid (Roche Diagnostics, Mannheim, Germany).Chemicals Gases were supplied by AGA-Linde (Berlin, Germany). 1,2- bis (2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid  tetrakis (acetoxymethyl) ester (BAPTA-AM) was purchased from Molecular Probes, (Leiden, The Netherlands), and all standard chemical products were obtained from Merck (Berlin, Germany).
Chemicals
Gases were supplied by AGA-Linde (Berlin, Germany). 1,2- bis (2-Aminophenoxy)ethane-N,N,N',N'-tetraacetic acid  tetrakis (acetoxymethyl) ester (BAPTA-AM) was purchased from Molecular Probes, (Leiden, The Netherlands), and all standard chemical products were obtained from Merck (Berlin, Germany).bistetrakisStatistical analysis All experiments were repeated at least five times, i.e. in five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups were analyzed using one-way ANOVA with Dunnett's multiple comparison post test or two-way ANOVA with Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with  p  values less than 0.05 were considered statistically significant.
Statistical analysis
All experiments were repeated at least five times, i.e. in five different plates on five different days. The data were presented as means ± SEM. The results of multiple groups were analyzed using one-way ANOVA with Dunnett's multiple comparison post test or two-way ANOVA with Bonferroni posttests using GraphPad Prism version 3.00 for Windows, GraphPad Software, San Diego California USA. Differences with  p  values less than 0.05 were considered statistically significant.pAuthors' contributions CP conceived the study, participated in its design and coordination, carried out the cellular studies involving the various gas treatments, participated in the preparation of the cells, and drafted the manuscript. PB and WS participated in the design of the experiments and the gas applications, JM performed neurotransmitter analysis and LDH determinations, and WK participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.
Authors' contributions
CP conceived the study, participated in its design and coordination, carried out the cellular studies involving the various gas treatments, participated in the preparation of the cells, and drafted the manuscript. PB and WS participated in the design of the experiments and the gas applications, JM performed neurotransmitter analysis and LDH determinations, and WK participated in the design of the study and performed the statistical analysis. All authors read and approved the final manuscript.


Background Microarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results.
Background
Microarrays have emerged as the preferred platform for high throughput gene expression analysis. Cross-hybridization among genes with high sequence similarities can be a source of error reducing the reliability of DNA microarray results.Results We have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at  .
Results
We have developed a tool called XHM (cross hybridization on microarrays) for assessment of the reliability of hybridization signals by detecting potential cross-hybridizations on DNA microarrays. This is done by comparing the sequences of the probes against an extensive database representing the transcriptome of the organism in question. XHM is available online at  .Conclusions Using XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results.
Conclusions
Using XHM with its user-adjustable parameters will enable scientists to check their lists of differentially expressed genes from microarray experiments for potential cross-hybridizations. This provides information that may be useful in the validation of the microarray results.

Background The development of DNA microarrays has revolutionized high throughput gene expression analysis. The two main platforms are: cDNA microarrays, where PCR products of individual cDNA fragments are immobilized on glass slides [ 1 ], and oligonucleotide microarrays, where oligonucleotides  in situ  synthesized or spotted on glass slides are used [ 2 , 3 ]. In a typical cDNA microarray experiment, total cellular RNA from two sources, a reference (or control) and an experimental sample are converted to cDNA by reverse transcription, labeled with two different fluorescent colors, and hybridized to an array of cDNA probes. One of the major concerns of cDNA microarrays is cross-hybridization of the labeled RNA (or cDNA) to non-target homologous probe sequences on the array [ 4 , 5 ]. Cross-hybridizations that may arise due to poly(A)-tail of mRNA or repetitive elements may be reduced or eliminated using hybridization blocking reagents like poly(A) oligonucleotides [ 6 ] or Cot1 DNA. Another major source of cross-hybridization may come from conserved sequences shared by two or more cDNAs, such as gene family members [ 4 , 7 ]. Analysis of sequenced multicellular eukaryotic genomes suggests that a large percentage of genes belong to gene families, some of which have high sequence similarities [ 8 ]. A recent study reported that approximately 17% of human transcripts in the UniGene database contain perfect match repeats of 20 bp minimum lengths [ 9 ]. From this study, however, the frequency of long, non-perfect match sequences such as those shared among paralogs, was not clear. Some studies have suggested that cross-hybridization can be a source of errors in cDNA microarray experiments [ 3 , 4 , 7 , 9 , 10 ]. In general, these studies indicate that cDNAs with nucleotide sequence identities higher than 70–80% over a certain length show significant levels of cross-hybridization. Analysis of chemokines, cytochrome P450 isozymes, G proteins and protease homologous gene families showed that cross-hybridization signals of 0.6–12% and 26–57% could arise from shared nucleotide identities of 55–80% and >80%, respectively [ 4 ]. Using synthetic 50 mer oligonucleotides, it was also shown that non-target sequences with >75–80% identity to 50 mer probes in microarrays could result in cross-hybridization [ 3 ]. In addition, cross-hybridization was also observed if a non-target sequence included stretches of ≥ 15 continuous bases identical to a 50 mer probe sequence [ 3 ]. Cross-hybridization is thought to contribute to discrepancies of results observed between oligonucleotide arrays and cDNA arrays [ 11 ]. Cross-hybridization may occur for both oligonucleotide and cDNA microarrays. An advantage with oligonucleotide arrays is that the complete sequences of the probes are known. Because cDNA arrays are constructed from PCR products of cDNA clones sequenced from 3'- or 5'-ends, the complete sequences of the spotted probes may not be available. We have not been able to identify programs or web servers capable of doing flexible cross-hybridization analysis. Programs with related functionality have been described, including ProbeWiz [ 12 ], OligoWiz [ 13 ] and PROBEmer [ 14 ], but these are for designing specific probes by avoiding regions of the cDNAs where there may be a cross-hybridization problem. For the assessment of the cross-hybridization potential in DNA microarray analysis, a more specific tool is required. In the present study we limit ourselves to the following problem: Given a probe (or a set of probes), identify the targets to which it can hybridize given user-defined criteria. The exact criteria to be used to decide whether two genes with high sequence similarity can cross-hybridize will depend on a number of factors, including the set of probes used and the experimental protocols – in particular hybridization and washing stringencies. We propose to use generic forms of criteria allowing the user to choose parameters in order to define the criteria for cross-hybridization. We investigate whether BLAST can be used to detect targets satisfying a set of criteria for cross-hybridization, based on previous experimental findings [ 3 , 4 ], and assess different (transcript) databases in order to evaluate their suitability for our purpose. A web tool is developed that allows the user to query precomputed results from several probe sets, or to analyze own probes with respect to a database representing transcripts in the organism where the microarray experiments are performed. The tool is applicable to analysis of both oligonucleotide and cDNA probes. Below we describe the developed XHM (cross hybridization on microarrays) system. We also include analysis performed in order to identify appropriate analysis methods and databases. Furthermore we describe the web server for XHM. In the final section we report some example results using our system on three cDNA probe sets for human, mouse and rat, and one oligonucleotide probe set for mouse.
Background
The development of DNA microarrays has revolutionized high throughput gene expression analysis. The two main platforms are: cDNA microarrays, where PCR products of individual cDNA fragments are immobilized on glass slides [ 1 ], and oligonucleotide microarrays, where oligonucleotides  in situ  synthesized or spotted on glass slides are used [ 2 , 3 ]. In a typical cDNA microarray experiment, total cellular RNA from two sources, a reference (or control) and an experimental sample are converted to cDNA by reverse transcription, labeled with two different fluorescent colors, and hybridized to an array of cDNA probes.in situOne of the major concerns of cDNA microarrays is cross-hybridization of the labeled RNA (or cDNA) to non-target homologous probe sequences on the array [ 4 , 5 ]. Cross-hybridizations that may arise due to poly(A)-tail of mRNA or repetitive elements may be reduced or eliminated using hybridization blocking reagents like poly(A) oligonucleotides [ 6 ] or Cot1 DNA. Another major source of cross-hybridization may come from conserved sequences shared by two or more cDNAs, such as gene family members [ 4 , 7 ]. Analysis of sequenced multicellular eukaryotic genomes suggests that a large percentage of genes belong to gene families, some of which have high sequence similarities [ 8 ]. A recent study reported that approximately 17% of human transcripts in the UniGene database contain perfect match repeats of 20 bp minimum lengths [ 9 ]. From this study, however, the frequency of long, non-perfect match sequences such as those shared among paralogs, was not clear.Some studies have suggested that cross-hybridization can be a source of errors in cDNA microarray experiments [ 3 , 4 , 7 , 9 , 10 ]. In general, these studies indicate that cDNAs with nucleotide sequence identities higher than 70–80% over a certain length show significant levels of cross-hybridization. Analysis of chemokines, cytochrome P450 isozymes, G proteins and protease homologous gene families showed that cross-hybridization signals of 0.6–12% and 26–57% could arise from shared nucleotide identities of 55–80% and >80%, respectively [ 4 ]. Using synthetic 50 mer oligonucleotides, it was also shown that non-target sequences with >75–80% identity to 50 mer probes in microarrays could result in cross-hybridization [ 3 ]. In addition, cross-hybridization was also observed if a non-target sequence included stretches of ≥ 15 continuous bases identical to a 50 mer probe sequence [ 3 ]. Cross-hybridization is thought to contribute to discrepancies of results observed between oligonucleotide arrays and cDNA arrays [ 11 ].Cross-hybridization may occur for both oligonucleotide and cDNA microarrays. An advantage with oligonucleotide arrays is that the complete sequences of the probes are known. Because cDNA arrays are constructed from PCR products of cDNA clones sequenced from 3'- or 5'-ends, the complete sequences of the spotted probes may not be available.We have not been able to identify programs or web servers capable of doing flexible cross-hybridization analysis. Programs with related functionality have been described, including ProbeWiz [ 12 ], OligoWiz [ 13 ] and PROBEmer [ 14 ], but these are for designing specific probes by avoiding regions of the cDNAs where there may be a cross-hybridization problem. For the assessment of the cross-hybridization potential in DNA microarray analysis, a more specific tool is required.In the present study we limit ourselves to the following problem: Given a probe (or a set of probes), identify the targets to which it can hybridize given user-defined criteria.The exact criteria to be used to decide whether two genes with high sequence similarity can cross-hybridize will depend on a number of factors, including the set of probes used and the experimental protocols – in particular hybridization and washing stringencies. We propose to use generic forms of criteria allowing the user to choose parameters in order to define the criteria for cross-hybridization.We investigate whether BLAST can be used to detect targets satisfying a set of criteria for cross-hybridization, based on previous experimental findings [ 3 , 4 ], and assess different (transcript) databases in order to evaluate their suitability for our purpose. A web tool is developed that allows the user to query precomputed results from several probe sets, or to analyze own probes with respect to a database representing transcripts in the organism where the microarray experiments are performed. The tool is applicable to analysis of both oligonucleotide and cDNA probes.Below we describe the developed XHM (cross hybridization on microarrays) system. We also include analysis performed in order to identify appropriate analysis methods and databases. Furthermore we describe the web server for XHM. In the final section we report some example results using our system on three cDNA probe sets for human, mouse and rat, and one oligonucleotide probe set for mouse.Implementation The XHM system queries the given nucleotide sequences (for example, a list of differentially regulated genes from DNA microarray experiments) against a database representing all possible targets (transcripts) in the organism being studied, and produces a list of all targets that can hybridize to each input sequence under the defined criteria. In this section we evaluate databases for use in the XHM system and we evaluate whether BLAST can be used to identify potential cross-hybridizing genes. Definitions For the calculation of melting temperature, the following formula is used [ 15 ]: Tm =  81.5 + 16.6 *  log  [ Na + ] + 41 * ( numG + numC )/ n  - 500/ n where [ Na + ] is the  Na + concentration,  numG  and  numC  is the number of Gs and Cs in the given alignment, and  n  is the total number of nucleotides aligned. We differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays [ 3 ]: •  Type A  similarities for sequence segments of a certain minimum length with a defined minimum percentage identity (long alignments with mismatch). •  Type B  similarities for identical segments of some minimum length (short perfect match). To perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001) [ 16 ] was used. The default settings of  blastn  are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions. Databases and sequences In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k  Homo sapiens  (Hs) and the 14 k  Rattus norvegicus  (Rn) clone sets are I.M.A.G.E. Consortium [LLNL] cDNA clones [ 17 , 18 ] obtained from Research Genetics (Huntsville, AL). The 15 k  Mus musculus  (Mm) clone set is from NIA [ 19 ]. In addition, oligonucleotide sequences (Mouse Genome Array Ready Oligonucleotide Set) from QIAGEN Operon [ 20 ] have been used to test the application. The details of the databases used to search for possible cross-hybridizations are as follows: • RefSeq Version 2 – RefSeq mRNA collection [ 21 ]. Number of sequences: 25377/21200/21724 (mouse/rat/human). • UniGene – UniGene Unique, build 129/123/164 (mouse/rat/human) [ 22 ]. Number of sequences: 87495/50137/118326 (mouse/rat/human). • TIGR gene index [ 23 ] – Only tentative consensus sequences (TCs), version 020403/042503/020503 (mouse/rat/human). Number of sequences 58129/51330/187287 (mouse/rat/human). • BeGIn – Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat). BeGIn is an alternative database we have developed (using tools described in [ 24 ]), by producing consensus sequences from each cluster in UniGene (build 129/123 for mouse/rat). In order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study. Overview of the XHM system The basis of the XHM system is a BLAST search (see Figure  1 ). The BLAST search provides one or several alignments between the probe sequence and each of the possible cross-hybridizing target sequences. The alignments are analyzed with respect to whether they fulfill the user-defined criteria for  type A  or  type B  matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3' or 5' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect. Appropriateness of BLAST Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets ( T 1 ... T n ) for a number of probes ( P 1 ... P n ). The simulated targets were ensured to satisfy the criteria described in [ 3 ] (50 bp sequences with at least 75% identity or 15 bp identical sequence). BLAST was then used to query the original probes against a large database containing the designed targets. It was then checked whether or not  T i  was contained in  P i 's hitlist. We found that  type A  matches can be missed if they do not contain any one stretch identical to the probe of length larger than BLAST's initial word length. Our experiments also showed that, depending on the probe type and the criteria used, we sometimes had to allow BLAST to generate very long lists of hits and using very high (permissive) cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server. A web server A web interface has been designed   with two entry points for accessing cross-hybridization information (see Figure  1 ). The user may either query a database of precomputed alignments, or compute new alignments using a real-time BLAST search. Both versions have parameters for the different thresholds and output alternatives. For ease of use, it is possible to run queries with no parameters explicitly specified. When querying with a large number of clones against a large database, running BLAST may be time consuming. Therefore, we pre-run BLAST with different clone-sets against different databases, and store the results. In this way the user may experiment with different thresholds for a set of clones repeatedly, and get the results within seconds. Nucleotide sequences or GenBank accession numbers may be used as input to the system when running the real-time BLAST searches. Searching using the precomputed BLAST alignments does not accept nucleotide sequences as input. User-adjustable input parameters Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for  type A  hits (defaults are 75% over 50 bp) and minimum length for  type B  hits (default is 15 bp), based on the findings described in [ 3 ], Na +  concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na +  concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence. In addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments. Output The output from the XHM system consists of different parts (see Figure  2 ). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected. For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST – number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B).
Implementation
The XHM system queries the given nucleotide sequences (for example, a list of differentially regulated genes from DNA microarray experiments) against a database representing all possible targets (transcripts) in the organism being studied, and produces a list of all targets that can hybridize to each input sequence under the defined criteria.In this section we evaluate databases for use in the XHM system and we evaluate whether BLAST can be used to identify potential cross-hybridizing genes.Definitions For the calculation of melting temperature, the following formula is used [ 15 ]: Tm =  81.5 + 16.6 *  log  [ Na + ] + 41 * ( numG + numC )/ n  - 500/ n where [ Na + ] is the  Na + concentration,  numG  and  numC  is the number of Gs and Cs in the given alignment, and  n  is the total number of nucleotides aligned. We differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays [ 3 ]: •  Type A  similarities for sequence segments of a certain minimum length with a defined minimum percentage identity (long alignments with mismatch). •  Type B  similarities for identical segments of some minimum length (short perfect match). To perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001) [ 16 ] was used. The default settings of  blastn  are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions.
Definitions
For the calculation of melting temperature, the following formula is used [ 15 ]:Tm =  81.5 + 16.6 *  log  [ Na + ] + 41 * ( numG + numC )/ n  - 500/ nTm =logNanumG + numCnnwhere [ Na + ] is the  Na + concentration,  numG  and  numC  is the number of Gs and Cs in the given alignment, and  n  is the total number of nucleotides aligned.NaNanumGnumCnWe differentiate between two kinds of sequence similarity cutoffs for cross-hybridization, based on the situations described for oligonucleotide microarrays [ 3 ]:•  Type A  similarities for sequence segments of a certain minimum length with a defined minimum percentage identity (long alignments with mismatch).Type A•  Type B  similarities for identical segments of some minimum length (short perfect match).Type BTo perform the searches in the different databases, NCBI BLAST version 2.2.1 (Mon Jul 9 14:02:00 EDT 2001) [ 16 ] was used. The default settings of  blastn  are used unless specified otherwise. This includes using the DUST filter for masking lowcomplexity regions.blastnDatabases and sequences In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k  Homo sapiens  (Hs) and the 14 k  Rattus norvegicus  (Rn) clone sets are I.M.A.G.E. Consortium [LLNL] cDNA clones [ 17 , 18 ] obtained from Research Genetics (Huntsville, AL). The 15 k  Mus musculus  (Mm) clone set is from NIA [ 19 ]. In addition, oligonucleotide sequences (Mouse Genome Array Ready Oligonucleotide Set) from QIAGEN Operon [ 20 ] have been used to test the application. The details of the databases used to search for possible cross-hybridizations are as follows: • RefSeq Version 2 – RefSeq mRNA collection [ 21 ]. Number of sequences: 25377/21200/21724 (mouse/rat/human). • UniGene – UniGene Unique, build 129/123/164 (mouse/rat/human) [ 22 ]. Number of sequences: 87495/50137/118326 (mouse/rat/human). • TIGR gene index [ 23 ] – Only tentative consensus sequences (TCs), version 020403/042503/020503 (mouse/rat/human). Number of sequences 58129/51330/187287 (mouse/rat/human). • BeGIn – Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat). BeGIn is an alternative database we have developed (using tools described in [ 24 ]), by producing consensus sequences from each cluster in UniGene (build 129/123 for mouse/rat). In order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study.
Databases and sequences
In this study we used three cDNA clone sets (corresponding to three cDNA sets spotted on microarrays). The 40 k  Homo sapiens  (Hs) and the 14 k  Rattus norvegicus  (Rn) clone sets are I.M.A.G.E. Consortium [LLNL] cDNA clones [ 17 , 18 ] obtained from Research Genetics (Huntsville, AL). The 15 k  Mus musculus  (Mm) clone set is from NIA [ 19 ]. In addition, oligonucleotide sequences (Mouse Genome Array Ready Oligonucleotide Set) from QIAGEN Operon [ 20 ] have been used to test the application. The details of the databases used to search for possible cross-hybridizations are as follows:Homo sapiensRattus norvegicusMus musculus• RefSeq Version 2 – RefSeq mRNA collection [ 21 ]. Number of sequences: 25377/21200/21724 (mouse/rat/human).• UniGene – UniGene Unique, build 129/123/164 (mouse/rat/human) [ 22 ]. Number of sequences: 87495/50137/118326 (mouse/rat/human).• TIGR gene index [ 23 ] – Only tentative consensus sequences (TCs), version 020403/042503/020503 (mouse/rat/human). Number of sequences 58129/51330/187287 (mouse/rat/human).• BeGIn – Bergen Gene Index version 1.0. Number of sequences: 100654/49285 (mouse/rat).BeGIn is an alternative database we have developed (using tools described in [ 24 ]), by producing consensus sequences from each cluster in UniGene (build 129/123 for mouse/rat).In order to achieve high specificity and sensitivity, we favored the databases where the highest number of probes were represented (completeness) and multiple matches per probe were minimized (minimum redundancy). Our criteria for choosing an appropriate database for a given organism was based on the results obtained for our probe sets. The presence of alternative splice forms in either the probe or the target set may complicate analysis. We do not consider this explicitly in the current study.Overview of the XHM system The basis of the XHM system is a BLAST search (see Figure  1 ). The BLAST search provides one or several alignments between the probe sequence and each of the possible cross-hybridizing target sequences. The alignments are analyzed with respect to whether they fulfill the user-defined criteria for  type A  or  type B  matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3' or 5' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect.
Overview of the XHM system
The basis of the XHM system is a BLAST search (see Figure  1 ). The BLAST search provides one or several alignments between the probe sequence and each of the possible cross-hybridizing target sequences. The alignments are analyzed with respect to whether they fulfill the user-defined criteria for  type A  or  type B  matches. The XHM system presents to the user a list of matches satisfying the criteria, and in addition information on GC-content in similar regions, estimated melting temperature (Tm) and position on the sequence (proximity to 3' or 5' end). Although the estimated Tm may not be completely accurate under microarray hybridization conditions, particularly on solid surfaces, it can give a relative measure of the impact of the potential cross-hybridization detected. An expert user may use the information to assess the likelihood and importance of the cross-hybridization effect.type Atype BAppropriateness of BLAST Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets ( T 1 ... T n ) for a number of probes ( P 1 ... P n ). The simulated targets were ensured to satisfy the criteria described in [ 3 ] (50 bp sequences with at least 75% identity or 15 bp identical sequence). BLAST was then used to query the original probes against a large database containing the designed targets. It was then checked whether or not  T i  was contained in  P i 's hitlist. We found that  type A  matches can be missed if they do not contain any one stretch identical to the probe of length larger than BLAST's initial word length. Our experiments also showed that, depending on the probe type and the criteria used, we sometimes had to allow BLAST to generate very long lists of hits and using very high (permissive) cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server.
Appropriateness of BLAST
Experiments were performed to assess whether BLAST is able to identify potentially cross-hybridizing targets. The analysis was done by generating simulated targets ( T 1 ... T n ) for a number of probes ( P 1 ... P n ). The simulated targets were ensured to satisfy the criteria described in [ 3 ] (50 bp sequences with at least 75% identity or 15 bp identical sequence). BLAST was then used to query the original probes against a large database containing the designed targets. It was then checked whether or not  T i  was contained in  P i 's hitlist.TT nPP nT iP iWe found that  type A  matches can be missed if they do not contain any one stretch identical to the probe of length larger than BLAST's initial word length. Our experiments also showed that, depending on the probe type and the criteria used, we sometimes had to allow BLAST to generate very long lists of hits and using very high (permissive) cutoffs on E-value in order to cover the intended targets. Part of the reason for this is that BLAST is designed to identify homologous sequences and the sequences satisfying the cross-hybridization criteria need not necessarily receive significant scores or E-values. Our conclusion was that BLAST can be used, but to achieve optimal results, non-default parameters for initial word size and mismatch penalty should be applied. This was taken into consideration in the web-server.type AA web server A web interface has been designed   with two entry points for accessing cross-hybridization information (see Figure  1 ). The user may either query a database of precomputed alignments, or compute new alignments using a real-time BLAST search. Both versions have parameters for the different thresholds and output alternatives. For ease of use, it is possible to run queries with no parameters explicitly specified. When querying with a large number of clones against a large database, running BLAST may be time consuming. Therefore, we pre-run BLAST with different clone-sets against different databases, and store the results. In this way the user may experiment with different thresholds for a set of clones repeatedly, and get the results within seconds. Nucleotide sequences or GenBank accession numbers may be used as input to the system when running the real-time BLAST searches. Searching using the precomputed BLAST alignments does not accept nucleotide sequences as input. User-adjustable input parameters Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for  type A  hits (defaults are 75% over 50 bp) and minimum length for  type B  hits (default is 15 bp), based on the findings described in [ 3 ], Na +  concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na +  concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence. In addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments. Output The output from the XHM system consists of different parts (see Figure  2 ). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected. For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST – number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B).
A web server
A web interface has been designed   with two entry points for accessing cross-hybridization information (see Figure  1 ). The user may either query a database of precomputed alignments, or compute new alignments using a real-time BLAST search. Both versions have parameters for the different thresholds and output alternatives. For ease of use, it is possible to run queries with no parameters explicitly specified.When querying with a large number of clones against a large database, running BLAST may be time consuming. Therefore, we pre-run BLAST with different clone-sets against different databases, and store the results. In this way the user may experiment with different thresholds for a set of clones repeatedly, and get the results within seconds.Nucleotide sequences or GenBank accession numbers may be used as input to the system when running the real-time BLAST searches. Searching using the precomputed BLAST alignments does not accept nucleotide sequences as input.User-adjustable input parameters Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for  type A  hits (defaults are 75% over 50 bp) and minimum length for  type B  hits (default is 15 bp), based on the findings described in [ 3 ], Na +  concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na +  concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence. In addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments.
User-adjustable input parameters
Some of the parameters are shared among the two main versions of the XHM system. These include minimum length and minimum percentage similarity for  type A  hits (defaults are 75% over 50 bp) and minimum length for  type B  hits (default is 15 bp), based on the findings described in [ 3 ], Na +  concentration (default is 0.1 M), and size of GC-clamp (default is 10). The Na +  concentration is used to calculate melting temperature, and the size of the GC-clamp is used to plot the GC-content throughout the query sequence.type Atype BIn addition, the real-time BLAST version also contains BLAST specific user-adjustable parameters. Main parameters include threshold on E-value (default is 10), number of alignments in the output from BLAST and whether DUST (low complexity) filter should be used. The user can adjust other BLAST specific parameters including gap opening and extension penalties, initial word size, mismatch penalty and whether or not to allow gapped alignments.Output The output from the XHM system consists of different parts (see Figure  2 ). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected. For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST – number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B).
Output
The output from the XHM system consists of different parts (see Figure  2 ). If there are several input sequences (batch query), the results are given for each sequence individually. For each probe the system generates a plot of GC-content along the sequence. This GC-plot may help the user to identify areas in the sequence with high GC content in order to evaluate the importance of the potential cross-hybridization detected.For each hit (possible hybridization), XHM presents the name and identifier of the hit, identity tuples (from BLAST – number of identical bases and number of bases in total in the BLAST alignment, or in a sub-alignment), start and end position on the probe, calculated Tm, percentage GC in alignment and type of hit (A or B).Results Choice of database Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table  1 . Using a 70% identity threshold for the  type A  similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the "full" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown). Application to microarray probes As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for  type A  similarities, and 25 nucleotides for the  type B  similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence ( type A ) and 20 nucleotides perfect identity ( type B ). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends. Results are shown in Table  1 . Using RefSeq, we observed that even though a substantial number of the 14 k rat cDNA probes (63.3%) had no  type A  hit, 809 (about 16.3% of the probes actually represented in RefSeq) had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%. For the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more  type A  hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits. Even though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database. The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available. The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy. The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%.
Results
Choice of database Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table  1 . Using a 70% identity threshold for the  type A  similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the "full" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown).
Choice of database
Experiments using the three cDNA sets and the oligonucleotide set as input to the XHM system yielded the results shown in Table  1 . Using a 70% identity threshold for the  type A  similarities, in the rat clone set the Rat Gene Index from TIGR appears to be the best choice. For the mouse clone set it appears that the BeGIn database is the best choice, and for the human clone set it seems that RefSeq or UniGene Unique may be two good choices. These evaluations were based on completeness (high number of probes represented) and minimum redundancy of the database. We used only the tentative consensuses (TCs) of the Gene Indices databases from TIGR. Using the "full" versions decreased the number of probes having zero hits, but substantially increased the number of probes having two or more hits (results not shown).type AApplication to microarray probes As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for  type A  similarities, and 25 nucleotides for the  type B  similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence ( type A ) and 20 nucleotides perfect identity ( type B ). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends. Results are shown in Table  1 . Using RefSeq, we observed that even though a substantial number of the 14 k rat cDNA probes (63.3%) had no  type A  hit, 809 (about 16.3% of the probes actually represented in RefSeq) had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%. For the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more  type A  hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits. Even though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database. The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available. The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy. The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%.
Application to microarray probes
As a practical experiment, we tested the XHM system on the cDNA probe sets for human, mouse and rat, and the mouse oligonucleotide set from QIAGEN. For the cDNA sets we used the following thresholds: 70% over 200 nucleotides for  type A  similarities, and 25 nucleotides for the  type B  similarities. For the oligonucleotides we used 70% identity over the whole oligonucleotide sequence ( type A ) and 20 nucleotides perfect identity ( type B ). Choosing the parameters is not trivial, and these thresholds, which are in the lower range of % similarities leading to cross-hybridizations, are meant as examples of a possible configuration. The XHM tool allows for full flexibility to experiment with the settings. The reason why we chose to consider a 200 nucleotides stretch for the cDNA probes is that sometimes these sequences contain errors, especially toward the ends.type Atype Btype Atype BResults are shown in Table  1 . Using RefSeq, we observed that even though a substantial number of the 14 k rat cDNA probes (63.3%) had no  type A  hit, 809 (about 16.3% of the probes actually represented in RefSeq) had two or more hits, indicating potential cross-hybridization. In most cases, a single hit represents the probe sequence itself, and does not represent a potential cross-hybridization. Looking at a more complete database, the TIGR Rat Gene Index, only 700 (5.2%) probes had no hits, and 2712 (21.3% of the probes represented in the database) had two or more hits. A conclusion from this was that the number of rat probes having potential cross-hybridizing partners was between 16% and 21%.type AFor the mouse cDNA probes the cross-hybridization numbers appeared to be higher. Using RefSeq, the number of probes having two or more  type A  hits was 3101 or 34.2% of the probes represented in the database. RefSeq is relatively incomplete, so this number may be a conservative estimate on the cross-hybridization occurrence. For the BeGIn database only 725 probes had no hits (5.3%) and 5211 probes (about 40% of the probes in the database) had two or more hits.type AEven though as many as 47.3% of the mouse probes were not found in the TIGR Gene Index, the proportion of probes found in the database that had two or more hits was 59%. This number is most likely an artifact caused by redundancy in the database.The difference in number of possible hybridizations found in mouse versus rat is most likely due to the fact that more mouse sequences are available.The human cDNA probes seems to have a lower cross-hybridization potential than the mouse probes. In UniGene Unique and RefSeq, looking only at the human probes that were found in the databases, just above 30% of them had two or more hits. The results using TIGR Human Gene Index suggested that almost 70% of the probes found in the database could cross-hybridize, but this is most likely an artifact caused by redundancy.The mouse oligonucleotide set is clearly less prone to cross-hybridization. Using the UniGene Unique database, we observed that 14% of the probes found in the database had two or more hits, whereas the corresponding number for the 15 k mouse cDNA probe set using UniGene Unique was 45%.Discussion A flexible tool for assessing the cross-hybridization potential of microarray probes has been developed and made available. Several transcriptome databases can be used for searching, and more may be added upon request. Using the XHM tool, analysis of three cDNA microarray probe sets and one oligonucleotide probe set revealed that a high proportion of the cDNA probes can potentially cross-hybridize with one or more other transcripts in the organism. As expected, compared to the cDNA probes, a smaller percentage of the mouse oligonucleotide probes showed a potential for cross-hybridization. This is because the oligonucleotide probe sequences are much shorter (69-mers), designed from specific regions of cDNAs to minimize cross-hybridizations. Despite increasing use of the more specific oligonucleotide arrays, cDNA sequences including full-length clone sets [ 25 , 26 ] are widely used in production of microarrays. Depending on the completeness and redundancy levels of the transcriptome databases used, with the chosen cutoff for  type A  similarities (at least 70% identity), 15–45% of the cDNA probes showed hybridization with two or more apparently different transcripts (disregarding the results using the TIGR Gene Indices). This high percentage of potentially cross-hybridizing genes suggests that it is essential to carefully validate results from microarray experiments, particularly where cDNA clones are used to prepare arrays. Although cross-hybridization is known as one of the main sources of errors of cDNA microarrays, the high proportions of cross-hybridizing genes detected in our test are likely to be overstated by the possible redundancies in the databases (not all entries represent unique genes). Also one may argue that the 70% identity threshold is somewhat low. Whether two different genes that are candidates for cross-hybridization actually lead to erroneous results in hybridization experiments will depend on factors such as the level of sequence identity, the stringency of the hybridization and the relative abundances of the transcripts. For example, two potentially cross-hybridizing genes do not necessarily pose a problem unless both are expressed in the tissue or cell-line analyzed. Quick inspection of the hit-list produced by the XHM tool for a typical input of differentially regulated genes will help in identifying significant noise from cross-hybridizations. A main advantage of the XHM tool is that it allows the user to perform searches at various stringencies to detect potential cross-hybridizations. Candidate genes from microarray analysis that show potential cross-hybridizations using the database search may then be further checked using other methods such as RT-PCR and Northern blot.
Discussion
A flexible tool for assessing the cross-hybridization potential of microarray probes has been developed and made available. Several transcriptome databases can be used for searching, and more may be added upon request. Using the XHM tool, analysis of three cDNA microarray probe sets and one oligonucleotide probe set revealed that a high proportion of the cDNA probes can potentially cross-hybridize with one or more other transcripts in the organism. As expected, compared to the cDNA probes, a smaller percentage of the mouse oligonucleotide probes showed a potential for cross-hybridization. This is because the oligonucleotide probe sequences are much shorter (69-mers), designed from specific regions of cDNAs to minimize cross-hybridizations. Despite increasing use of the more specific oligonucleotide arrays, cDNA sequences including full-length clone sets [ 25 , 26 ] are widely used in production of microarrays.Depending on the completeness and redundancy levels of the transcriptome databases used, with the chosen cutoff for  type A  similarities (at least 70% identity), 15–45% of the cDNA probes showed hybridization with two or more apparently different transcripts (disregarding the results using the TIGR Gene Indices). This high percentage of potentially cross-hybridizing genes suggests that it is essential to carefully validate results from microarray experiments, particularly where cDNA clones are used to prepare arrays.type AAlthough cross-hybridization is known as one of the main sources of errors of cDNA microarrays, the high proportions of cross-hybridizing genes detected in our test are likely to be overstated by the possible redundancies in the databases (not all entries represent unique genes). Also one may argue that the 70% identity threshold is somewhat low.Whether two different genes that are candidates for cross-hybridization actually lead to erroneous results in hybridization experiments will depend on factors such as the level of sequence identity, the stringency of the hybridization and the relative abundances of the transcripts. For example, two potentially cross-hybridizing genes do not necessarily pose a problem unless both are expressed in the tissue or cell-line analyzed. Quick inspection of the hit-list produced by the XHM tool for a typical input of differentially regulated genes will help in identifying significant noise from cross-hybridizations. A main advantage of the XHM tool is that it allows the user to perform searches at various stringencies to detect potential cross-hybridizations. Candidate genes from microarray analysis that show potential cross-hybridizations using the database search may then be further checked using other methods such as RT-PCR and Northern blot.Conclusions We have shown that a significant proportion of probes used in cDNA microarray analysis may show cross-hybridization with non-target sequences. We have developed a flexible tool, XHM, suitable for detecting potential cross-hybridization artifacts during microarray data analysis. The tool may also be used to select specific probes for preparation of microarrays.
Conclusions
We have shown that a significant proportion of probes used in cDNA microarray analysis may show cross-hybridization with non-target sequences. We have developed a flexible tool, XHM, suitable for detecting potential cross-hybridization artifacts during microarray data analysis. The tool may also be used to select specific probes for preparation of microarrays.Availability The XHM system is freely available at  . Program code for academic use can be supplied upon request to the author.
Availability
The XHM system is freely available at  . Program code for academic use can be supplied upon request to the author.Author's contributions KF implemented the system and made a draft of the manuscript. FY and AL contributed with ideas and proofread the manuscript. IJ has formulated and supervised the work, and edited the manuscript. All authors have read and approved the final manuscript.
Author's contributions
KF implemented the system and made a draft of the manuscript. FY and AL contributed with ideas and proofread the manuscript. IJ has formulated and supervised the work, and edited the manuscript. All authors have read and approved the final manuscript.