BACKGROUND
non-coding rnas have recently been reported as more common, more diverse, and accredited more important functions than previously anticipated  <cit> .
among the most abundant non-coding transcripts, there is a group called natural antisense transcripts  that carries regions of perfect complementarity to protein coding  rnas  <cit> .
in silico studies of available transcript sequence data have found that up to 24% of human protein coding loci also encode cis-nats  <cit> .
however, antisense transcripts tend to be poly negative and nuclear localized  <cit> .
if this is true, the abundance of nats  may be higher yet, since nuclear non-polyadenylated transcripts are underrepresented in transcript sequence databases.
this fact may have important implications for researchers, not only because of their potential biological function but they may also turn out to be influential on the interpretation of large experimental data sets.
for instance, the cdna microarray technique has been used in genome-wide expression studies to address basic questions about gene function and in the pursuit of a more precise molecular classification of tumors.
in this case, the ability to monitor the expression of thousands of genes simultaneously has allowed the identification of disease-specific subsets of genes useful to improve diagnosis and disease management  <cit> .
the majority of the more than  <dig>  microarray expression profiles released through ncbi was obtained with double stranded cdna capture probes and is assumed to reflect the pure expression of the sense transcripts used as templates for cdna synthesis.
however, the widespread expression of natural antisense transcripts  invalidates this assumption since double-stranded probes will show the combined expression of both the intended sense target and any nat with complementary sequence  <cit> .
still, for nine out of ten cases, signals from double-stranded cdna probes correlates with those obtained from sense specific oligonucleotide platforms  <cit> .
based on these observations, we reasoned that antisense transcripts are either not efficiently detected by conventional cdna capture probes or that important information must be hidden behind this paradox.
therefore, we modeled a typical cdna microarray tumor-classification analysis and compared the results from conventional double-stranded cdna capture probes with single stranded cdna capture probes capable of monitoring opposite strands of each cdna independently.
we detected a number of antisense signals that exceed by far the number of known antisense transcripts.
the detected signals showed a clear cell specific expression pattern with a common core group of antisenses expressed in all analyzed materials.
moreover, antisense transcripts displayed a prevalent tendency to be positively correlated with the expression of their corresponding sense counterparts.
this confirms the idea that a large part of the data obtained from conventional double-stranded cdna microarrays are in fact compounded signals product of both sense and antisense hybridization.
yet, detection of antisense transcription by conventional double-stranded cdna microarrays does not strongly distort the relationship between expression profiles of the analyzed samples compared with those obtained from pure sense signals.
this is most likely due to the observed coordinate regulation of senses and antisenses and a more efficient hybridization of sense strands because a different exon structure of antisense transcripts and the sense transcripts used for cdna synthesis.
RESULTS
production of single-stranded microarrays
we generated strand specific cdna probes in situ after covalently binding nh2-modified cdna inserts onto cross-linked n-hydroxysuccinamide slides in a strand specific manner.
specific binding of 5' dna ends serves two different but additive purposes.
first, 5' end-specific binding provide protection against in situ enzymatic attack of highly processive 5'-3' exonucleases; unbound strands could then be exposed to enzymatic degradation.
second, only 5'-end modified strands will be covalently bound, rendering non-modified strands vulnerable to easy removal by heat denaturation.
we found that the most reliable method for processing double stranded cdnas into single stranded capture probes was the sequential application of both approaches.
the procedure is schematically depicted in figure 1a.
to validate the method, microarrays containing single-stranded sense and antisense probes and double-stranded probes  were generated from a  <dig> kb fragment containing the β-lactamase gene.
hybridizations were performed with equimolar amounts of cy3- or cy5- direct-labelled sense and antisense β-lactamase transcripts .
sense probes showed strong signal from cy <dig> labelled sense cdna  and signal equivalent to background level in the cy <dig> channel .
contrary, antisense probes detected strong signal from cy5-labeled antisense cdna while cy <dig> signal remained equivalent to background .
we conclude that our method can produce selective single stranded dna capture probes from double-stranded pcr products in situ with an efficacy and specificity sufficient to eliminate detectable levels of complementary strand, thus producing a selective array of strand-specific capture probes.
strand-specific cdna microarrays
following this test, we prepared strand-specific sense and antisense cdna probes, and double-stranded probes, from  <dig> randomly selected full-length cdnas from the mgc clone collection.
both strand specifically 5' nh2-modified pcr products for sense and antisense probe generation and nh2-modified products at both 5' ends were printed on the same surface.
in the following, sense probes are termed c for coding and antisense probes n for non-coding.
double-stranded probes are termed cn.
to verify the specificity of our cdna capture probes, we hybridized the arrays with cy <dig> or cy <dig> labeled universal primers used to amplify all cdna inserts .
data from the test hybridization shows that cn probes produce log <dig> ratio intensities centered on  <dig>  demonstrating that they captured both labeled targets.
conversely, strand specific c and n probes produced log <dig> intensity ratios centered approximately on  <dig> and - <dig> respectively .
figure 2b show log <dig> ratios for c, n, and cn probe for each cdna clone sorted on ascending basis for respective cn probes.
with very few exceptions, each probe set consistently produced strand specific probes equally well across all cdnas.
antisense detection of n probes hybridized with total rnas
next, we modeled an experimental design typically used for tumor classification studies.
we performed hybridizations using direct-labeled randomly primed total rna extracted from eight breast-cancer cell lines and one derived from normal breast epithelium against universal human reference rna as a common reference following standard protocols .
to control for even processing into strand-specific probes we printed internal β-lactamase dna control spots in each block and included β-lactamase spike-in sense and antisense control targets in either samples or reference.
their hybridization signals confirmed that detected n signals reflect hybridization to the n probe over the entire array .
we initially analyzed the detection capacity of n probes.
we applied a stringent filter criterion including signals with signal-to-noise  ratios above  <dig>  across all nine hybridizations, we detected  <dig> n signals in the cell lines and  <dig> n signals in the reference, out of the set of  <dig> probes .
we filtered for non-specific cross-hybridization with repetitive elements in the probe sequence  <cit> , extended self-complementarity between the c and n probes, and c and n probes with sequence complementarity to the same transcript.
we identified repeat sequences of  <dig> or more continuous bases in  <dig> of the  <dig> cdna used for probe synthesis,  <dig> cases with extended self-complementarity between c and n probes and  <dig> cases for which the c and n probes matched the same refseq mrna  <cit> .
after filtering for potentially spurious signals,  <dig> n signals from the cell lines and  <dig> from the reference were interpreted as detecting putative antisense transcripts.
the n signals detected in the cell lines vary from 27%  to 74%  of the cdnas that also showed sense transcription.
we compared these signals with nats identified by the most comprehensive study published  <cit> .
despite significant variations in the number of n signals, the percentage of detected reported nats remains unchanged in each cell line .
in the reference channel, an average of  <dig> % cdnas detecting c signals also produced n signals .
as many as  <dig> putative antisense transcripts in the cell lines and  <dig> in the reference were detected in all nine hybridizations and, again, a similar proportion of reported antisense transcripts in each group were noted .
we observe greater variation in the number of detected n signals for the cell lines compared to the reference, indicating that biological variation is larger than variation resulting from technical replication of measurements .
here, we aimed to gain insight into the occurrence of nats and to analyze their influence in the context of microarray hybridizations rather than to rigorously screen for novel antisense transcripts.
however, n signals show biological variation that tend to be sample specific, yet reveal a common core detected in all experiments.
this group includes genes involved in apopotosis and programmed cell death.
if a significant proportion of these detected unannotated transcripts were random artifacts, we would expect the common core to show a lower proportion of known annotated transcripts.
nevertheless, it is remarkable that the proportion of annotated antisense transcripts for the common core is similar to that estimated for each cell line separately .
to verify the nature of the detected unannotated transcripts we radio-labeled sense strands from ten randomly selected cdnas by in vitro transcription and used them as hybridization riboprobes in northern blot hybridizations.
seven of the ten tested riboprobes were able to identify complementary transcripts in the total rna mixture .
these observations strengthen the view that most detected n signals might represent true nats .
this impression is reinforced by the fact that hybridization with tilling arrays predicted that only one tenth of the total number of transcription units is known in human  <cit>  and the observed well-defined expression pattern of n signals described subsequently.
we find no reasons to conceive that this extended antisense transcriptional activity is associated with the nature of the analyzed materials  as mcf- <dig>  derived from normal breast epithelia detected a number of antisense signals second closet to average.
in summary, as much as 88% of the tested conventional cdna probes could potentially produce microarray signals reflecting differences in sense or antisense expression, or both, in one sample or both of sample and reference.
*number of image clones where n probe snr > 10
**number of image clones previously reported  as transcribed from antisense
***number of image clones with > =  <dig> continuous bases homologous to repeats
****number of image clones with > =  <dig> continuous bases on both sense- and antisense strand that match refseq
+number of image clones with > =  <dig> continuous bases complementary between sense- and antisense transcript
++number of image clones with either repeats***, double hits****, or self-complementarity+
+++number of image clones that overlap between masked++and clones priviously reported**
++++number of image clones with putative antisense transcription, i.e.
those clones that are not masked++
$number of image clones previously reported as transcribed from antisense out of, putative antisense signals total++++
$$number of image clones not masked++ and not privously reported** as transcribed from antisense
$$$number of image clones with putative sense transcription, i.e.
where c probe snr >  <dig>  excluding clones that are masked++
$$$$percent of image clones displaying n signal out of those that display c signals excluding clones that are masked++
note: values are paired per clone, i.e.
the number of image clones with c signal and n signal as percentage of the number of image clones with c signal
relationship between c and n signals
to analyze closely antisense expression and to explore the extent to which antisense signals might influence protein-coding profiles detected by double-stranded microarrays, we jointly investigated the relationship between signal from c, n, and cn probes.
first, we visualized distances between expression profiles derived from separate probes using unsupervised cluster analysis.
we regarded expression data from different probes as individual data sets, separating hybridizations into one c, one n, and one cn profile matched on clone ids.
cluster analysis using probes derived from  <dig> cdna clones show that c and cn profiles co-segregate within each cell line .
the majority of n profiles form a cohesive cluster well separated from c and cn profiles for the same group of cell lines.
for two cell lines, the n, c and cn profiles separate from all other cell lines but within each group the c and cn profiles co-segregate with the n profile clearly separated.
visual inspection revealed that c and n signals seem to follow coordinated variation.
to corroborate this observation we calculated pair wise correlation between c signal and n signal across the nine hybridizations for corresponding cdna clones.
figure 4b shows the distribution of pearson correlations for  <dig> c and n signal pairs, evidencing a tendency for positive correlation between them.
the same coordinated pattern was apparent when only probe pairs from cdna clones with previously known nats were considered.
overall, only a minority of clones showed an inverse relationship between senses and antisense expression levels.
these probes seem to be responsible for the separation between c and n profiles.
a tendency for positively correlated co-expression of sense and antisense transcripts has been observed in large-scale studies performed in mouse  <cit> .
however, studies on human material claimed that expression of members of sense-antisense pairs follow an inverse relation which was supposed to agree with a mechanistic model according to which increasing expression of one of the members downregulates transcription of the other  <cit> .
our results contradict this observation.
to verify our observation by an alternative method, we selected the gene encoding human ketohexokinase as representative example .
in our microarray measurements, khk's transcription displayed a high positive correlation between n and c signals .
khk exons  <dig>   <dig> and  <dig> fully overlap clone cr <dig> which transcription proceeds in the antisense orientation.
we designed a forward primer on khk exon  <dig> and reverse primer on exon  <dig> targeting the regions of sequence complementarity in both cases.
due to different splicing, this primer pair would allow simultaneous amplification of a  <dig> bp khk fragment and a  <dig> bp product from the cr <dig> transcript .
figure 4d shows the outcome of these rt-pcr experiments.
a tight coordinated variation between expression levels of khk and its antisense transcript was obtained across all tested cell lines  confirming our microarray results.
thus, the analyzed genes appear to follow a pattern of more or less coordinated up- or down-regulation of both strands in parallel.
supported on previously reported data  <cit> , we interpret the coordinated expression of n with c signals as evidence for tight sense-antisense regulation rather than the effect of a stochastic transcriptional noise.
moreover, if sense/antisense expressions tend to be correlated for protein-coding loci, we speculated that profiles should not substantially differ, regardless of whether the array probes are single- or double-stranded.
single-stranded compared to conventional double stranded cdna arrays
to investigate further the connection between c and cn profiles we calculated pairwise euclidean distances between c, n and cn signals across cell lines.
taken together, these data indicate that c and cn profiles are more similar across the hybridized samples than either is to n profiles .
to exclude the possibility that a small number of outliers caused the difference, we investigated the distribution of n to c distance per cdna clone within hybridizations .
we found a very similar distribution for each hybridization suggesting that antisense transcription – while it is genome-wide – does not significantly distance profiles obtained from pure sense probes from those derived from dsdna.
to test this conjecture, we treated c, n and cn profiles from each cell line as part of an independent experiment.
expression data from each probe set were median-centered separately and their expression profiles compared by cluster analysis .
all cell lines segregate in a similar manner, independent of the nature of the probe.
moreover, the relationship between expression patterns detected by c, n and cn capture probes for each one of the nine samples was similar for all of them.
c and cn segregated consistently close to each other leaving n probes as outliers.
we observed a closer distance between c and cn expression than between either of these profiles and n, across all performed experiments.
this could be explained by the fact that n probes are derived from sense transcripts cdnas and are not a mirror copy of nats.
therefore, mature antisense transcripts and n probes would not be perfectly complementary but, assuming that all sense and antisense immature transcripts undergo splicing, will display restricted regions of exon overlap.
this situation should favor the hybridization dynamics of c probes since renaturation kinetic is directly dependent on the length of the complementarity with the immobilized probes  <cit> .
CONCLUSIONS
in spite of stringent signal-to-noise ratio criteria, our strand-specific cdna microarrays detected a number of antisense transcripts that exceeds by far the number of previously annotated antisense genes in humans.
partial validation and observed well defined expression patterns suggest that a considerable fraction of these signals might represent bona-fide unannotated antisense transcripts .
these nats are expressed in a cell specific manner and displayed a strong tendency to follow the expression pattern of their sense counterparts.
since antisense transcription data is embedded in double stranded cdna array experiments, it is expected to affect signals and gene clusters, and would make data validation difficult of array results by other means.
we analyzed this issue and found that even if antisense transcription is genome-wide it exerts a restricted influence on the interpretation of conventional cdna microarray data.
today, these problems can be circumvented in well-annotated genomes because strand specific expression can be discriminated by the use of oligonucleotide capture probes.
however, strand-specific oligo design would be hampered today by the limited access to antisense sequence data, as shown here.
although reassuring the overall validity of cdna microarrays in previous tumour-classification studies, our results emphasize the need for further development of methods that accurately measure strand-specific expression.

 non-coding rnas have recently been reported as more common, more diverse, and accredited more important functions than previously anticipated. among the most abundant non-coding transcripts, there is a group called natural antisense transcripts that carries regions of perfect complementarity to protein coding rnas. in silico studies of available transcript sequence data have found that up to 24% of human protein coding loci also encode cis-nats. however, antisense transcripts tend to be poly negative and nuclear localized. if this is true, the abundance of nats may be higher yet, since nuclear non-polyadenylated transcripts are underrepresented in transcript sequence databases. this fact may have important implications for researchers, not only because of their potential biological function but they may also turn out to be influential on the interpretation of large experimental data sets. for instance, the cdna microarray technique has been used in genome-wide expression studies to address basic questions about gene function and in the pursuit of a more precise molecular classification of tumors. in this case, the ability to monitor the expression of thousands of genes simultaneously has allowed the identification of disease-specific subsets of genes useful to improve diagnosis and disease management. the majority of the more than microarray expression profiles released through ncbi was obtained with double stranded cdna capture probes and is assumed to reflect the pure expression of the sense transcripts used as templates for cdna synthesis. however, the widespread expression of natural antisense transcripts invalidates this assumption since double-stranded probes will show the combined expression of both the intended sense target and any nat with complementary sequence. still, for nine out of ten cases, signals from double-stranded cdna probes correlates with those obtained from sense specific oligonucleotide platforms. based on these observations, we reasoned that antisense transcripts are either not efficiently detected by conventional cdna capture probes or that important information must be hidden behind this paradox. therefore, we modeled a typical cdna microarray tumor-classification analysis and compared the results from conventional double-stranded cdna capture probes with single stranded cdna capture probes capable of monitoring opposite strands of each cdna independently. we detected a number of antisense signals that exceed by far the number of known antisense transcripts. the detected signals showed a clear cell specific expression pattern with a common core group of antisenses expressed in all analyzed materials. moreover, antisense transcripts displayed a prevalent tendency to be positively correlated with the expression of their corresponding sense counterparts. this confirms the idea that a large part of the data obtained from conventional double-stranded cdna microarrays are in fact compounded signals product of both sense and antisense hybridization. yet, detection of antisense transcription by conventional double-stranded cdna microarrays does not strongly distort the relationship between expression profiles of the analyzed samples compared with those obtained from pure sense signals. this is most likely due to the observed coordinate regulation of senses and antisenses and a more efficient hybridization of sense strands because a different exon structure of antisense transcripts and the sense transcripts used for cdna synthesis.  production of single-stranded microarrays we generated strand specific cdna probes in situ after covalently binding nh2-modified cdna inserts onto cross-linked n-hydroxysuccinamide slides in a strand specific manner. specific binding of 5' dna ends serves two different but additive purposes. first, 5' end-specific binding provide protection against in situ enzymatic attack of highly processive 5'-3' exonucleases; unbound strands could then be exposed to enzymatic degradation. second, only 5'-end modified strands will be covalently bound, rendering non-modified strands vulnerable to easy removal by heat denaturation. we found that the most reliable method for processing double stranded cdnas into single stranded capture probes was the sequential application of both approaches. the procedure is schematically depicted in figure 1a. to validate the method, microarrays containing single-stranded sense and antisense probes and double-stranded probes were generated from a kb fragment containing the β-lactamase gene. hybridizations were performed with equimolar amounts of cy3- or cy5- direct-labelled sense and antisense β-lactamase transcripts. sense probes showed strong signal from cy labelled sense cdna and signal equivalent to background level in the cy channel. contrary, antisense probes detected strong signal from cy5-labeled antisense cdna while cy signal remained equivalent to background. we conclude that our method can produce selective single stranded dna capture probes from double-stranded pcr products in situ with an efficacy and specificity sufficient to eliminate detectable levels of complementary strand, thus producing a selective array of strand-specific capture probes. strand-specific cdna microarrays following this test, we prepared strand-specific sense and antisense cdna probes, and double-stranded probes, from randomly selected full-length cdnas from the mgc clone collection. both strand specifically 5' nh2-modified pcr products for sense and antisense probe generation and nh2-modified products at both 5' ends were printed on the same surface. in the following, sense probes are termed c for coding and antisense probes n for non-coding. double-stranded probes are termed cn. to verify the specificity of our cdna capture probes, we hybridized the arrays with cy or cy labeled universal primers used to amplify all cdna inserts. data from the test hybridization shows that cn probes produce log ratio intensities centered on demonstrating that they captured both labeled targets. conversely, strand specific c and n probes produced log intensity ratios centered approximately on and - respectively. figure 2b show log ratios for c, n, and cn probe for each cdna clone sorted on ascending basis for respective cn probes. with very few exceptions, each probe set consistently produced strand specific probes equally well across all cdnas. antisense detection of n probes hybridized with total rnas next, we modeled an experimental design typically used for tumor classification studies. we performed hybridizations using direct-labeled randomly primed total rna extracted from eight breast-cancer cell lines and one derived from normal breast epithelium against universal human reference rna as a common reference following standard protocols. to control for even processing into strand-specific probes we printed internal β-lactamase dna control spots in each block and included β-lactamase spike-in sense and antisense control targets in either samples or reference. their hybridization signals confirmed that detected n signals reflect hybridization to the n probe over the entire array. we initially analyzed the detection capacity of n probes. we applied a stringent filter criterion including signals with signal-to-noise ratios above across all nine hybridizations, we detected n signals in the cell lines and n signals in the reference, out of the set of probes. we filtered for non-specific cross-hybridization with repetitive elements in the probe sequence, extended self-complementarity between the c and n probes, and c and n probes with sequence complementarity to the same transcript. we identified repeat sequences of or more continuous bases in of the cdna used for probe synthesis, cases with extended self-complementarity between c and n probes and cases for which the c and n probes matched the same refseq mrna. after filtering for potentially spurious signals, n signals from the cell lines and from the reference were interpreted as detecting putative antisense transcripts. the n signals detected in the cell lines vary from 27% to 74% of the cdnas that also showed sense transcription. we compared these signals with nats identified by the most comprehensive study published. despite significant variations in the number of n signals, the percentage of detected reported nats remains unchanged in each cell line. in the reference channel, an average of % cdnas detecting c signals also produced n signals. as many as putative antisense transcripts in the cell lines and in the reference were detected in all nine hybridizations and, again, a similar proportion of reported antisense transcripts in each group were noted. we observe greater variation in the number of detected n signals for the cell lines compared to the reference, indicating that biological variation is larger than variation resulting from technical replication of measurements. here, we aimed to gain insight into the occurrence of nats and to analyze their influence in the context of microarray hybridizations rather than to rigorously screen for novel antisense transcripts. however, n signals show biological variation that tend to be sample specific, yet reveal a common core detected in all experiments. this group includes genes involved in apopotosis and programmed cell death. if a significant proportion of these detected unannotated transcripts were random artifacts, we would expect the common core to show a lower proportion of known annotated transcripts. nevertheless, it is remarkable that the proportion of annotated antisense transcripts for the common core is similar to that estimated for each cell line separately. to verify the nature of the detected unannotated transcripts we radio-labeled sense strands from ten randomly selected cdnas by in vitro transcription and used them as hybridization riboprobes in northern blot hybridizations. seven of the ten tested riboprobes were able to identify complementary transcripts in the total rna mixture. these observations strengthen the view that most detected n signals might represent true nats. this impression is reinforced by the fact that hybridization with tilling arrays predicted that only one tenth of the total number of transcription units is known in human and the observed well-defined expression pattern of n signals described subsequently. we find no reasons to conceive that this extended antisense transcriptional activity is associated with the nature of the analyzed materials as mcf- derived from normal breast epithelia detected a number of antisense signals second closet to average. in summary, as much as 88% of the tested conventional cdna probes could potentially produce microarray signals reflecting differences in sense or antisense expression, or both, in one sample or both of sample and reference. *number of image clones where n probe snr > 10 **number of image clones previously reported as transcribed from antisense ***number of image clones with > = continuous bases homologous to repeats ****number of image clones with > = continuous bases on both sense- and antisense strand that match refseq +number of image clones with > = continuous bases complementary between sense- and antisense transcript ++number of image clones with either repeats***, double hits****, or self-complementarity+ +++number of image clones that overlap between masked++and clones priviously reported** ++++number of image clones with putative antisense transcription, i.e. those clones that are not masked++ $number of image clones previously reported as transcribed from antisense out of, putative antisense signals total++++ $$number of image clones not masked++ and not privously reported** as transcribed from antisense $$$number of image clones with putative sense transcription, i.e. where c probe snr > excluding clones that are masked++ $$$$percent of image clones displaying n signal out of those that display c signals excluding clones that are masked++ note: values are paired per clone, i.e. the number of image clones with c signal and n signal as percentage of the number of image clones with c signal relationship between c and n signals to analyze closely antisense expression and to explore the extent to which antisense signals might influence protein-coding profiles detected by double-stranded microarrays, we jointly investigated the relationship between signal from c, n, and cn probes. first, we visualized distances between expression profiles derived from separate probes using unsupervised cluster analysis. we regarded expression data from different probes as individual data sets, separating hybridizations into one c, one n, and one cn profile matched on clone ids. cluster analysis using probes derived from cdna clones show that c and cn profiles co-segregate within each cell line. the majority of n profiles form a cohesive cluster well separated from c and cn profiles for the same group of cell lines. for two cell lines, the n, c and cn profiles separate from all other cell lines but within each group the c and cn profiles co-segregate with the n profile clearly separated. visual inspection revealed that c and n signals seem to follow coordinated variation. to corroborate this observation we calculated pair wise correlation between c signal and n signal across the nine hybridizations for corresponding cdna clones. figure 4b shows the distribution of pearson correlations for c and n signal pairs, evidencing a tendency for positive correlation between them. the same coordinated pattern was apparent when only probe pairs from cdna clones with previously known nats were considered. overall, only a minority of clones showed an inverse relationship between senses and antisense expression levels. these probes seem to be responsible for the separation between c and n profiles. a tendency for positively correlated co-expression of sense and antisense transcripts has been observed in large-scale studies performed in mouse. however, studies on human material claimed that expression of members of sense-antisense pairs follow an inverse relation which was supposed to agree with a mechanistic model according to which increasing expression of one of the members downregulates transcription of the other. our results contradict this observation. to verify our observation by an alternative method, we selected the gene encoding human ketohexokinase as representative example. in our microarray measurements, khk's transcription displayed a high positive correlation between n and c signals. khk exons and fully overlap clone cr which transcription proceeds in the antisense orientation. we designed a forward primer on khk exon and reverse primer on exon targeting the regions of sequence complementarity in both cases. due to different splicing, this primer pair would allow simultaneous amplification of a bp khk fragment and a bp product from the cr transcript. figure 4d shows the outcome of these rt-pcr experiments. a tight coordinated variation between expression levels of khk and its antisense transcript was obtained across all tested cell lines confirming our microarray results. thus, the analyzed genes appear to follow a pattern of more or less coordinated up- or down-regulation of both strands in parallel. supported on previously reported data, we interpret the coordinated expression of n with c signals as evidence for tight sense-antisense regulation rather than the effect of a stochastic transcriptional noise. moreover, if sense/antisense expressions tend to be correlated for protein-coding loci, we speculated that profiles should not substantially differ, regardless of whether the array probes are single- or double-stranded. single-stranded compared to conventional double stranded cdna arrays to investigate further the connection between c and cn profiles we calculated pairwise euclidean distances between c, n and cn signals across cell lines. taken together, these data indicate that c and cn profiles are more similar across the hybridized samples than either is to n profiles. to exclude the possibility that a small number of outliers caused the difference, we investigated the distribution of n to c distance per cdna clone within hybridizations. we found a very similar distribution for each hybridization suggesting that antisense transcription – while it is genome-wide – does not significantly distance profiles obtained from pure sense probes from those derived from dsdna. to test this conjecture, we treated c, n and cn profiles from each cell line as part of an independent experiment. expression data from each probe set were median-centered separately and their expression profiles compared by cluster analysis. all cell lines segregate in a similar manner, independent of the nature of the probe. moreover, the relationship between expression patterns detected by c, n and cn capture probes for each one of the nine samples was similar for all of them. c and cn segregated consistently close to each other leaving n probes as outliers. we observed a closer distance between c and cn expression than between either of these profiles and n, across all performed experiments. this could be explained by the fact that n probes are derived from sense transcripts cdnas and are not a mirror copy of nats. therefore, mature antisense transcripts and n probes would not be perfectly complementary but, assuming that all sense and antisense immature transcripts undergo splicing, will display restricted regions of exon overlap. this situation should favor the hybridization dynamics of c probes since renaturation kinetic is directly dependent on the length of the complementarity with the immobilized probes.  in spite of stringent signal-to-noise ratio criteria, our strand-specific cdna microarrays detected a number of antisense transcripts that exceeds by far the number of previously annotated antisense genes in humans. partial validation and observed well defined expression patterns suggest that a considerable fraction of these signals might represent bona-fide unannotated antisense transcripts. these nats are expressed in a cell specific manner and displayed a strong tendency to follow the expression pattern of their sense counterparts. since antisense transcription data is embedded in double stranded cdna array experiments, it is expected to affect signals and gene clusters, and would make data validation difficult of array results by other means. we analyzed this issue and found that even if antisense transcription is genome-wide it exerts a restricted influence on the interpretation of conventional cdna microarray data. today, these problems can be circumvented in well-annotated genomes because strand specific expression can be discriminated by the use of oligonucleotide capture probes. however, strand-specific oligo design would be hampered today by the limited access to antisense sequence data, as shown here. although reassuring the overall validity of cdna microarrays in previous tumour-classification studies, our results emphasize the need for further development of methods that accurately measure strand-specific expression. 
here, we wanted to analyze experimentally the incidence of antisense transcription in human cells and to estimate their influence on protein coding expression patterns obtained by double-stranded microarrays. antisense transcription is cell specific and showed a strong tendency to be positively correlated to the expression of the sense counterparts. antisense transcription in humans can be far more common than previously estimated. this can be explained by a biological phenomena and a bias of the technique: a) a co-ordinate sense and antisense expression variation and b) a bias for sense-hybridization to occur with more efficiency, presumably due to variable exonic overlap between antisense transcripts. however, it has limited influence on expression profiles obtained with conventional cdna probes. even if their expression is wide-spread, detected antisense signals seem to have a limited distorting effect on sense profiles obtained with double-stranded probes. therefore, we profiled transcription of sense and antisense independently by using strand-specific cdna microarrays. this phenomenon raises questions regarding the validity of data obtained from double-stranded cdna microarrays since hybridization to both strands may occur. recent studies revealed that many mammalian protein-coding genes also transcribe their complementary strands. up to 88% of expressed protein coding loci displayed concurrent expression from the complementary strand.
 recent studies revealed that many mammalian protein-coding genes also transcribe their complementary strands. this phenomenon raises questions regarding the validity of data obtained from double-stranded cdna microarrays since hybridization to both strands may occur. here, we wanted to analyze experimentally the incidence of antisense transcription in human cells and to estimate their influence on protein coding expression patterns obtained by double-stranded microarrays. therefore, we profiled transcription of sense and antisense independently by using strand-specific cdna microarrays.  up to 88% of expressed protein coding loci displayed concurrent expression from the complementary strand. antisense transcription is cell specific and showed a strong tendency to be positively correlated to the expression of the sense counterparts. even if their expression is wide-spread, detected antisense signals seem to have a limited distorting effect on sense profiles obtained with double-stranded probes.  antisense transcription in humans can be far more common than previously estimated. however, it has limited influence on expression profiles obtained with conventional cdna probes. this can be explained by a biological phenomena and a bias of the technique: a) a co-ordinate sense and antisense expression variation and b) a bias for sense-hybridization to occur with more efficiency, presumably due to variable exonic overlap between antisense transcripts. 