BACKGROUND
gene expression analysis provides a view of transcriptional activity and regulation, which is useful as an indicator of likely protein expression and potential function.
the current approaches to measuring gene expression vary from parallel semi-quantitative measurements of large numbers of mrna transcripts in a single sample, using gene chip technologies, to focussed sensitive approaches, such as qrt-pcr, that enable quantitative evaluation of the expression of single genes in multiple samples  <cit> .
while gene chip technology offers the means to carry out hypothesis-free investigation of the expression of a large number of genes, a major disadvantage of this technology is its lack of sensitivity.
in contrast, qrt-pcr is currently capable of measuring single genes in a uniquely robust and sensitive fashion being capable of detecting specific mrnas at levels as low as  <dig> copy per  <dig>  cells .
for some time, there have been attempts to identify technologies that can facilitate more quantitative and sensitive expression profiling of a number of genes in single samples simultaneously.
in this present study, the potential of one such technology  has been assessed.
lda technology is based on the qrt-pcr platform, therefore, while retaining the sensitivity of qrt-pcr, ldas are claimed to allow the simultaneous quantification of large numbers of target genes in single samples  <cit> .
consequently, lda technology could be used to compare levels of expression of panels of specific genes in treated and untreated cells in vitro, and also to look at the relative expression levels of panels of genes in diseased versus non-diseased tissues.
in the current study, the potential of ldas for the evaluation of gene expression profiles in human cells and tissues has been assessed.
key features of the assessment included ease of use, sensitivity and reproducibility.
in addition, to evaluate the ability of ldas to allow the accurate measurement of changes in the expression of multiple genes, the technique was applied to thrombin-stimulated human endothelial cells, and the results compared with those generated using conventional qrt-pcr.
RESULTS
variability of gene expression data derived from ldas
a single sample of cdna from human liver was run down all eight channels of three identical ldas from the same production batch.
the data derived across these ldas were virtually identical, with correlation coefficients approximating unity .
these data therefore demonstrate negligible variation between arrays from the same batch.
representative data are displayed graphically in figure  <dig>  ct values  for lda  <dig> have been plotted against ct values for lda  <dig>  the results are more reproducible at the lower ct values  when compared to the higher ct values, where detection limits of the system are being approached.
the data generated for ldas 1– <dig> were used to assess the quadruplicated values for each gene on each lda, and also to look at the variation of the  <dig> values across the three ldas.
the data showed low coefficient of variation  between wells within a single lda, and between ldas.
as expected, the cv% increased with lower gene expression levels i.e.
higher cts.
combined data for all targets across the three identical arrays are shown in table  <dig>
fidelity and dynamic range of ldas
for the liver cdna, 2-fold dilution plots are shown  for two genes, 18s-ribosomal rna , which showed high expression in the liver, and bcl <dig>  which exhibited much lower expression.
these results demonstrate that these two primer probe sets generate linear results over the expression range studied, with r <dig> values close to  <dig> being obtained.
these data demonstrate that using the ldas it is possible to accurately detect 2-fold changes in target copy number.
they also demonstrate that these small changes can be detected at both low cts  and high cts , within the approximate range of  <dig> ×  <dig> –  <dig> ×  <dig> copies.
for the 10-fold dilution series of cdna , the r2values were also determined, figure  <dig>  shows examples of the plots generated for two genes, hla-dra, which was highly expressed in the tonsil, and cd26e, which had lower expression.
for cd26e, with a higher starting ct value, it was not possible to plot all the points in the dilution series, as the sample was diluted beyond the level of detection.
however, for both genes, we determined a clear linear relationship between the gene expression data points and for hla-dra linearity was obtained over  <dig> log base  <dig> dilutions.
comparing efficiencies of the  <dig> primer probe sets
for the liver cdna 2-fold dilution series, r <dig> values and slopes were determined by linear regression analysis.
data for all genes are shown in table  <dig>  the ct values of the undiluted cdna for the targets range from  <dig>  to  <dig> with good linearity for most of the targets over the 2-fold dilution series, most with r <dig> values close to  <dig>  however over the range of ct values the slopes vary, 18s  has a slope of - <dig>  whereas bcl <dig>  has a slope of - <dig> .
this data indicates different amplification efficiencies of the primer probe sets.
comparison of gene expression profiles derived using ldas with those derived using conventional qrt-pcr
the levels of il- <dig>  mcp- <dig> and rantes were quantified using conventional qrt-pcr and lda-based qrt-pcr in control and thrombin-treated human aortic endothelial cells  at  <dig> and  <dig> hour time points .
both approaches produced qualitatively and quantitatively similar data in that the expression of all three genes was up-regulated by thrombin to similar degrees.
thus, conventional qrt-pcr showed that il- <dig> was up-regulated by 10-fold at  <dig> hours, and by 13-fold at  <dig> hours, while the lda-derived data demonstrated a 9-fold increase in il- <dig> at  <dig> hours, and 10-fold at  <dig> hours.
mcp- <dig> expression data showed similar trends whether measured by either system, with  <dig> and 5-fold increases in the levels of transcript at  <dig> and  <dig> hours respectively, following incubation with thrombin.
the level of basal rantes expression was low, however, it was still possible using conventional and lda-based qrt-pcr to demonstrate up-regulation in expression  <dig> hours post-thrombin.
as the ldas contained  <dig> primer probe sets in quadruplicate, it was possible to assess the effects of thrombin treatment on many more genes in a single sample than would be possible with conventional qrt-pcr.
of the  <dig> genes investigated,  <dig> at two hours and  <dig> at  <dig> hours were changed by more than 50% from the control.
for all targets for which gene expression was changed by more than 50% the majority were up-regulated in response to thrombin, with only two genes being down-regulated .
where target expression was compared between treatment and control groups, the expression levels of housekeeping genes , were comparable in the treated and control samples .
discussion
gene expression provides a view of transcriptional activity and regulation, and is often used as a surrogate marker for protein levels.
analysis of gene expression is used in a variety of studies, which range from identifying target genes that are altered in diseased tissues, to the screening of chemical libraries in phenotypic assays.
the current approaches to measuring gene expression vary from technologies that involve parallel quantification of large numbers of mrna transcripts in single samples  to very focussed approaches  that look at the expression of single genes in multiple samples.
for some time, a technology has been sought that combines the ability to measure the expression of many genes in a single sample as with the chip array approach, while retaining the sensitivity and quantitative capacity offered by qrt-pcr.
we have now evaluated the potential of lda as such a technology.
the aim of the study was therefore to assess the lda technology, and to determine whether it is capable of detecting and measuring changes in a large number of genes simultaneously in a sensitive, robust and accurate manner.
initially, we set out to investigate the variability of the technique.
cdna from one common liver sample was loaded in all channels across three arrays.
each gene was investigated in quadruplicate on each of the three arrays .
this facilitated investigating the variability across the quadruplicate values for each array and between triplicate arrays.
the resulting data indicate that the variability between ldas within a batch is low, with correlation coefficients for between array comparisons all being greater than  <dig> .
additionally, analysis of the quadruplicate value for each gene on individual arrays revealed a low % cv, indicating a high level of reproducibility of data within each array.
however, the current study has not addressed variability between cards from different batches and so the possibility exists of larger variation occurring between ldas from different batches.
having determined that the variability within and between the arrays was low, we next wanted to determine how capable the ldas are of accurately quantifying small changes in gene expression.
linear regression analysis of the 2-fold serial dilution of liver cdna across  <dig> ldas showed for most genes, r <dig> values close to  <dig>  indicating a clear linear relationship of the gene expression data points over the range of the dilution series.
the data demonstrate that ldas are able to accurately and quantitatively detect 2-fold changes in target copy number as was demonstrated using a serial dilution of cdna.
it is hoped that the lda cards will demonstrate the same fidelity in other experimental scenarios including those where  <dig> different rna preparations are used from treated cells and untreated control cells.
the data in figures  <dig> and  <dig> support this, whereby modest changes were observed.
in addition to assessing the accuracy and ability of the ldas to detect small changes in gene expression, the linear regression analysis enabled the assessment of the slopes for each gene, with the slope indicating the efficiency of the primer probe set.
the efficiencies of the primer probe sets used in this study did vary and so the delta delta ct analysis system utilised by the abi software for analysing the lda data may not always be appropriate  as it works on the principle that the efficiencies of the different primer probe sets are comparable.
next, we sought to assess whether ldas are equally capable of detecting genes that are present at low and high copy numbers.
comparison of the dynamic range of ldas was carried out using a 10-fold serial dilution of cdna prepared from tonsil.
the same channels  of  <dig> separate lda cards were loaded with six concentrations of tonsil cdna.
for targets with a copy threshold of less than  <dig>  across the concentration range, a dynamic range of the system over  <dig> orders of magnitude was demonstrated, indicating that it is possible to accurately and quantitatively detect genes at high and low copy numbers.
this data is in agreement with a recent study using lda cards, which found the technology had a wide dynamic range, up to seven orders of magnitude  <cit> .
having built confidence regarding the sensitivity, reproducibility and dynamic range of ldas, the next step was to apply them to an experimental situation and compare the data with those generated using conventional qrt-pcr.
thus, both techniques were used to evaluate thrombin-induced changes in gene expression in human aortic endothelial cells.
the study focused on three genes, il- <dig>  mcp- <dig> and rantes, which are implicated in atherosclerosis  <cit> .
the resulting data indicate that ldas produced qualitatively and quantitatively similar data to conventional qrt-pcr.
both techniques indicated that thrombin caused up-regulation in the three key genes of interest .
this suggests that ldas are suitable for examining changes in gene expression in response to pharmacological stimuli.
a key advantage of ldas over conventional qrt-pcr is their ability to assess changes in multiple genes in a single sample.
therefore, in the current study in thrombin-treated cells, in addition to demonstrating that il- <dig>  mcp- <dig> and rantes are up-regulated, the ldas provided data on a further  <dig> genes.
of these genes, the expression levels of  <dig> at  <dig> hours and  <dig> at  <dig> hours were changed by more than 50% from the control.
from the genes whose expression was changed by more than 50%, the majority were up-regulated in response to thrombin, with only two genes being down-regulated.
these additional data demonstrate the power of the lda technology to investigate changes in expression of a large number of genes simultaneously in a single study in a sensitive and quantitative manner.
in the present study, in addition to demonstrating the expected thrombin-induced increase in il- <dig> and mcp- <dig> mrna, the ldas detected for the first time that thrombin regulates several other genes  in human endothelial cells.
other considerations that are also important in assessing this new technology are its ease of use and cost implications.
loading and setting up the lda cards was straightforward, with no requirement for robotic liquid handling.
the cost of conducting studies on the ldas is less than that of conventional qrt-pcr.
this primarily reflects the savings made in time and money through not having to design and purchase specific primer probe sets.
additionally, ldas require a very low reaction volume, which means that there are large savings in master mix, and a reduction in the use of rna per study.
in the current study involving  <dig> ldas, we calculated the savings relative to carrying out the study using conventional qrt-pcr to be in the order of 60% on time and 70% on consumable cost.
CONCLUSIONS
in conclusion, this study has shown that lda technology provides a sensitive and reproducible approach to study gene expression.
in addition, this new technique has been shown to produce comparable results to those produced by conventional qrt-pcr.
however, ldas have the advantage of allowing multiple genes to be studied from a single sample, and also offer savings in terms of costs and materials.
therefore, ldas represent a valuable new approach to sensitive and quantitative gene expression profiling.

 gene expression analysis provides a view of transcriptional activity and regulation, which is useful as an indicator of likely protein expression and potential function. the current approaches to measuring gene expression vary from parallel semi-quantitative measurements of large numbers of mrna transcripts in a single sample, using gene chip technologies, to focussed sensitive approaches, such as qrt-pcr, that enable quantitative evaluation of the expression of single genes in multiple samples. while gene chip technology offers the means to carry out hypothesis-free investigation of the expression of a large number of genes, a major disadvantage of this technology is its lack of sensitivity. in contrast, qrt-pcr is currently capable of measuring single genes in a uniquely robust and sensitive fashion being capable of detecting specific mrnas at levels as low as copy per cells. for some time, there have been attempts to identify technologies that can facilitate more quantitative and sensitive expression profiling of a number of genes in single samples simultaneously. in this present study, the potential of one such technology has been assessed. lda technology is based on the qrt-pcr platform, therefore, while retaining the sensitivity of qrt-pcr, ldas are claimed to allow the simultaneous quantification of large numbers of target genes in single samples. consequently, lda technology could be used to compare levels of expression of panels of specific genes in treated and untreated cells in vitro, and also to look at the relative expression levels of panels of genes in diseased versus non-diseased tissues. in the current study, the potential of ldas for the evaluation of gene expression profiles in human cells and tissues has been assessed. key features of the assessment included ease of use, sensitivity and reproducibility. in addition, to evaluate the ability of ldas to allow the accurate measurement of changes in the expression of multiple genes, the technique was applied to thrombin-stimulated human endothelial cells, and the results compared with those generated using conventional qrt-pcr.  variability of gene expression data derived from ldas a single sample of cdna from human liver was run down all eight channels of three identical ldas from the same production batch. the data derived across these ldas were virtually identical, with correlation coefficients approximating unity. these data therefore demonstrate negligible variation between arrays from the same batch. representative data are displayed graphically in figure ct values for lda have been plotted against ct values for lda the results are more reproducible at the lower ct values when compared to the higher ct values, where detection limits of the system are being approached. the data generated for ldas 1– were used to assess the quadruplicated values for each gene on each lda, and also to look at the variation of the values across the three ldas. the data showed low coefficient of variation between wells within a single lda, and between ldas. as expected, the cv% increased with lower gene expression levels i.e. higher cts. combined data for all targets across the three identical arrays are shown in table fidelity and dynamic range of ldas for the liver cdna, 2-fold dilution plots are shown for two genes, 18s-ribosomal rna, which showed high expression in the liver, and bcl which exhibited much lower expression. these results demonstrate that these two primer probe sets generate linear results over the expression range studied, with r values close to being obtained. these data demonstrate that using the ldas it is possible to accurately detect 2-fold changes in target copy number. they also demonstrate that these small changes can be detected at both low cts and high cts, within the approximate range of × – × copies. for the 10-fold dilution series of cdna, the r2values were also determined, figure shows examples of the plots generated for two genes, hla-dra, which was highly expressed in the tonsil, and cd26e, which had lower expression. for cd26e, with a higher starting ct value, it was not possible to plot all the points in the dilution series, as the sample was diluted beyond the level of detection. however, for both genes, we determined a clear linear relationship between the gene expression data points and for hla-dra linearity was obtained over log base dilutions. comparing efficiencies of the primer probe sets for the liver cdna 2-fold dilution series, r values and slopes were determined by linear regression analysis. data for all genes are shown in table the ct values of the undiluted cdna for the targets range from to with good linearity for most of the targets over the 2-fold dilution series, most with r values close to however over the range of ct values the slopes vary, 18s has a slope of - whereas bcl has a slope of -. this data indicates different amplification efficiencies of the primer probe sets. comparison of gene expression profiles derived using ldas with those derived using conventional qrt-pcr the levels of il- mcp- and rantes were quantified using conventional qrt-pcr and lda-based qrt-pcr in control and thrombin-treated human aortic endothelial cells at and hour time points. both approaches produced qualitatively and quantitatively similar data in that the expression of all three genes was up-regulated by thrombin to similar degrees. thus, conventional qrt-pcr showed that il- was up-regulated by 10-fold at hours, and by 13-fold at hours, while the lda-derived data demonstrated a 9-fold increase in il- at hours, and 10-fold at hours. mcp- expression data showed similar trends whether measured by either system, with and 5-fold increases in the levels of transcript at and hours respectively, following incubation with thrombin. the level of basal rantes expression was low, however, it was still possible using conventional and lda-based qrt-pcr to demonstrate up-regulation in expression hours post-thrombin. as the ldas contained primer probe sets in quadruplicate, it was possible to assess the effects of thrombin treatment on many more genes in a single sample than would be possible with conventional qrt-pcr. of the genes investigated, at two hours and at hours were changed by more than 50% from the control. for all targets for which gene expression was changed by more than 50% the majority were up-regulated in response to thrombin, with only two genes being down-regulated. where target expression was compared between treatment and control groups, the expression levels of housekeeping genes, were comparable in the treated and control samples. discussion gene expression provides a view of transcriptional activity and regulation, and is often used as a surrogate marker for protein levels. analysis of gene expression is used in a variety of studies, which range from identifying target genes that are altered in diseased tissues, to the screening of chemical libraries in phenotypic assays. the current approaches to measuring gene expression vary from technologies that involve parallel quantification of large numbers of mrna transcripts in single samples to very focussed approaches that look at the expression of single genes in multiple samples. for some time, a technology has been sought that combines the ability to measure the expression of many genes in a single sample as with the chip array approach, while retaining the sensitivity and quantitative capacity offered by qrt-pcr. we have now evaluated the potential of lda as such a technology. the aim of the study was therefore to assess the lda technology, and to determine whether it is capable of detecting and measuring changes in a large number of genes simultaneously in a sensitive, robust and accurate manner. initially, we set out to investigate the variability of the technique. cdna from one common liver sample was loaded in all channels across three arrays. each gene was investigated in quadruplicate on each of the three arrays. this facilitated investigating the variability across the quadruplicate values for each array and between triplicate arrays. the resulting data indicate that the variability between ldas within a batch is low, with correlation coefficients for between array comparisons all being greater than. additionally, analysis of the quadruplicate value for each gene on individual arrays revealed a low % cv, indicating a high level of reproducibility of data within each array. however, the current study has not addressed variability between cards from different batches and so the possibility exists of larger variation occurring between ldas from different batches. having determined that the variability within and between the arrays was low, we next wanted to determine how capable the ldas are of accurately quantifying small changes in gene expression. linear regression analysis of the 2-fold serial dilution of liver cdna across ldas showed for most genes, r values close to indicating a clear linear relationship of the gene expression data points over the range of the dilution series. the data demonstrate that ldas are able to accurately and quantitatively detect 2-fold changes in target copy number as was demonstrated using a serial dilution of cdna. it is hoped that the lda cards will demonstrate the same fidelity in other experimental scenarios including those where different rna preparations are used from treated cells and untreated control cells. the data in figures and support this, whereby modest changes were observed. in addition to assessing the accuracy and ability of the ldas to detect small changes in gene expression, the linear regression analysis enabled the assessment of the slopes for each gene, with the slope indicating the efficiency of the primer probe set. the efficiencies of the primer probe sets used in this study did vary and so the delta delta ct analysis system utilised by the abi software for analysing the lda data may not always be appropriate as it works on the principle that the efficiencies of the different primer probe sets are comparable. next, we sought to assess whether ldas are equally capable of detecting genes that are present at low and high copy numbers. comparison of the dynamic range of ldas was carried out using a 10-fold serial dilution of cdna prepared from tonsil. the same channels of separate lda cards were loaded with six concentrations of tonsil cdna. for targets with a copy threshold of less than across the concentration range, a dynamic range of the system over orders of magnitude was demonstrated, indicating that it is possible to accurately and quantitatively detect genes at high and low copy numbers. this data is in agreement with a recent study using lda cards, which found the technology had a wide dynamic range, up to seven orders of magnitude. having built confidence regarding the sensitivity, reproducibility and dynamic range of ldas, the next step was to apply them to an experimental situation and compare the data with those generated using conventional qrt-pcr. thus, both techniques were used to evaluate thrombin-induced changes in gene expression in human aortic endothelial cells. the study focused on three genes, il- mcp- and rantes, which are implicated in atherosclerosis. the resulting data indicate that ldas produced qualitatively and quantitatively similar data to conventional qrt-pcr. both techniques indicated that thrombin caused up-regulation in the three key genes of interest. this suggests that ldas are suitable for examining changes in gene expression in response to pharmacological stimuli. a key advantage of ldas over conventional qrt-pcr is their ability to assess changes in multiple genes in a single sample. therefore, in the current study in thrombin-treated cells, in addition to demonstrating that il- mcp- and rantes are up-regulated, the ldas provided data on a further genes. of these genes, the expression levels of at hours and at hours were changed by more than 50% from the control. from the genes whose expression was changed by more than 50%, the majority were up-regulated in response to thrombin, with only two genes being down-regulated. these additional data demonstrate the power of the lda technology to investigate changes in expression of a large number of genes simultaneously in a single study in a sensitive and quantitative manner. in the present study, in addition to demonstrating the expected thrombin-induced increase in il- and mcp- mrna, the ldas detected for the first time that thrombin regulates several other genes in human endothelial cells. other considerations that are also important in assessing this new technology are its ease of use and cost implications. loading and setting up the lda cards was straightforward, with no requirement for robotic liquid handling. the cost of conducting studies on the ldas is less than that of conventional qrt-pcr. this primarily reflects the savings made in time and money through not having to design and purchase specific primer probe sets. additionally, ldas require a very low reaction volume, which means that there are large savings in master mix, and a reduction in the use of rna per study. in the current study involving ldas, we calculated the savings relative to carrying out the study using conventional qrt-pcr to be in the order of 60% on time and 70% on consumable cost.  in conclusion, this study has shown that lda technology provides a sensitive and reproducible approach to study gene expression. in addition, this new technique has been shown to produce comparable results to those produced by conventional qrt-pcr. however, ldas have the advantage of allowing multiple genes to be studied from a single sample, and also offer savings in terms of costs and materials. therefore, ldas represent a valuable new approach to sensitive and quantitative gene expression profiling. 
comparisons between ldas reveal low variability, with correlation coefficients close to by performing 2-fold and 10-fold serial dilutions of cdna samples in the ldas we determined a clear linear relationship between the gene expression data points over orders of magnitude. ldas represent a valuable new approach for sensitive and quantitative gene expression profiling. we also showed that it is possible to use ldas to accurately and quantitatively detect 2-fold changes in target copy number as well as measuring genes that are expressed with low and high copy numbers in the range of × – × copies. we have now evaluated ldas as a means of determining the expression of multiple genes simultaneously in human tissues and cells. furthermore, the data generated by the lda from a cell based pharmacological study were comparable to data generated by conventional qrt-pcr. low density arrays have recently been introduced as a novel approach to gene expression profiling. based on real time quantitative rt-pcr, these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to qrt-pcr.
 low density arrays have recently been introduced as a novel approach to gene expression profiling. based on real time quantitative rt-pcr, these arrays enable a more focused and sensitive approach to the study of gene expression than gene chips, while offering higher throughput than more established approaches to qrt-pcr. we have now evaluated ldas as a means of determining the expression of multiple genes simultaneously in human tissues and cells.  comparisons between ldas reveal low variability, with correlation coefficients close to by performing 2-fold and 10-fold serial dilutions of cdna samples in the ldas we determined a clear linear relationship between the gene expression data points over orders of magnitude. we also showed that it is possible to use ldas to accurately and quantitatively detect 2-fold changes in target copy number as well as measuring genes that are expressed with low and high copy numbers in the range of × – × copies. furthermore, the data generated by the lda from a cell based pharmacological study were comparable to data generated by conventional qrt-pcr.  ldas represent a valuable new approach for sensitive and quantitative gene expression profiling. 