the online version of this article ( doi:10.1007/s12263 - 008 - 0094 - 1 ) contains supplementary material , which is available to authorized users .
gene expression microarrays have become the leading technology to evaluate changes in genome wide gene expression under different conditions . to confirm results from microarray studies , a selection of differentially expressed genes of interest
is generally verified using a highly sensitive technique , mostly a quantitative real - time reverse transcriptase pcr ( rt - pcr / qpcr ) reaction [ 8 , 21 ] . even though qpcr is considered to be a
 golden standard  or a necessary validation [ 11 , 15 , 16 , 19 , 20 ] , it has also been stated that qpcr results are not always better   or validation is not always necessary . in a qpcr reaction
, cdna is synthesised from mrna molecules present in the sample , which is then amplified exponentially using a combination of gene - specific primers , labelled nucleotides and the dna polymerase enzyme , theoretically doubling the amount of cdna at every cycle . upon analysis ,
the expression level of the target gene is computed relative to the expression level of one or more reference genes , often housekeeping genes .
selecting proper housekeeping genes is one of the most critical aspects of the analysis , since they need to be constantly expressed between all samples and conditions in the experiment .
a survey of publications reveals a wide range in correlations between microarray and qpcr results [ 37 , 12 , 19 ] . to allow qpcr in a more high - throughput way ,
several companies have developed a practical solution to perform up to 384 qpcr reactions on one single plate , such as the taqman gene expression arrays from applied biosystems and rt profiler pcr array system from sabiosciences .
pcr - arrays have mainly been used in cancer research , but also in some other fields , for example , drug metabolism . at the time of writing , 74 rt profiler pcr - arrays
are catalogued that contain primer sequences for genes belonging to a specific function , structure or pathway ( e.g. , apoptosis , common cytokines , nf-b signalling pathway ) or disease ( e.g. , cancer ) .
every plate contains five housekeeping genes ( b2 m , hprt1 , rpl13a , gadph and actb ) , three positive pcr controls , three reverse transcriptase controls and one control to detect the presence of genomic cdna contamination .
correlations between results of several rt profiler pcr - arrays and qpcr have been reported to be good [ 3 , 13 ] . in human nutritional intervention studies ,
most publications in this field make use of qpcr to validate a few genes of interest [ 14 , 18 ] , but no larger scale approaches have been used .
the goal of this study is to evaluate if for this purpose a pcr - array can be used in a dietary intervention study where the expected changes are small .
this paper elaborates on a study investigating the relationship between diet and inflammation . for microarray analysis ,
mrnas from peripheral mononuclear blood cell ( pbmc ) samples of eight obese subjects were collected .
these subjects participated in both the fish oil ( fishoil ) and weight loss ( weightloss ) interventions .
rna was pooled with the control ( cy3 ) being the control diet and with either the weightloss or fishoil end - point as cy5 .
rna quantity and purity were determined using the nanodrop nd-2000 ( nanodrop technologies , wilmington , de , usa ) , and rna integrity was assessed by determining the rna 28s/18s ratio using the bioanalyzer 2100 ( agilent technologies , santa clara , ca , usa ) .
pooled rna samples were hybridised to agilent human 1a g4110b microarrays , containing 22,575 reporters .
this was done according to a dye - flip design , resulting in a total number of four arrays .
array quality control was performed in spotfire decisionsite 9.0 and r 2.5.1 using the limma library version 2.10.5 .
reporters passed quality control when ( 1 ) signals of both channels consisted of more than 55 pixels ; ( 2 ) both signals were higher than 2.6 times the standard deviation of the local background and ( 3 ) at least one signal was not saturated .
all further data analyses were performed in r. within - array normalisation was performed by using the loess algorithm .
next , a linear model was fitted for every reporter and a moderated t test was performed . to ensure microarray reporter specificity
microarray results were validated using a specific pcr - array ( sabiosciences , frederick , usa ) that met two criteria .
we selected all human pcr - arrays that contained a relatively large number of genes that were differentially expressed ( p value  <  0.05 and |fc|  >  1.2 ) by the two treatment comparisons performed in the microarray study and that contained a gene set that was related to the observed changes in plasma metabolic parameters . based on the outcome , the 96-well plate human common cytokines pcr - array ( pahs-021 ) was selected .
the mrna of each individual was converted into cdna using the rt first strand kit ( sabiosciences , frederick , usa ) .
this cdna was then added to the rt sybr green qpcr master mix ( sabiosciences , frederick , usa ) .
all steps were done according to the manufacturer s protocol for the abi prism 7000 sequence detection system .
rna quality was good , with 260/280 ratios slightly higher than 2.0 and 260/230 ratios slightly higher than 1.8 . to analyze the pcr - array data ,
an ms - excel sheet with macros was downloaded from the manufacturer s website ( http://www.sabiosciences.com/pcrarraydataanalysis.php ) .
we preferred to use offline calculations since it was not possible to save the results online , and even more importantly because it was easier to track the formulas and to understand what was being done .
for each pcr reaction , the excel sheet calculated two normalised average ct values , a paired t test p value and a fold change . to filter out undetectable genes ,
an extra quality check was added by us : for each intervention , the pcr gene product was flagged as either  good  or 
a gene was considered good when in both the control and the dietary intervention the difference between the measured genomic cdna ct value ( maximum 35 ) and the measured ct value of the gene was at least three cycles in the same five out of eight subjects for each comparison . for each included gene ,
this was done to correct the lack of robustness of the analysis spreadsheet , as provided by the manufacturer , and to take into consideration the outlying values that have a considerable influence on the calculated fold change and its associated p value .
data normalisation was based on correcting all ct values for the average ct values of several constantly expressed housekeeping genes ( hkgs ) present on the array .
pcr - array analysis results were evaluated and also compared to the outcome of the gene expression microarrays .
in our current analysis , only four hkgs ( hprt1 , rpl13a , gapdh and actb ) were used for normalisation .
beta-2-microglobulin ( b2 m ) was not included , since it was significantly altered on the pcr - array after the weight loss period . applying
the flagging criteria resulted in finding 29 and 33 detectable genes after the fishoil and the weightloss intervention , respectively .
all detected genes in the fishoil intervention were also measured accurately in the weightloss comparison .
the flagging procedure removed several observations that were most likely caused by failing amplifications , since only one value of one subject was undetectable ( ct  >  35 ) .
note however that in some cases this flagging may be somewhat conservative : for example , for two subjects in the fishoil experiment , the ifnk gene changed from baseline undetected ( ct  =  35 ) to ct values of 30.27 and 30.72 cycles .
all other subjects had a nearly constant ct value ranging between 29.5 and 29.8 cycles .
the same pattern occurred for two subjects , and therefore it could well be a real biological effect .
in general , the measurements obtained by the pcr - array were consistent between subjects and show very small variation , as in regular qpcr .
table  1 summarizes our findings with respect to the detectable genes on the pcr - array and their corresponding microarray values .
seven pcr genes were present more than once on the agilent microarray platform . for two of those genes ( faslg and tnf )
their corresponding reporters on the microarray were identical , specific , and showed only a slight difference in fold change .
the other five genes ( actb , csf1 , pdgfa , tfga and tfgb1 ) had two different microarray reporter sequences that align to different parts of the gene product .
for these seven genes , the fold change for the highest expressed reporter was chosen to represent the gene on the microarray .
it was not possible to measure the expression of five of the pcr - measured genes on the microarray : two genes ( b2 m , tnfsf14 ) were always saturated , whereas the other three ( bmp8b , csf1 , ifnk ) were filtered out during microarray quality control due to a too low background - corrected intensity value .
all other genes on the pcr - array , apart from ifna1 , were also present on the reannotated microarray , where ifna4 was only covered by reporters targeting several of its family members as well . within the complete list of genes ( cf .
supplementary table  1 ) , only ~10% of genes detected on the pcr - array could not be detected on the microarray .
conversely , ~50% of genes , for which primers were available on the pcr - arrays , could be detected on the microarray but not on the pcr - array .
this result contradicts the original hypothesis of a better sensitivity of qpcr in comparison to microarray [ 1 , 19 ] .
this could be explained by the different criteria used and may point to the fact that a required threefold difference in ct value compared to the genomic control , as suggested by the manufacturer , is too strict .
however , this can not offer an explanation for all the findings , illustrated by the inha gene , which was detectable on the microarray in low concentrations and significant for all comparisons , but had ct values of ~35 on the pcr - array . on the other hand , with highly expressed genes ,
thus , the reported lower dynamic range of microarray measurements [ 1 , 19 ] was confirmed for this end of the spectrum . in principle , a simple rescanning of the array at a lower scanning intensity could overcome this problem .
however , such rescanning procedures have to be done shortly after hybridisation , and in our experience , in practice , this is not often done .
we did not observe that array performance would be especially lower with lower expression levels or fold changes , as reported before [ 1 , 57 , 15 , 19 ] , though several authors also found acceptable detection levels of lower fold changes or expression levels or less correlation between expression and accuracy [ 6 , 19 , 20 ] . in our study ,
consistency in direction of change between both methods remained high down to the 10% change range for the qpcr .
finally , even though a trend of the microarray in estimating smaller fold changes as compared to qpcr was observed [ 6 , 22 ] , the difference was quite limited.table  1detectable reporters on rt profiler pcr - array in human pbmcsgenenumber of reporters on mafishoil versus controlweightloss versus controlfc  mafc  pcrfc  mafc  pcractb21.011.051.031.06b2m1n / a1.09n / a1.23*bmp611.021.391.17*1.15bmp8b1n / a1.42n / a1.11csf141.111.041.051.15faslg21.011.111.041.04gapdh11.011.081.071.06gdf1111.021.091.071.05hprt111.001.111.11*1.05ifng11.031.061.21*1.07ifnk1n / an / an / a1.38il1511.031.211.081.05il1611.031.071.13*1.08il1811.101.131.021.07il1b11.051.061.031.02il2411.20*1.141.29 * 1.05il71n / an / a1.19*1.43lta11.011.051.09*1.09ltb11.001.051.06*1.12pdgfa21.10*1.241.36*1.46rpl13a11.021.011.051.08tgfa21.031.031.15*1.07tgfb121.071.031.011.02tgfb31n / an / a1.061.32tnf21.09*1.101.071.12tnfsf1011.07*1.141.10*1.18tnfsf1211.031.061.031.03tnfsf1311.021.031.001.01tnfsf13b11.081.231.001.03tnfsf141n / a1.33n / a1.02tnfsf41n / an / a1.28*1.48tnfsf811.08*1.251.011.02txlna11.13 * 1.081.07*1.06calculated fold changes of 34 detectable pcr gene products as compared to the fold changes derived from the microarray experiment for each interventionfc fold change , ma microarray , n / a not available , pcr pcr - array *  p  <  0.05 detectable reporters on rt profiler pcr - array in human pbmcs calculated fold changes of 34 detectable pcr gene products as compared to the fold changes derived from the microarray experiment for each intervention fc fold change , ma microarray , n / a not available , pcr pcr - array with respect to significantly regulated genes , some changes were consistent while few were not ( cf . table  1 and supplementary table  1 ) .
the p values of the pcr - array were much higher than the p values of the microarray .
however , the microarray material was pooled , whereas the pcr samples were not , resulting in much lower variation for the first analysis technique . since this would logically lead to lower p values , this effect was not taken into account for the comparison .
the majority of genes on both the pcr - array and the microarray were not differentially expressed for either technology . among differentially expressed genes
the pdfga gene was detected as significant under both experimental conditions on the microarray and its computed fold changes were confirmed on the pcr - array . for tnfsf4 , the results also corresponded : for both platforms downregulated for weightloss and flagged for fishoil .
other genes were less concordant with il24 as most conflicting gene , showing opposite values for both comparisons .
bad annotation or crosshybridisation are unlikely causes for this inconsistency , since both arrays were very carefully annotated in our study , as the microarray contains specific probes for this gene , and the pcr primers were designed to be specific for all gene variants .
it may however be explained by a possible difference in splice variants detected by the two technologies , as il24 has more than one associated transcript .
it is known that between - platform consistency decreases with different primers [ 5 , 6 , 19 ] or increased distance on the sequence between them .
unfortunately , since the primer sequences of the pcr products are not publicly available , we can not verify this possible cause of discrepancy .
in conclusion , we have observed consistencies and differences between results obtained by two different technologies .
in general , we could not observe a better detection level of the pcr - array , though this may be caused by the chosen quality criteria set . especially with different probes / primers , it is hard to decide which technology gives a more accurate measurement .
especially , for nutritional studies , where effects can be expected to be small , one could argue that running a microarray with a broader coverage could be followed by using a pcr - array focused at a specific process , to possibly detect extra transcripts . however
, current and future costs will also play a role in the choice of the platforms to be utilized .
finally , we conclude that pcr - arrays should not be considered a perfect validation tool , since both array technologies have their own specific detection limits and neither can be considered to be the real golden standard .
below is the link to the electronic supplementary material . 

  supplementary table  1 ( xls 55 kb )
 microarray technology allows us to perform high - throughput screening of changes in gene expression . 
 the outcome of microarray experiments largely depends on the applied analysis methods and cut - off values chosen . 
 results are often required to be verified using a more sensitive detection technique , such as quantitative real - time pcr ( qpcr or rt - pcr ) . throughout the years 
 , this technique has become a de facto golden standard . 
 individual qpcrs are time - consuming , but the technology to perform high - throughput qpcr reactions has become available through pcr - arrays that allow up to 384 pcr reactions simultaneously . 
 our current aim was to investigate the usability of a rt2 profiler pcr - array as validation in a nutritional intervention study , where the measured changes in gene expression were low . 
 for some differentially expressed genes , the pcr - array confirmed the microarray prediction , though not for all . 
 furthermore , the pcr - array allowed picking up the expression of genes that were not measurable on the microarray platform but also vice versa . 
 we conclude that both techniques have their own ( dis)advantages and specificities , and for less pronounced changes using both technologies may be useful as complementation rather than validation.electronic supplementary materialthe online version of this article ( doi:10.1007/s12263 - 008 - 0094 - 1 ) contains supplementary material , which is available to authorized users . 
Electronic supplementary material
Introduction
Materials and methods
Results and discussion
Electronic supplementary material