BACKGROUND
gene expression profiling investigations involving postmortem brain tissue of cases with neuropsychiatric disorders such as autism have been limited due to tissue availability and tissue quality  <cit> .
such investigations, however, are critical for understanding uniquely human disorders  <cit> .
while experimenters cannot control tissue availability, novel technologies can be employed to utilize the precious and scarce tissue resources available from brain banks even if preservation quality is not ideal  <cit> .
the cdna-mediated annealing, selection, and ligation  gene expression assay has been shown to produce highly reliable results when applied to formalin-fixed, paraffin-embedded tissues  <cit> .
to overcome the difficulties associated with poly a/oligo-dt-based priming in special experimental conditions such as with profiling partially degraded rna, the dasl-based assay uses random priming at the cdna synthesis step.
it generates first strand cdna to minimize variation during random priming and avoids biases associated with sample amplification and labeling from multiple rounds of random priming  <cit> .
furthermore, the assay requires only a ~ <dig> nucleotide target sequence for query oligonucleotide annealing, which makes it effective for quantifying partially degraded rna samples.
the dasl-based methodology has already been applied in the study of human liver, esophagus, breast, prostate, ovarian, and other biopsy and autopsy tissues  <cit> .
it can be extended for use in a genome-wide format, which may be of value in the elucidation of genes mediating neuropsychiatric diseases  <cit> .
however, it has not yet been applied to postmortem frozen and formalin-fixed brain tissue, despite its potential benefits when assaying samples with low rna quality.
in this study, we investigated the utility of the standard ivt- and dasl- based genome-wide expression profiling assays in the context of a clinically important neuropsychiatric disorder, autism.
our objectives were to: 1) compare the quality of microarray data from ivt-based and dasl-based platforms on artificially degraded reference rna; and 2) compare the quality of microarray data from these two rna profiling platforms on postmortem frozen and formalin-fixed brain tissue.
RESULTS
dasl-based expression profiling is more reliable than ivt-based profiling on artificially degraded brain reference rna
to first assess the reliability of ivt- and dasl-based platforms in expression profiling of artificially degraded reference rna samples, we performed these two assays on brain and pooled reference rna heated at 95°c for  <dig> ,  <dig>   <dig>  and  <dig> minutes .
heating fragments the reference rna, which simulates rna degradation conditions in vivo.
the more time that rna is subjected to heating, the more fragmented the rna becomes .
fold change differences between brain reference rna and pooled reference rna were used to assess the reliability of gene expression profiles at different levels of rna fragmentation .
the ivt-based assay yielded low correlations between expression profiles detected in intact rna, which was used as the standard, and degraded rna.
correlation with intact rna yielded a correlation coefficient r <dig> =  <dig>  at  <dig> minutes of heating, r <dig> =  <dig>  at  <dig> minutes, and r <dig> =  <dig>  at  <dig> minutes.
in contrast, even extremely degraded rna profiled by the dasl-based assay yielded higher correlations with intact rna.
correlation with intact rna yielded a correlation coefficient r <dig> =  <dig>  at  <dig> minutes of heating, r <dig> =  <dig>  at  <dig> minutes, and r <dig> =  <dig>  at  <dig> minutes.
high direct correlations were also achieved using the dasl-based assay between detected genes of intact reference brain rna and degraded reference brain rna samples only .
correlation with intact rna yielded a correlation of  <dig>  at  <dig> minutes,  <dig>  at  <dig> minutes, and  <dig>  at  <dig> minutes of degradation.
similarity between degraded and intact samples achieved by the ivt-based assay, however, was much lower.
correlations of  <dig>  at  <dig> minutes,  <dig>  at  <dig> minutes, and  <dig>  at  <dig> minutes with intact rna were found.
in general, partial rna degradation affected both assays , but the ivt-based assay was affected more severely than the dasl-based assay  as evidenced by lower correlations of degraded reference samples with standard intact conditions.
dasl-based platform is more reliable than ivt-based platform on rna from postmortem frozen brain tissue but not formalin-fixed tissue
we next assessed the performance of dasl- and ivt-based assays on rna extracted from postmortem frozen and formalin-fixed brain tissue .
generally, average probes detected, average probe concordance, and average signal were higher for the dasl-based assay than the ivt-based assay for rna extracted from frozen tissue and formalin fixed tissue .
average self-reproducibility was also higher for the dasl-based assay than for the ivt-based assay in frozen tissue, but not formalin fixed tissue.
upon examination of scatterplots of the formalin fixed genome-wide data, however, it was apparent that the high correlation between technical replicates in the ivt-based assay was due to detected expression only at background levels for these samples .
b5223
umb1670
umb1796
btb-3859
case id, diagnosis, age, gender, cause of death , postmortem interval , rna integrity number , and preservation method of the postmortem human brain samples assayed in this study are listed.
through examining a small number of samples assayed on both ivt-based and dasl-based platforms, we observed that the ivt-based assay was more severely affected by rna degradation than the dasl-based assay with rna extracted from brain tissues also, in accordance with the reference rna experiments .
for example, though correlations between detected probes decreased with decreasing rin in both assays, correlation between a sample with rin  <dig>  and a sample with rin  <dig>  was  <dig>  in dasl-based assay, but only  <dig>  in ivt-based assay.
with few exceptions, correlations between the same degraded samples and relatively intact rna samples from frozen tissue and reference rna were higher between those assayed by the dasl-based assay than by ivt-based assay.
in addition, distributions of detected probes were clearly affected for ivt-based profiling of samples with low rin, but this was not the case in dasl-based profiling .
in general, correlations of detected probes from the same samples between platforms were low .
correlations of samples from the same case , frozen and formalin fixed, within and between platforms were also low .
nonetheless, neither platform appeared to produce reliable results using rna from formalin-fixed brain tissue .
few genes across samples were detected above baseline on either platform, but a subset of probes detected above baseline levels in these samples on the dasl-based assay may show some reproducibility .
predictors of variance in tissue dataset
we next analyzed variance prediction to understand important experimental and subject factors of gene expression differences in our dataset.
these analyses can help determine statistical preprocessing that must be performed to prepare the dataset for differential expression analysis, and can indicate experimental considerations for future experiments.
hierarchical clustering by average linkage showed grouping of samples by assay type  and tissue preservation .
to quantify these and other predictors of variance in the dataset, we performed multivariate distance matrix regression  analysis  <cit> .
multiple regression mdmr  showed that tissue preservation method  accounted for 27% of the variance in the dataset, followed, in significance, by assay type , batch , age of the case , diagnosis , gender , and postmortem interval .
in a small sample of cases with available rin  and assayed on both ivt- and dasl-based assays, rin also explained a large percentage of variability in the expression data.
however, the influence of rin on the ivt-processed samples  in single regression mdmr was greater than that on the dasl-processed samples .
these results confirm that the ivt-based assay is more greatly influenced by differences in rna degradation than the dasl-based assay.
validation of microarray results by qpcr
qpcr validation of the dasl-based microarray data was performed on a subset of genes.
we compared the log <dig> fold changes of the qpcr data with the log <dig> transformed, quantile normalized data.
using a spearman's rank correlation, the log <dig> fold changes of these  <dig> genes  across qpcr and dasl-based microarray platforms were found to be correlated at r =  <dig>  .
forward and reverse primer sequences for  <dig> experimental and  <dig> reference genes used for rt-pcr to validate microarray results are shown.
discussion
recent advances in gene expression technology have made genome-wide expression profiling possible in partially degraded rna samples  <cit> .
dasl technology has been applied reliably to even formalin fixed, paraffin-embedded tissues from a range of tissue sources  <cit> .
it has, however, not yet been utilized to profile expression in brain tissue, in which rna degradation levels are known to be high and availability is limited  <cit> .
we have shown that the dasl-based genome-wide expression-profiling approaches applied to partially degraded brain-specific reference rna and postmortem brain tissue-extracted rna may confer some advantages over traditional ivt-based methods.
our observations raise questions about the reliability of the assays that could impact interpretation of association analyses involving gene expression levels.
the greater the degradation of rna, the less reliable results from ivt- and dasl-based platforms become.
therefore, although the dasl-based approach appeared to recover more reliable gene expression values from partially degraded rna samples, reliable results may still not be gleaned from the most severely degraded and chemically modified rna samples such as from formalin-fixed brain tissue.
the main cause of failure using dasl in formalin-fixed brain tissue may be difficulty with performing the reverse transcription step.
unfortunately, even though the dasl-based assay performed better than the ivt-based assay in limited brain frozen tissues, our sample sizes were too small to make conclusive arguments about the relative efficacy of dasl- and ivt-based platforms on rna extracted from frozen and formalin fixed tissues.
our results, however, suggest that the dasl-based platform may confer some advantages for profiling partially degraded rna from frozen brain tissue.
through the examination of the expression datasets in tissue-based rna samples from control and autistic cases, we explored important factors to consider in postmortem brain tissue expression profiling.
the most important explanatory variable of gene expression profile variance across samples in our dataset was in fact not the platform on which the rna was assayed, but how the tissue was preserved.
this result has important implications for brain banks in the methods used for preserving rna  <cit>  and for interpreting and comparing across brain gene expression studies, especially in neuropsychiatric disorders  <cit>  with a spectrum of phenotypes like autism.
though in general the dasl-based approach is more reliable than the ivt-based approach for profiling degraded mrna samples, our mdmr analyses suggest that rna degradation and other factors still play important roles in determining dataset variance, thereby potentially confounding differential expression analyses.
these additional factors may be dealt with during data preprocessing steps using such statistical tools as combat  <cit> , and is described in chow and pramparo et al.
: early brain gene expression and copy number anomalies in autism, submitted.
CONCLUSIONS
nonetheless, the study of neuropsychiatric disorders may benefit from dasl-based expression profiling technology, especially when investigating molecular pathways involved in diseases that cannot be modeled by animals  <cit> .
this platform and other expression profiling methods will be vital in helping make use of scarce and precious brain tissue to elucidate uniquely human genetic pathogenic mechanisms.

 gene expression profiling investigations involving postmortem brain tissue of cases with neuropsychiatric disorders such as autism have been limited due to tissue availability and tissue quality. such investigations, however, are critical for understanding uniquely human disorders. while experimenters cannot control tissue availability, novel technologies can be employed to utilize the precious and scarce tissue resources available from brain banks even if preservation quality is not ideal. the cdna-mediated annealing, selection, and ligation gene expression assay has been shown to produce highly reliable results when applied to formalin-fixed, paraffin-embedded tissues. to overcome the difficulties associated with poly a/oligo-dt-based priming in special experimental conditions such as with profiling partially degraded rna, the dasl-based assay uses random priming at the cdna synthesis step. it generates first strand cdna to minimize variation during random priming and avoids biases associated with sample amplification and labeling from multiple rounds of random priming. furthermore, the assay requires only a ~ nucleotide target sequence for query oligonucleotide annealing, which makes it effective for quantifying partially degraded rna samples. the dasl-based methodology has already been applied in the study of human liver, esophagus, breast, prostate, ovarian, and other biopsy and autopsy tissues. it can be extended for use in a genome-wide format, which may be of value in the elucidation of genes mediating neuropsychiatric diseases. however, it has not yet been applied to postmortem frozen and formalin-fixed brain tissue, despite its potential benefits when assaying samples with low rna quality. in this study, we investigated the utility of the standard ivt- and dasl- based genome-wide expression profiling assays in the context of a clinically important neuropsychiatric disorder, autism. our objectives were to: 1) compare the quality of microarray data from ivt-based and dasl-based platforms on artificially degraded reference rna; and 2) compare the quality of microarray data from these two rna profiling platforms on postmortem frozen and formalin-fixed brain tissue.  dasl-based expression profiling is more reliable than ivt-based profiling on artificially degraded brain reference rna to first assess the reliability of ivt- and dasl-based platforms in expression profiling of artificially degraded reference rna samples, we performed these two assays on brain and pooled reference rna heated at 95°c for, and minutes. heating fragments the reference rna, which simulates rna degradation conditions in vivo. the more time that rna is subjected to heating, the more fragmented the rna becomes. fold change differences between brain reference rna and pooled reference rna were used to assess the reliability of gene expression profiles at different levels of rna fragmentation. the ivt-based assay yielded low correlations between expression profiles detected in intact rna, which was used as the standard, and degraded rna. correlation with intact rna yielded a correlation coefficient r = at minutes of heating, r = at minutes, and r = at minutes. in contrast, even extremely degraded rna profiled by the dasl-based assay yielded higher correlations with intact rna. correlation with intact rna yielded a correlation coefficient r = at minutes of heating, r = at minutes, and r = at minutes. high direct correlations were also achieved using the dasl-based assay between detected genes of intact reference brain rna and degraded reference brain rna samples only. correlation with intact rna yielded a correlation of at minutes, at minutes, and at minutes of degradation. similarity between degraded and intact samples achieved by the ivt-based assay, however, was much lower. correlations of at minutes, at minutes, and at minutes with intact rna were found. in general, partial rna degradation affected both assays, but the ivt-based assay was affected more severely than the dasl-based assay as evidenced by lower correlations of degraded reference samples with standard intact conditions. dasl-based platform is more reliable than ivt-based platform on rna from postmortem frozen brain tissue but not formalin-fixed tissue we next assessed the performance of dasl- and ivt-based assays on rna extracted from postmortem frozen and formalin-fixed brain tissue. generally, average probes detected, average probe concordance, and average signal were higher for the dasl-based assay than the ivt-based assay for rna extracted from frozen tissue and formalin fixed tissue. average self-reproducibility was also higher for the dasl-based assay than for the ivt-based assay in frozen tissue, but not formalin fixed tissue. upon examination of scatterplots of the formalin fixed genome-wide data, however, it was apparent that the high correlation between technical replicates in the ivt-based assay was due to detected expression only at background levels for these samples. b5223 umb1670 umb1796 btb-3859 case id, diagnosis, age, gender, cause of death, postmortem interval, rna integrity number, and preservation method of the postmortem human brain samples assayed in this study are listed. through examining a small number of samples assayed on both ivt-based and dasl-based platforms, we observed that the ivt-based assay was more severely affected by rna degradation than the dasl-based assay with rna extracted from brain tissues also, in accordance with the reference rna experiments. for example, though correlations between detected probes decreased with decreasing rin in both assays, correlation between a sample with rin and a sample with rin was in dasl-based assay, but only in ivt-based assay. with few exceptions, correlations between the same degraded samples and relatively intact rna samples from frozen tissue and reference rna were higher between those assayed by the dasl-based assay than by ivt-based assay. in addition, distributions of detected probes were clearly affected for ivt-based profiling of samples with low rin, but this was not the case in dasl-based profiling. in general, correlations of detected probes from the same samples between platforms were low. correlations of samples from the same case, frozen and formalin fixed, within and between platforms were also low. nonetheless, neither platform appeared to produce reliable results using rna from formalin-fixed brain tissue. few genes across samples were detected above baseline on either platform, but a subset of probes detected above baseline levels in these samples on the dasl-based assay may show some reproducibility. predictors of variance in tissue dataset we next analyzed variance prediction to understand important experimental and subject factors of gene expression differences in our dataset. these analyses can help determine statistical preprocessing that must be performed to prepare the dataset for differential expression analysis, and can indicate experimental considerations for future experiments. hierarchical clustering by average linkage showed grouping of samples by assay type and tissue preservation. to quantify these and other predictors of variance in the dataset, we performed multivariate distance matrix regression analysis. multiple regression mdmr showed that tissue preservation method accounted for 27% of the variance in the dataset, followed, in significance, by assay type, batch, age of the case, diagnosis, gender, and postmortem interval. in a small sample of cases with available rin and assayed on both ivt- and dasl-based assays, rin also explained a large percentage of variability in the expression data. however, the influence of rin on the ivt-processed samples in single regression mdmr was greater than that on the dasl-processed samples. these results confirm that the ivt-based assay is more greatly influenced by differences in rna degradation than the dasl-based assay. validation of microarray results by qpcr qpcr validation of the dasl-based microarray data was performed on a subset of genes. we compared the log fold changes of the qpcr data with the log transformed, quantile normalized data. using a spearman's rank correlation, the log fold changes of these genes across qpcr and dasl-based microarray platforms were found to be correlated at r =. forward and reverse primer sequences for experimental and reference genes used for rt-pcr to validate microarray results are shown. discussion recent advances in gene expression technology have made genome-wide expression profiling possible in partially degraded rna samples. dasl technology has been applied reliably to even formalin fixed, paraffin-embedded tissues from a range of tissue sources. it has, however, not yet been utilized to profile expression in brain tissue, in which rna degradation levels are known to be high and availability is limited. we have shown that the dasl-based genome-wide expression-profiling approaches applied to partially degraded brain-specific reference rna and postmortem brain tissue-extracted rna may confer some advantages over traditional ivt-based methods. our observations raise questions about the reliability of the assays that could impact interpretation of association analyses involving gene expression levels. the greater the degradation of rna, the less reliable results from ivt- and dasl-based platforms become. therefore, although the dasl-based approach appeared to recover more reliable gene expression values from partially degraded rna samples, reliable results may still not be gleaned from the most severely degraded and chemically modified rna samples such as from formalin-fixed brain tissue. the main cause of failure using dasl in formalin-fixed brain tissue may be difficulty with performing the reverse transcription step. unfortunately, even though the dasl-based assay performed better than the ivt-based assay in limited brain frozen tissues, our sample sizes were too small to make conclusive arguments about the relative efficacy of dasl- and ivt-based platforms on rna extracted from frozen and formalin fixed tissues. our results, however, suggest that the dasl-based platform may confer some advantages for profiling partially degraded rna from frozen brain tissue. through the examination of the expression datasets in tissue-based rna samples from control and autistic cases, we explored important factors to consider in postmortem brain tissue expression profiling. the most important explanatory variable of gene expression profile variance across samples in our dataset was in fact not the platform on which the rna was assayed, but how the tissue was preserved. this result has important implications for brain banks in the methods used for preserving rna and for interpreting and comparing across brain gene expression studies, especially in neuropsychiatric disorders with a spectrum of phenotypes like autism. though in general the dasl-based approach is more reliable than the ivt-based approach for profiling degraded mrna samples, our mdmr analyses suggest that rna degradation and other factors still play important roles in determining dataset variance, thereby potentially confounding differential expression analyses. these additional factors may be dealt with during data preprocessing steps using such statistical tools as combat, and is described in chow and pramparo et al.: early brain gene expression and copy number anomalies in autism, submitted.  nonetheless, the study of neuropsychiatric disorders may benefit from dasl-based expression profiling technology, especially when investigating molecular pathways involved in diseases that cannot be modeled by animals. this platform and other expression profiling methods will be vital in helping make use of scarce and precious brain tissue to elucidate uniquely human genetic pathogenic mechanisms. 
although data associated with a small sample of formalin-fixed rna samples were poor when obtained from both assays, the dasl-based platform exhibited greater reliability in a subset of probes and samples. gene expression assays have been shown to yield high quality genome-wide data from partially degraded rna samples. we compared cdna-mediated annealing, selection, and ligation - and in vitro transcription -based genome-wide expression profiling assays on rna samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue. we ultimately consider the implications of these results on investigations of neuropsychiatric disorders. the dasl-based platform produced expression results of greater reliability than the ivt-based platform in artificially degraded reference brain rna and rna from frozen tissue-based samples. our results suggest that the dasl-based gene expression-profiling platform may confer some advantages on mrna assays of the brain over traditional ivt-based methods. however, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor rna quality and other technical limitations inherent in many assays.
 gene expression assays have been shown to yield high quality genome-wide data from partially degraded rna samples. however, these methods have not yet been applied to postmortem human brain tissue, despite their potential to overcome poor rna quality and other technical limitations inherent in many assays. we compared cdna-mediated annealing, selection, and ligation - and in vitro transcription -based genome-wide expression profiling assays on rna samples from artificially degraded reference pools, frozen brain tissue, and formalin-fixed brain tissue.  the dasl-based platform produced expression results of greater reliability than the ivt-based platform in artificially degraded reference brain rna and rna from frozen tissue-based samples. although data associated with a small sample of formalin-fixed rna samples were poor when obtained from both assays, the dasl-based platform exhibited greater reliability in a subset of probes and samples.  our results suggest that the dasl-based gene expression-profiling platform may confer some advantages on mrna assays of the brain over traditional ivt-based methods. we ultimately consider the implications of these results on investigations of neuropsychiatric disorders. braingene expressiondaslivt 