BACKGROUND
dilated cardiomyopathy  is a heterogeneous disease of multifactorial etiopathogenesis.
however, investigations on endomyocardial biopsies  have revealed that chronic t-cell infiltration , induced and maintained by cardiotropic viral infections, as well as by secondary autoimmune pathways, is identifiable in a significant proportion of patients presenting with dcm  <cit> .
in addition, functional characterization of infiltrates and expression investigations of further immunocompetent factors may be decisively important for a profound understanding of the pathogenesis of dcmi, as well as for immunomodulatory treatment strategies  <cit> .
furthermore, expression of genes involved in general heart failure pathways such as the remodeling of the extracellular matrix is altered in embs from dcm patients  <cit> .
gene expression investigations of embs are promising approaches for the investigation novel pathogenic pathways involved in dcm/dcmi  <cit> , and may reveal profiles with prognostic impact for the natural course of the disease as well as under immunomodulatory treatment  <cit> .
real-time rt-pcr is a sensitive, precise and reproducible tool to determine gene expression in tissues.
however, the rna amounts extractable from embs are low, thereby limiting the number of analyzable target genes by real-time rt-pcr.
preamplification  techniques may enhance sensitivity of real-time rt-pcr especially for low abundance genes, and can establish substantially higher cdna amounts, which can then expand the number of the analyzable target genes.
however, maintenance of gene expression profiles as well as a broad applicability to many target genes with a feasible additional effort is a further intriguing aspect of preamp procedures.
we here report the application of the taqman® preamp master mix technique  to embs' cdna, which, as in pbmcs' cdna, enables a robust preamp of multiple pooled taqman® abi inventoried gene expression assays and primers of self-designed gene expression assays, including wobbled primer designs, with acceptable preamp uniformity values , and low intra- and inter-assay variance coefficients.
we compared this technique with a sequence-specific reverse transcription  technique followed by a multiplex preamp step  in accordance to the strategy by peixoto et al.
<cit>  in a set of self-designed  <dig> gene assays for t-cell specific genes, which performed with a partly higher preamp efficacy , however resulted in substantial variations of preamp uniformity, and higher inter-assay variation coefficients , and moreover did not offer the possibility of expanding the possible real-time rt-pcr analyses out of the limited embs' cdna.
furthermore, we show preliminary data on the differential expression of  <dig> out of  <dig>  investigated target genes in patients with embs-confirmed dcmi compared with embs from dcm patients.
moreover, we provide evidence that cdkn1b  as well as hprt  is not differentially expressed in dcm versus donor hearts, and may therefore be a relevant housekeeping gene candidate for real-time rt-pcr analyses of myocardial tissues.
RESULTS
preamp real-time rt-pcr from pbmcs' cdna
t-preamp
albeit the ct values obtained from cdna from both pbmcs and embs in conventional direct real-time pcr were virtually equivalent  with respect to the hypoxanthine-guanine phosphoribosyltransferase  gene assay designed by the institute of medical immunology at the charité-campus mitte  and the abi inventoried taqman® gene assay for hprt  in initial investigations, t-preamp cdna revealed differences in preamp uniformity values of these two hprt gene assays.
whereas the ct improvement for hprt-ccm was  <dig> , the gene assay hprt-abi achieved a ct improvement of only  <dig>  cycles.
this resulted in a preamp uniformity related to cdkn1b of  <dig>  for hprt-ccm and  <dig>  for hprt-abi, respectively.
in light of this minor t-preamp performance of the hprt-abi gene assay, we used further on only the hprt-ccm as housekeeping gene assay.
direct real-time pcr from the pooled pbmcs' cdna revealed a mean ct value  <dig>  ±  <dig>  , and in t-preamp cdna a mean ct value of  <dig>  ±  <dig>   for the investigated gene assays excluding cd <dig>  t-preamp yielded a mean improvement of  <dig>  ±  <dig>  cycles  for the investigated gene assays .
there was no traceable gene expression neither in the non-preamplified nor in the t-preamp cdna with respect to il- <dig>  il- <dig>  il- <dig>  il- <dig>  ifn-b, cyr <dig>  cxcl <dig>  cx3cl <dig>  apn, grail, collagen types i, iii and iv, laminin, mmp <dig>  timp- <dig>  gdf <dig> and cd62e.
considering the threshold ct for low expression levels published by canales et al.
<cit> , 13/ <dig>  of the target genes showed ct values ±  <dig> in direct real-time rt-pcr.
the mean preamp uniformity for the investigated gene assays in pbmcs  related to cdkn1b was  <dig>  ±  <dig> , ranging from - <dig>  to  <dig> , and the preamp uniformity in pbmcs related to hprt-ccm was  <dig>  ±  <dig> , ranging from - <dig>  to  <dig> , respectively.
there was no significant difference between the self-designed  and the taqman® abi inventoried gene assays .
with respect to cd <dig>  preamp uniformity related to cdkn1b was  <dig> , and  <dig>  related to hprt-ccm, respectively.
there was no association between the preamp uniformity related to cdkn1b of target gene ct values in direct real-time rt-pcr <  <dig>  or ≥  <dig> .
the same was true for the preamp uniformity values related to hprt-ccm .
in light of the disproportionate preamp uniformities of hprt-ccm and hprt-abi, whilst yielding comparable ct values in conventional real-time rt-pcr, we hypothesized that pcr efficiency during preamp might be a pivotal issue.
the comparison of the two gene assays showing minor preamp performance  to candidate  gene assays with t-preamp uniformities ranging between - <dig>  and + <dig>   over serial dilutions of pbmcs' cdna revealed a non-significant tendency for increased pcr efficiency by t-preamp .
this was also true for hprt-abi.
preamp uniformity for these gene assays except for cd <dig> showed comparable preamp uniformities over the serial cdna dilutions .
the cd <dig> gene assay, however, demonstrated a substantially decreased pcr efficiency by t-preamp compared with direct real-time rt-pcr , and a gradually increasing preamp uniformity at serial cdna dilutions to  <dig>  at the ratio 1: <dig> .
ssrt-preamp
using the ssrt-preamp system on hprt-ccm as housekeeping gene and further  <dig> t-cell related self-designed gene assays, a mean improvement of  <dig>  ±  <dig>  cycles  was obtained.
the measured ct values in direct real-time rt-pcr were  <dig>  ±  <dig>  , and in ssrt-preamp  <dig>  ±  <dig>  .
considering the threshold ct for low expression levels published by canales et al.
<cit> , 1/ <dig>  of the target genes showed ct values ≥  <dig> in direct real-time rt-pcr.
the mean preamp uniformity for the investigated gene assays related to hprt-ccm was - <dig>  ±  <dig>  , which was significantly  lower compared with the t-preamp data.
the following gene assays were below a preamp uniformity of - <dig> : cd3z, ifng, t-bet and perforin.
there was no association between the preamp uniformity related to hprt-ccm of target gene ct values in direct real-time rt-pcr <  <dig>  or ≥  <dig> .
preamp real-time rt-pcr from embs
t-preamp
t-preamp of embs' cdna with the investigated target gene assays resulted in a mean ct improvement of  <dig>  ±  <dig>   for cdkn1b, and in mean improvement of  <dig>  ±  <dig>  ct values  for hprt-ccm, respectively.
the mean preamp uniformity for hprt-ccm related to cdkn1b was - <dig>  ±  <dig>  .
the mean ΔΔct  – Δct  for hprt-ccm and cdkn1b was  <dig>  ±  <dig>  .
there was a high association between the ct values of hprt-ccm and cdkn1b both in cdna  and in preamp cdna .
moreover, the Δct values of cdkn1b and hprt-ccm of  versus  showed a high correlation .
the inter-assay cvs were  <dig> % for cdkn1b, and  <dig> % for hprt-ccm, respectively.
with respect to the investigated target gene assays , t-preamp yielded a mean improvement of  <dig>  ±  <dig>  ct values .
the cd <dig> gene assay demonstrated a mean ct improvement of  <dig>  ±  <dig> .
the preamp uniformity of the investigated gene assays  related to cdkn1b was - <dig>  ±  <dig>  , and related to hprt-ccm - <dig>  ±  <dig> , , respectively.
as in cdna from pbmcs, there was no significant difference between the self-designed  and the inventoried abi gene assays .
considering the threshold ct for low expression levels published by canales et al  <cit> , 48/ <dig>  of the target genes showed ct values ≥  <dig> in direct real-time rt-pcr, whereas ct values for hprt-ccm demonstrated < <dig>  and for cdkn1b <  <dig> in each case of the  <dig> investigated embs.
there was no association between the preamp uniformity related to cdkn1b of ct values below  or above  the threshold ct for low expression levels .
the same was true for the preamp uniformity values related to hprt-ccm .
the inter-assay variance of ct improvement for the investigated gene assays preamplified in each  <dig> different embs was below  <dig>  ct values , and the resulting mean cv was  <dig>  ±  <dig> % , including the cd <dig> gene assay, which did not perform differently compared to the remaining gene assays in this respect .
abi inventoried gene assays demonstrated a higher inter-assay cv  compared with the self-designed gene assays .
noticeably, there was no traceable gene expression in some of the investigated each embs per gene group with respect to trbv <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  il <dig>  il <dig>  il <dig>  ifnb and apn neither in the direct nor in the preamp real-time rt-pcr .
the  <dig> different preamp procedures from the cdna from  <dig> emb resulted in minor differences of ct values .
the mean intra-assay cv was  <dig>  ±  <dig> % , including cd <dig>  which did not perform differently compared to the remaining gene assays with regard to the intra-assay cv .
abi inventoried gene assays demonstrated a lower intra-assay cv  compared with the self-designed gene assays  .
ssrt-preamp
employing ssrt-preamp, the investigated gene assays yielded a mean improvement of  <dig>  ±  <dig>  ct values .
the mean ct values obtained by direct real-time rt-pcr were  <dig>  ±  <dig>  , and the ssrt-preamp ct values were  <dig>  ±  <dig>  , respectively.
in one emb, no traceable ifng expression was observed neither by direct nor by ssrt-preamp real-time rt-pcr.
considering the threshold ct for low expression levels published by canales et al  <cit> , 12/ <dig>  of the target genes showed ct values ≥  <dig> in direct real-time rt-pcr, whereas ct values for hprt-ccm demonstrated < <dig> in each case of the  <dig> investigated embs.
the mean preamp uniformity of the investigated gene assays related to hprt-ccm was - <dig>  ±  <dig>  , which was significantly  lower compared with the t-preamp data.
as in pbmcs, the gene assays for cd3z, ifng, t-bet and perforin performed below the preamp uniformity value of - <dig> .
the mean inter-assay cv using the ssrt-preamp technique was higher compared with t-preamp .
there was no association between the preamp uniformity related to hprt-ccm of target gene ct values in direct real-time rt-pcr <  <dig>  or ≥  <dig> .
however, the ssrt-preamp yielded comparably low intra-assay cvs as obtained by t-preamp .
the real-time rt-pcr results from each  <dig> dcmi- and dcm-patients revealed differential expression with respect to  <dig> out of the  <dig>  investigated target genes.
these differential gene expressions were related to both hprt-ccm and cdkn1b.
significantly different gene expressions related to hprt-ccm are shown in table  <dig>  the data of all investigated target genes related to hprt-ccm, including the non differentially expressed genes, are listed in the additional file  <dig>  gene expression data related to cdkn1b are listed in the additional file  <dig>  significantly increased gene expression was determined with respect to several t-cell related genes , interleukin  <dig> , tumor necrosis factor alpha , the chemokine cx3cl <dig> , cysteine-rich angiogenic inducer  <dig> , th <dig> associated genes , genes expressed by cytotoxic t-lymphocytes , β1-integrins , the selectin cd62e  and growth-differentiation factor  <dig> .
the significantly down-regulated genes in embs from dcmi-patients were the chemokine cxcl <dig> , adiponectin  and tissue factor .
significantly different gene expression  t-preamp real-time rt-pcr results in embs from dcm patients compared with embs from patients with immunohistologically confirmed dcmi .
the values are given as means ± sd, followed by the respective p values in significantly different comparisons.
hprt and cdkn1b are not regulated in dcm compared with donor hearts
the value of hprt as housekeeping gene is well acknowledged in various tissues.
the close association between hprt-ccm and cdkn1b ct values as well as the equally significant differential expression of genes related to both hprt-ccm and cdkn1b in embs from dcm versus dcmi patients infer a possible relevance of cdkn1b as a housekeeping gene.
to address this issue, we determined the ct values of hprt  and cdkn1b by direct real-time rt-pcr in each  <dig> explanted donor and terminally failing dcm hearts, from which enough tissue amounts were accessible to adjust rna to  <dig> ng/μl before cdna synthesis.
as stated above for pbmcs, hprt-abi and hprt-ccm demonstrated a difference around  <dig>  ct values.
there was no statistical difference of ct values of hprt-abi, hprt-ccm and cdkn1b in donor and dcm hearts .
discussion
it is widely accepted that conventional histological diagnostics are not sufficient to elucidate the complex processes critically involved in the pathogenesis of dcmi, that they clearly lack prognostic impact, and that they are not sufficient to select dcm/dcmi patients who will likely benefit from immunomodulatory treatment  <cit> .
gene expression investigations are promising approaches for a profound understanding of the pathogenesis of dcm/dcmi, the prognostic impact of gene expression profiles in the natural course of the disease as well as under immunomodulatory treatment  <cit> .
however, the amount of tissue and rna/cdna available from embs is limited, which restricts the number of analyzable target genes per emb.
preamp procedures may enhance sensitivity of real-time rt-pcr especially for low abundance expressed genes, and can establish substantially higher cdna amounts, which can then expand the number of the analyzable target genes from a limited cdna.
however, maintenance of gene expression profiles as well as a broad applicability to many target genes with a feasible additional effort is a further intriguing aspect of preamp procedures.
the t-preamp technique enables a relatively simple workflow, which basically consists of setting up the primer/gene assay pool and t-preamp of cdna over  <dig> cycles in a special master mix, maintaining robust preamp conditions for several gene assays, which results in mostly stable preamp uniformity values.
identical primers are used for both the t-preamp reaction and the succeeding real-time rt-pcr.
this is substantially easier to accomplish compared with the ssrt-preamp, which requires design of specific primers for both the ssrt and the following preamplification step  <cit> .
according to the manufacturer's specifications, up to  <dig> gene assays can be preamplified simultaneously in each t-preamp reaction, which is confirmed by our data on the simultaneously preamplified  <dig> gene assays both in pbmcs' and embs' cdna.
a further advantage of this technique is that out of  <dig> μl cdna obtained from the herein applied rna extraction and cdna synthesis methods for embs,  <dig> different preamp reactions are feasible from one single emb, which would hypothetically enable gene expression analysis of  <dig> different target genes .
this is a substantially higher number of quantifiable target genes compared with the direct conventional real-time pcr, by which a maximum of  <dig> different target genes would be applicable in duplicate real-time rt-pcr analyses and  <dig> μl cdna used in each reaction, and also compared with the ssrt-preamp procedure, which equally does not grant the possibility of expanding the maximum possible real-time rt-pcr analyses out of a limited amount of cdna.
this fact implies a further advantage of a negligible sampling error for this high putative number of real-time rt-pcr analyses carried out from one emb, as opposed to the situation in which gene expression analyses would have to be split into different groups of target genes on cdna from several embs from patients, given the limited rna/cdna amount available from each emb for direct real-time rt-pcr analyses.
noticeably, sampling error is a critical issue for the histological assessment of myocardial inflammation  <cit> .
the t-preamp technique with  <dig> preamp cycles results in a substantial increase of sensitivity of the real-time rt-pcr by around  <dig> ct values, with preamp uniformity values ranging between - <dig>  to + <dig>  both in pbmcs' and embs' cdna .
the low intra- and interassay cvs in embs cdna  imply a high precision and repeatability of the t-preamp procedure.
furthermore, our data show that the preamp uniformity is maintained over a broad range of ct values in direct real-time pcr, including low abundance ct ranges   <cit> .
this applies to both taqman® abi inventoried gene assays as well as to self-designed gene assays adhering to abi recommendations.
as shown herein, only the forward/reverse primers of self-designed gene assays need to be included in the preamp master mix reaction, and do not perform significantly different compared with the abi inventoried taqman® gene assays regarding the preamp uniformity, of which both primers and probes have to be pooled in the t-preamp reaction.
moreover, we determined that the preamp uniformity is also maintained with regard to gene assays using common reverse primers and common probes but diverse forward primers  as well as to wobbled primer designs .
these numerous advantages cannot be met by the ssrt-preamp procedure.
first, this technique is not feasible for abi inventoried taqman® gene expression assays, since the primer sequences of these assays are not accessible, and therefore primer design for the ssrt-preamp steps is not possible.
secondly, ssrt-preamp results on both pbmcs' and embs' cdna demonstrated highly diverging preamp uniformity, with values below the suggested range of - <dig>  to  <dig>  with regard to cd3z, ifng, t-bet and perforin, albeit the same gene assays performed within the suggested preamp uniformity range of - <dig>  to  <dig>  using the taqman® preamp master mix.
however, our data show that both preamp procedures perform without significantly different preamp uniformity values over a broad range of ct values in direct real-time pcr, including low abundance ct ranges   <cit> , which implies a potential application in preclinical diagnostics especially for the t-preamp procedure due to its numerous advantages .
one further important finding is the lack of traceable expression in both cdna and t-preamp cdna for target genes and samples, in which no detectable ct values were obtained neither by direct real-time rt-pcr in cdna from both pbmcs and embs.
this infers that both t- and ssrt-preamp techniques yield reliable results and do not produce erroneous values.
as a limitation of our study, the comparison of preamp uniformity values of the investigated gene assays between t-preamp and ssrt-preamp was based on hprt-ccm only, and not on cdkn1b, since the latter taqman® abi inventoried gene assay is not applicable to ssrt-preamp.
however, t-preamp data on hprt-ccm indicate that this gene assay can be equally used as referral gene assay for the calculation of the preamp uniformity.
this infers that not the design characteristics of these gene assays , but rather the preamp procedure per se is the decisively important issue leading to the observed discrepancies of preamp uniformity values.
it is possible that interactions of the primers of the different gene assays during the ssrt-preamp procedure may lead to the observed bias in preamp uniformity.
this is in line with our observations on the more pronounced distortion of the preamp uniformity values when adding more gene assays into the ssrt-preamp reactions .
due to the higher complexity of the ssrt-preamp procedure and the expected higher chance of primer interactions during preamp, we did not investigate the preamp characteristics of the gene assays using common reverse primers/probes and diverse forward primers, nor wobbled primer designs , with the ssrt-preamp procedure.
nested pcr techniques have a higher specificity compared with non-nested pcr  <cit> , and real-time rt-pcr is known to have a higher specificity compared with nested pcr  <cit> .
thus, the advantage of the ssrt-preamp approach might be the higher specificity due to the nested primer design.
since the recipe of the t-preamp master mix® is not accessible, we cannot speculate on the decisively important factors responsible for the observed disparity of the preamp uniformity of the t-preamp and the ssrt-preamp, so far.
possibly, the ssrt before the multiplex nested preamp may lead to the high diversity of preamp uniformity of the ssrt-preamp procedure, and this distortion is more pronounced regarding the gene assays cd3z, ifng, t-bet and perforin.
nonetheless, the equally low intra-assay cvs using both ssrt-preamp and t-preamp indicate that the ssrt-preamp is an equally reproducible approach, albeit potentially leading to more pronounced skewing of the calculation of relative gene expressions.
however, the higher inter-assay cvs obtained by ssrt-preamp, although still <10%, indicate that the t-preamp technique leads to a higher precision for the direct comparison in the relative quantification of gene expression analyses between samples.
our data show that the preamp uniformity of the t-preamp technique depends on the respective gene assay both in pbmcs' and embs' cdna, and implies that gene assays should be tested for preamp uniformity before setting up serial t-preamp investigations.
the two gene assays, which did not perform comparably to the suggested t-preamp uniformity reference gene assay cdkn1b, were hprt-abi and cd <dig> .
as a consequence, calculation of expression profiles of all target genes would be substantially altered, if hprt-abi would be used as a housekeeping gene assay in t-preamp real-time rt-pcr analyses, which emphasizes the value of preamp uniformity testing referring especially to a possible housekeeping gene candidate.
in line with our observations, denning et al.
previously identified that the gapdh taqman® abi inventoried gene assay does not maintain the suggested preamp uniformity values.
therefore, these authors also normalized gene expression to cdkn1b, as well  <cit> .
nonetheless, as shown for cd <dig>  the low inter- and intra-assay variations of the preamp technique  warrants a high reproducibility of the preamp real-time rt-pcr results, even for the gene assays identified to show substantially altered preamp uniformity values out of the suggested range between - <dig>  and + <dig> .
however, in the context of the relatively robust preamp uniformity performance of the t-preamp technique , one should also consider the pcr efficiency of the respective gene assays, since the "true" range of preamp uniformity hypothetically rises with decreasing pcr efficiency.
furthermore, the impact of contaminating dna can be theoretically significant in such low-level expression levels of the target transcripts.
the intron spanning primer design avoiding co-amplification of genomic dna of both the self-designed and of the taqman® abi inventoried gene assays, however, and the missing cross reactivity with genomic dna tested in dna from pbmcs largely excludes an impact of this theoretically problematic issue in our investigations.
our investigations on possible causes for disproportionate t-preamp uniformities, although not elucidating the concrete reason, reveal that the t-preamp performance does not depend on the amplicon length and on the pcr efficiency of the respective gene assays.
denning et al.
deduced from their investigations on gapdh  and  <dig> further gene assays with shorter amplicon lengths, that the amplicon length might be crucially important for t-preamp uniformity  <cit> .
however, our data cannot confirm this hypothesis, especially since the trbv <dig> and trbv <dig> gene assays yielding amplicons around  <dig> bp and  <dig> bp  resulted in preamp uniformities in the range between - <dig>  and + <dig> , performing comparably to further self-designed gene assays , and taqman® abi inventoried gene assays .
there was no evidence for an impact of the pcr efficiency of these gene assays on the preamp uniformity.
in contrast, the taqman® abi inventoried gene assays hprt-abi  and cd <dig>  showed substantially lower improvement of ct values under the same conditions.
noticeably, this distortion of preamp uniformity was stable at the serial cdna dilutions regarding hprt-abi, whereas the cd <dig> gene assay showed an increasing preamp uniformity with decreasing pcr efficiency at serial cdna dilutions.
we therefore conclude that distortion of t-preamp uniformity is gene assay specific and not a general problem of the t-preamp technique.
these findings add clarity, however, are troublesome at the same time, since the concrete reason for disproportionate t-preamp performance of certain gene assays still remains unclear.
nonetheless, these data again highlight the importance of preamp uniformity pretests before setting up serial investigations using the t-preamp technique.
to gather, compare and discuss the experiences especially on the obvious distortion of t-preamp uniformity with regard to particular gene expression assays, we propose the constitution of a central open access forum.
in light of the possible impact of primer interactions during the t-preamp reaction, of rna quality and dna contamination, this forum should not only refer to the t-preamp performance of single gene assays, but also incorporate the precise gene assay mix in the respective t-preamp reaction, the applied rna extraction technique, rna quality and dna contamination issues.
hopefully, the decisively important design characteristics of gene assays leading to a substantial deviation of the t-preamp uniformity can be identified, eventually.
these insights might lead to additional rules of gene assay design, by which the t-preamp uniformity can be predicted.
several gene expression analyses have been reported comparing heart failure to non-failing hearts, or different cardiomyopathy entities.
in this preliminary analysis, we compared embs from  <dig> dcm patients with  <dig> patients with immunohistologically confirmed dcmi.
both patient groups had comparably depressed lvef, which alleviates a possible bias of secondary general heart-failure associated mechanisms.
our preliminary data from these t-preamp real-time rt-pcr analyses revealed differential gene expression with respect to 27/ <dig>  of the investigated target genes.
in brief, these investigations confirmed that the immunohistologically diagnosed dcmi is accompanied by a significantly increased expression of t-cell related genes .
up-regulation of distinct trbv families  in embs from dcmi patients indicates a selective recruitment and/or expansion, and therefore restriction of trbv usage of the t-cell infiltrates in dcmi  <cit> .
the increased expression of il <dig>  tnfa and cx3cl <dig> infer that t-cell infiltration in dcmi is paralleled by differential increment of these cytokines, whereas a further chemokine, cxcl <dig>  was confirmed to be downregulated in dcmi patients as recently reported for dcmi patients with slightly impaired lvef by microarray analyses  <cit> .
furthermore, in line with these microarray findings, our investigations confirmed a down-regulation of apn, while its receptors apn-r <dig> and apn-r <dig> were not differentially expressed, and an up-regulation of cyr <dig>  <cit> .
the results on markers of t-cell activation reveal a yet not recognized potential role of granzymes a and b, granulysin, as well as t-bet and eomesodermin as markers for cytotoxic and th1-polarized t-cells, respectively, in dcmi.
in contrast, markers for th2-polarized t-cells, anergic and regulatory t-cells  were not increased in embs from patients with immunohistologically confirmed dcmi, which might indicate a missing counter-balance of the t-cellular response in dcmi.
the lack of differential expression of toll-like receptors  and signaling factors involved in tlr downstream pathways do not favor the hypothesis of a paramount role of tlrs in dcmi, so far.
our analyses furthermore confirmed downregulation of intramyocardial tf expression in dcmi versus dcm patients  <cit> .
whilst altered expression of several components of the extracellular matrix is well known in dcm and further non-dcm cardiomyopathies  <cit> , no direct link to inflammation or cardiotropic viral infection has been described, so far.
our preliminary data do not show any differential expression of collagen types i, iii and iv, mmps  <dig>   <dig>   <dig>  timps  <dig>   <dig> and upa in embs from dcmi versus dcm patients.
increased expression of β1-integrins and further adhesion molecules such as cd62e have been confirmed by immunohistological analyses  <cit> , which is in line with our data on av5b <dig> and cd62e.
gdf <dig> is a newly recognized marker of heart failure, and our data implicate an additional role of dcmi for increased gdf <dig> expression  <cit> .
in summary, these preliminary analyses confirm that the t-preamp procedure, in addition to its technical applicability to embs, thereby expanding the number of analyzable target genes in embs investigations, is capable of revealing significantly different gene expression profiles in dcmi versus dcm patients.
albeit numerous reports have confirmed close associations between rna and protein gene expression, a discrepancy between these two levels of gene expression was elucidated in some investigations, i.e.
due to posttranslational modification  <cit> .
we show here a significant association between expression of t-cellular markers  and dcmi as determined by immunohistologically quantified cd3+ infiltrates.
although it would be beyond the scope of this methodology paper, significantly different gene expression analyses as determined by preamp real-time pcr should be ideally confirmed by protein expression analyses in future investigations.
further investigations on larger patient cohorts are warranted to explore associations with the various cardiotropic viruses  <cit> , the impact of lvef, the acuity of the disease , the prognostic role of gene expression for the diverse natural course of the disease   <cit> , as well as the changes of gene expression in dcmi patients under immunomodulatory treatment modalities .
the application of the t-preamp procedure might be especially useful in light of the new emb guidelines  <cit> .
moreover, these promising results of t-preamp real-time rt-pcr in embs might be stimulating for researchers analyzing biopsies or comparably small sized tissue samples from other organs, and for preamp real-time rt-pcr analyses from limited cell amounts such as those obtained by laser capture microdissection  <cit> .
finally, we here provide first evidence that cdkn1b, in addition to its function as the suggested reference gene for the preamp uniformity, can be used as a relevant housekeeping gene for the relative quantification of gene expression in real-time rt-pcr in myocardial tissues, equivalent to hprt.
whereas the tumor suppressor gene function of cdkn1b is well understood in other tissues and pathologic conditions  <cit> , the physiological role and expression pattern of cdkn1b in myocardial tissues has not been investigated, yet.
congruent with our observations, denning et al.
have previously confirmed that cdkn1b can also serve as housekeeping gene in thyroid tissues  <cit> .
CONCLUSIONS
in comparison to the ssrt-preamp, t-preamp enables a relatively simple workflow, and results in a robust preamp of multiple target genes  by a mean ct improvement around  <dig> cycles, and in a lower inter-assay variance in rna derived from embs.
preliminary analyses comparing embs from dcm and dcmi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that t-preamp is a suitable tool to perform gene expression analyses in embs, expanding gene expression investigations with the limited rna/cdna amounts derived from embs.
cdkn1b, in addition to its function as a reference gene for the calculation of preamp uniformity, might serve as a suitable housekeeping gene for real-time rt-pcr analyses of myocardial tissues.

 dilated cardiomyopathy is a heterogeneous disease of multifactorial etiopathogenesis. however, investigations on endomyocardial biopsies have revealed that chronic t-cell infiltration, induced and maintained by cardiotropic viral infections, as well as by secondary autoimmune pathways, is identifiable in a significant proportion of patients presenting with dcm. in addition, functional characterization of infiltrates and expression investigations of further immunocompetent factors may be decisively important for a profound understanding of the pathogenesis of dcmi, as well as for immunomodulatory treatment strategies. furthermore, expression of genes involved in general heart failure pathways such as the remodeling of the extracellular matrix is altered in embs from dcm patients. gene expression investigations of embs are promising approaches for the investigation novel pathogenic pathways involved in dcm/dcmi, and may reveal profiles with prognostic impact for the natural course of the disease as well as under immunomodulatory treatment. real-time rt-pcr is a sensitive, precise and reproducible tool to determine gene expression in tissues. however, the rna amounts extractable from embs are low, thereby limiting the number of analyzable target genes by real-time rt-pcr. preamplification techniques may enhance sensitivity of real-time rt-pcr especially for low abundance genes, and can establish substantially higher cdna amounts, which can then expand the number of the analyzable target genes. however, maintenance of gene expression profiles as well as a broad applicability to many target genes with a feasible additional effort is a further intriguing aspect of preamp procedures. we here report the application of the taqman® preamp master mix technique to embs' cdna, which, as in pbmcs' cdna, enables a robust preamp of multiple pooled taqman® abi inventoried gene expression assays and primers of self-designed gene expression assays, including wobbled primer designs, with acceptable preamp uniformity values, and low intra- and inter-assay variance coefficients. we compared this technique with a sequence-specific reverse transcription technique followed by a multiplex preamp step in accordance to the strategy by peixoto et al. in a set of self-designed gene assays for t-cell specific genes, which performed with a partly higher preamp efficacy, however resulted in substantial variations of preamp uniformity, and higher inter-assay variation coefficients, and moreover did not offer the possibility of expanding the possible real-time rt-pcr analyses out of the limited embs' cdna. furthermore, we show preliminary data on the differential expression of out of investigated target genes in patients with embs-confirmed dcmi compared with embs from dcm patients. moreover, we provide evidence that cdkn1b as well as hprt is not differentially expressed in dcm versus donor hearts, and may therefore be a relevant housekeeping gene candidate for real-time rt-pcr analyses of myocardial tissues.  preamp real-time rt-pcr from pbmcs' cdna t-preamp albeit the ct values obtained from cdna from both pbmcs and embs in conventional direct real-time pcr were virtually equivalent with respect to the hypoxanthine-guanine phosphoribosyltransferase gene assay designed by the institute of medical immunology at the charité-campus mitte and the abi inventoried taqman® gene assay for hprt in initial investigations, t-preamp cdna revealed differences in preamp uniformity values of these two hprt gene assays. whereas the ct improvement for hprt-ccm was, the gene assay hprt-abi achieved a ct improvement of only cycles. this resulted in a preamp uniformity related to cdkn1b of for hprt-ccm and for hprt-abi, respectively. in light of this minor t-preamp performance of the hprt-abi gene assay, we used further on only the hprt-ccm as housekeeping gene assay. direct real-time pcr from the pooled pbmcs' cdna revealed a mean ct value ±, and in t-preamp cdna a mean ct value of ± for the investigated gene assays excluding cd t-preamp yielded a mean improvement of ± cycles for the investigated gene assays. there was no traceable gene expression neither in the non-preamplified nor in the t-preamp cdna with respect to il- il- il- il- ifn-b, cyr cxcl cx3cl apn, grail, collagen types i, iii and iv, laminin, mmp timp- gdf and cd62e. considering the threshold ct for low expression levels published by canales et al., 13/ of the target genes showed ct values ± in direct real-time rt-pcr. the mean preamp uniformity for the investigated gene assays in pbmcs related to cdkn1b was ±, ranging from - to, and the preamp uniformity in pbmcs related to hprt-ccm was ±, ranging from - to, respectively. there was no significant difference between the self-designed and the taqman® abi inventoried gene assays. with respect to cd preamp uniformity related to cdkn1b was, and related to hprt-ccm, respectively. there was no association between the preamp uniformity related to cdkn1b of target gene ct values in direct real-time rt-pcr < or ≥. the same was true for the preamp uniformity values related to hprt-ccm. in light of the disproportionate preamp uniformities of hprt-ccm and hprt-abi, whilst yielding comparable ct values in conventional real-time rt-pcr, we hypothesized that pcr efficiency during preamp might be a pivotal issue. the comparison of the two gene assays showing minor preamp performance to candidate gene assays with t-preamp uniformities ranging between - and + over serial dilutions of pbmcs' cdna revealed a non-significant tendency for increased pcr efficiency by t-preamp. this was also true for hprt-abi. preamp uniformity for these gene assays except for cd showed comparable preamp uniformities over the serial cdna dilutions. the cd gene assay, however, demonstrated a substantially decreased pcr efficiency by t-preamp compared with direct real-time rt-pcr, and a gradually increasing preamp uniformity at serial cdna dilutions to at the ratio 1:. ssrt-preamp using the ssrt-preamp system on hprt-ccm as housekeeping gene and further t-cell related self-designed gene assays, a mean improvement of ± cycles was obtained. the measured ct values in direct real-time rt-pcr were ±, and in ssrt-preamp ±. considering the threshold ct for low expression levels published by canales et al., 1/ of the target genes showed ct values ≥ in direct real-time rt-pcr. the mean preamp uniformity for the investigated gene assays related to hprt-ccm was - ±, which was significantly lower compared with the t-preamp data. the following gene assays were below a preamp uniformity of -: cd3z, ifng, t-bet and perforin. there was no association between the preamp uniformity related to hprt-ccm of target gene ct values in direct real-time rt-pcr < or ≥. preamp real-time rt-pcr from embs t-preamp t-preamp of embs' cdna with the investigated target gene assays resulted in a mean ct improvement of ± for cdkn1b, and in mean improvement of ± ct values for hprt-ccm, respectively. the mean preamp uniformity for hprt-ccm related to cdkn1b was - ±. the mean ΔΔct – Δct for hprt-ccm and cdkn1b was ±. there was a high association between the ct values of hprt-ccm and cdkn1b both in cdna and in preamp cdna. moreover, the Δct values of cdkn1b and hprt-ccm of versus showed a high correlation. the inter-assay cvs were % for cdkn1b, and % for hprt-ccm, respectively. with respect to the investigated target gene assays, t-preamp yielded a mean improvement of ± ct values. the cd gene assay demonstrated a mean ct improvement of ±. the preamp uniformity of the investigated gene assays related to cdkn1b was - ±, and related to hprt-ccm - ±,, respectively. as in cdna from pbmcs, there was no significant difference between the self-designed and the inventoried abi gene assays. considering the threshold ct for low expression levels published by canales et al, 48/ of the target genes showed ct values ≥ in direct real-time rt-pcr, whereas ct values for hprt-ccm demonstrated < and for cdkn1b < in each case of the investigated embs. there was no association between the preamp uniformity related to cdkn1b of ct values below or above the threshold ct for low expression levels. the same was true for the preamp uniformity values related to hprt-ccm. the inter-assay variance of ct improvement for the investigated gene assays preamplified in each different embs was below ct values, and the resulting mean cv was ± %, including the cd gene assay, which did not perform differently compared to the remaining gene assays in this respect. abi inventoried gene assays demonstrated a higher inter-assay cv compared with the self-designed gene assays. noticeably, there was no traceable gene expression in some of the investigated each embs per gene group with respect to trbv il il il ifnb and apn neither in the direct nor in the preamp real-time rt-pcr. the different preamp procedures from the cdna from emb resulted in minor differences of ct values. the mean intra-assay cv was ± %, including cd which did not perform differently compared to the remaining gene assays with regard to the intra-assay cv. abi inventoried gene assays demonstrated a lower intra-assay cv compared with the self-designed gene assays. ssrt-preamp employing ssrt-preamp, the investigated gene assays yielded a mean improvement of ± ct values. the mean ct values obtained by direct real-time rt-pcr were ±, and the ssrt-preamp ct values were ±, respectively. in one emb, no traceable ifng expression was observed neither by direct nor by ssrt-preamp real-time rt-pcr. considering the threshold ct for low expression levels published by canales et al, 12/ of the target genes showed ct values ≥ in direct real-time rt-pcr, whereas ct values for hprt-ccm demonstrated < in each case of the investigated embs. the mean preamp uniformity of the investigated gene assays related to hprt-ccm was - ±, which was significantly lower compared with the t-preamp data. as in pbmcs, the gene assays for cd3z, ifng, t-bet and perforin performed below the preamp uniformity value of -. the mean inter-assay cv using the ssrt-preamp technique was higher compared with t-preamp. there was no association between the preamp uniformity related to hprt-ccm of target gene ct values in direct real-time rt-pcr < or ≥. however, the ssrt-preamp yielded comparably low intra-assay cvs as obtained by t-preamp. the real-time rt-pcr results from each dcmi- and dcm-patients revealed differential expression with respect to out of the investigated target genes. these differential gene expressions were related to both hprt-ccm and cdkn1b. significantly different gene expressions related to hprt-ccm are shown in table the data of all investigated target genes related to hprt-ccm, including the non differentially expressed genes, are listed in the additional file gene expression data related to cdkn1b are listed in the additional file significantly increased gene expression was determined with respect to several t-cell related genes, interleukin, tumor necrosis factor alpha, the chemokine cx3cl, cysteine-rich angiogenic inducer, th associated genes, genes expressed by cytotoxic t-lymphocytes, β1-integrins, the selectin cd62e and growth-differentiation factor. the significantly down-regulated genes in embs from dcmi-patients were the chemokine cxcl, adiponectin and tissue factor. significantly different gene expression t-preamp real-time rt-pcr results in embs from dcm patients compared with embs from patients with immunohistologically confirmed dcmi. the values are given as means ± sd, followed by the respective p values in significantly different comparisons. hprt and cdkn1b are not regulated in dcm compared with donor hearts the value of hprt as housekeeping gene is well acknowledged in various tissues. the close association between hprt-ccm and cdkn1b ct values as well as the equally significant differential expression of genes related to both hprt-ccm and cdkn1b in embs from dcm versus dcmi patients infer a possible relevance of cdkn1b as a housekeeping gene. to address this issue, we determined the ct values of hprt and cdkn1b by direct real-time rt-pcr in each explanted donor and terminally failing dcm hearts, from which enough tissue amounts were accessible to adjust rna to ng/μl before cdna synthesis. as stated above for pbmcs, hprt-abi and hprt-ccm demonstrated a difference around ct values. there was no statistical difference of ct values of hprt-abi, hprt-ccm and cdkn1b in donor and dcm hearts. discussion it is widely accepted that conventional histological diagnostics are not sufficient to elucidate the complex processes critically involved in the pathogenesis of dcmi, that they clearly lack prognostic impact, and that they are not sufficient to select dcm/dcmi patients who will likely benefit from immunomodulatory treatment. gene expression investigations are promising approaches for a profound understanding of the pathogenesis of dcm/dcmi, the prognostic impact of gene expression profiles in the natural course of the disease as well as under immunomodulatory treatment. however, the amount of tissue and rna/cdna available from embs is limited, which restricts the number of analyzable target genes per emb. preamp procedures may enhance sensitivity of real-time rt-pcr especially for low abundance expressed genes, and can establish substantially higher cdna amounts, which can then expand the number of the analyzable target genes from a limited cdna. however, maintenance of gene expression profiles as well as a broad applicability to many target genes with a feasible additional effort is a further intriguing aspect of preamp procedures. the t-preamp technique enables a relatively simple workflow, which basically consists of setting up the primer/gene assay pool and t-preamp of cdna over cycles in a special master mix, maintaining robust preamp conditions for several gene assays, which results in mostly stable preamp uniformity values. identical primers are used for both the t-preamp reaction and the succeeding real-time rt-pcr. this is substantially easier to accomplish compared with the ssrt-preamp, which requires design of specific primers for both the ssrt and the following preamplification step. according to the manufacturer's specifications, up to gene assays can be preamplified simultaneously in each t-preamp reaction, which is confirmed by our data on the simultaneously preamplified gene assays both in pbmcs' and embs' cdna. a further advantage of this technique is that out of μl cdna obtained from the herein applied rna extraction and cdna synthesis methods for embs, different preamp reactions are feasible from one single emb, which would hypothetically enable gene expression analysis of different target genes. this is a substantially higher number of quantifiable target genes compared with the direct conventional real-time pcr, by which a maximum of different target genes would be applicable in duplicate real-time rt-pcr analyses and μl cdna used in each reaction, and also compared with the ssrt-preamp procedure, which equally does not grant the possibility of expanding the maximum possible real-time rt-pcr analyses out of a limited amount of cdna. this fact implies a further advantage of a negligible sampling error for this high putative number of real-time rt-pcr analyses carried out from one emb, as opposed to the situation in which gene expression analyses would have to be split into different groups of target genes on cdna from several embs from patients, given the limited rna/cdna amount available from each emb for direct real-time rt-pcr analyses. noticeably, sampling error is a critical issue for the histological assessment of myocardial inflammation. the t-preamp technique with preamp cycles results in a substantial increase of sensitivity of the real-time rt-pcr by around ct values, with preamp uniformity values ranging between - to + both in pbmcs' and embs' cdna. the low intra- and interassay cvs in embs cdna imply a high precision and repeatability of the t-preamp procedure. furthermore, our data show that the preamp uniformity is maintained over a broad range of ct values in direct real-time pcr, including low abundance ct ranges. this applies to both taqman® abi inventoried gene assays as well as to self-designed gene assays adhering to abi recommendations. as shown herein, only the forward/reverse primers of self-designed gene assays need to be included in the preamp master mix reaction, and do not perform significantly different compared with the abi inventoried taqman® gene assays regarding the preamp uniformity, of which both primers and probes have to be pooled in the t-preamp reaction. moreover, we determined that the preamp uniformity is also maintained with regard to gene assays using common reverse primers and common probes but diverse forward primers as well as to wobbled primer designs. these numerous advantages cannot be met by the ssrt-preamp procedure. first, this technique is not feasible for abi inventoried taqman® gene expression assays, since the primer sequences of these assays are not accessible, and therefore primer design for the ssrt-preamp steps is not possible. secondly, ssrt-preamp results on both pbmcs' and embs' cdna demonstrated highly diverging preamp uniformity, with values below the suggested range of - to with regard to cd3z, ifng, t-bet and perforin, albeit the same gene assays performed within the suggested preamp uniformity range of - to using the taqman® preamp master mix. however, our data show that both preamp procedures perform without significantly different preamp uniformity values over a broad range of ct values in direct real-time pcr, including low abundance ct ranges, which implies a potential application in preclinical diagnostics especially for the t-preamp procedure due to its numerous advantages. one further important finding is the lack of traceable expression in both cdna and t-preamp cdna for target genes and samples, in which no detectable ct values were obtained neither by direct real-time rt-pcr in cdna from both pbmcs and embs. this infers that both t- and ssrt-preamp techniques yield reliable results and do not produce erroneous values. as a limitation of our study, the comparison of preamp uniformity values of the investigated gene assays between t-preamp and ssrt-preamp was based on hprt-ccm only, and not on cdkn1b, since the latter taqman® abi inventoried gene assay is not applicable to ssrt-preamp. however, t-preamp data on hprt-ccm indicate that this gene assay can be equally used as referral gene assay for the calculation of the preamp uniformity. this infers that not the design characteristics of these gene assays, but rather the preamp procedure per se is the decisively important issue leading to the observed discrepancies of preamp uniformity values. it is possible that interactions of the primers of the different gene assays during the ssrt-preamp procedure may lead to the observed bias in preamp uniformity. this is in line with our observations on the more pronounced distortion of the preamp uniformity values when adding more gene assays into the ssrt-preamp reactions. due to the higher complexity of the ssrt-preamp procedure and the expected higher chance of primer interactions during preamp, we did not investigate the preamp characteristics of the gene assays using common reverse primers/probes and diverse forward primers, nor wobbled primer designs, with the ssrt-preamp procedure. nested pcr techniques have a higher specificity compared with non-nested pcr, and real-time rt-pcr is known to have a higher specificity compared with nested pcr. thus, the advantage of the ssrt-preamp approach might be the higher specificity due to the nested primer design. since the recipe of the t-preamp master mix® is not accessible, we cannot speculate on the decisively important factors responsible for the observed disparity of the preamp uniformity of the t-preamp and the ssrt-preamp, so far. possibly, the ssrt before the multiplex nested preamp may lead to the high diversity of preamp uniformity of the ssrt-preamp procedure, and this distortion is more pronounced regarding the gene assays cd3z, ifng, t-bet and perforin. nonetheless, the equally low intra-assay cvs using both ssrt-preamp and t-preamp indicate that the ssrt-preamp is an equally reproducible approach, albeit potentially leading to more pronounced skewing of the calculation of relative gene expressions. however, the higher inter-assay cvs obtained by ssrt-preamp, although still <10%, indicate that the t-preamp technique leads to a higher precision for the direct comparison in the relative quantification of gene expression analyses between samples. our data show that the preamp uniformity of the t-preamp technique depends on the respective gene assay both in pbmcs' and embs' cdna, and implies that gene assays should be tested for preamp uniformity before setting up serial t-preamp investigations. the two gene assays, which did not perform comparably to the suggested t-preamp uniformity reference gene assay cdkn1b, were hprt-abi and cd. as a consequence, calculation of expression profiles of all target genes would be substantially altered, if hprt-abi would be used as a housekeeping gene assay in t-preamp real-time rt-pcr analyses, which emphasizes the value of preamp uniformity testing referring especially to a possible housekeeping gene candidate. in line with our observations, denning et al. previously identified that the gapdh taqman® abi inventoried gene assay does not maintain the suggested preamp uniformity values. therefore, these authors also normalized gene expression to cdkn1b, as well. nonetheless, as shown for cd the low inter- and intra-assay variations of the preamp technique warrants a high reproducibility of the preamp real-time rt-pcr results, even for the gene assays identified to show substantially altered preamp uniformity values out of the suggested range between - and +. however, in the context of the relatively robust preamp uniformity performance of the t-preamp technique, one should also consider the pcr efficiency of the respective gene assays, since the "true" range of preamp uniformity hypothetically rises with decreasing pcr efficiency. furthermore, the impact of contaminating dna can be theoretically significant in such low-level expression levels of the target transcripts. the intron spanning primer design avoiding co-amplification of genomic dna of both the self-designed and of the taqman® abi inventoried gene assays, however, and the missing cross reactivity with genomic dna tested in dna from pbmcs largely excludes an impact of this theoretically problematic issue in our investigations. our investigations on possible causes for disproportionate t-preamp uniformities, although not elucidating the concrete reason, reveal that the t-preamp performance does not depend on the amplicon length and on the pcr efficiency of the respective gene assays. denning et al. deduced from their investigations on gapdh and further gene assays with shorter amplicon lengths, that the amplicon length might be crucially important for t-preamp uniformity. however, our data cannot confirm this hypothesis, especially since the trbv and trbv gene assays yielding amplicons around bp and bp resulted in preamp uniformities in the range between - and +, performing comparably to further self-designed gene assays, and taqman® abi inventoried gene assays. there was no evidence for an impact of the pcr efficiency of these gene assays on the preamp uniformity. in contrast, the taqman® abi inventoried gene assays hprt-abi and cd showed substantially lower improvement of ct values under the same conditions. noticeably, this distortion of preamp uniformity was stable at the serial cdna dilutions regarding hprt-abi, whereas the cd gene assay showed an increasing preamp uniformity with decreasing pcr efficiency at serial cdna dilutions. we therefore conclude that distortion of t-preamp uniformity is gene assay specific and not a general problem of the t-preamp technique. these findings add clarity, however, are troublesome at the same time, since the concrete reason for disproportionate t-preamp performance of certain gene assays still remains unclear. nonetheless, these data again highlight the importance of preamp uniformity pretests before setting up serial investigations using the t-preamp technique. to gather, compare and discuss the experiences especially on the obvious distortion of t-preamp uniformity with regard to particular gene expression assays, we propose the constitution of a central open access forum. in light of the possible impact of primer interactions during the t-preamp reaction, of rna quality and dna contamination, this forum should not only refer to the t-preamp performance of single gene assays, but also incorporate the precise gene assay mix in the respective t-preamp reaction, the applied rna extraction technique, rna quality and dna contamination issues. hopefully, the decisively important design characteristics of gene assays leading to a substantial deviation of the t-preamp uniformity can be identified, eventually. these insights might lead to additional rules of gene assay design, by which the t-preamp uniformity can be predicted. several gene expression analyses have been reported comparing heart failure to non-failing hearts, or different cardiomyopathy entities. in this preliminary analysis, we compared embs from dcm patients with patients with immunohistologically confirmed dcmi. both patient groups had comparably depressed lvef, which alleviates a possible bias of secondary general heart-failure associated mechanisms. our preliminary data from these t-preamp real-time rt-pcr analyses revealed differential gene expression with respect to 27/ of the investigated target genes. in brief, these investigations confirmed that the immunohistologically diagnosed dcmi is accompanied by a significantly increased expression of t-cell related genes. up-regulation of distinct trbv families in embs from dcmi patients indicates a selective recruitment and/or expansion, and therefore restriction of trbv usage of the t-cell infiltrates in dcmi. the increased expression of il tnfa and cx3cl infer that t-cell infiltration in dcmi is paralleled by differential increment of these cytokines, whereas a further chemokine, cxcl was confirmed to be downregulated in dcmi patients as recently reported for dcmi patients with slightly impaired lvef by microarray analyses. furthermore, in line with these microarray findings, our investigations confirmed a down-regulation of apn, while its receptors apn-r and apn-r were not differentially expressed, and an up-regulation of cyr. the results on markers of t-cell activation reveal a yet not recognized potential role of granzymes a and b, granulysin, as well as t-bet and eomesodermin as markers for cytotoxic and th1-polarized t-cells, respectively, in dcmi. in contrast, markers for th2-polarized t-cells, anergic and regulatory t-cells were not increased in embs from patients with immunohistologically confirmed dcmi, which might indicate a missing counter-balance of the t-cellular response in dcmi. the lack of differential expression of toll-like receptors and signaling factors involved in tlr downstream pathways do not favor the hypothesis of a paramount role of tlrs in dcmi, so far. our analyses furthermore confirmed downregulation of intramyocardial tf expression in dcmi versus dcm patients. whilst altered expression of several components of the extracellular matrix is well known in dcm and further non-dcm cardiomyopathies, no direct link to inflammation or cardiotropic viral infection has been described, so far. our preliminary data do not show any differential expression of collagen types i, iii and iv, mmps timps and upa in embs from dcmi versus dcm patients. increased expression of β1-integrins and further adhesion molecules such as cd62e have been confirmed by immunohistological analyses, which is in line with our data on av5b and cd62e. gdf is a newly recognized marker of heart failure, and our data implicate an additional role of dcmi for increased gdf expression. in summary, these preliminary analyses confirm that the t-preamp procedure, in addition to its technical applicability to embs, thereby expanding the number of analyzable target genes in embs investigations, is capable of revealing significantly different gene expression profiles in dcmi versus dcm patients. albeit numerous reports have confirmed close associations between rna and protein gene expression, a discrepancy between these two levels of gene expression was elucidated in some investigations, i.e. due to posttranslational modification. we show here a significant association between expression of t-cellular markers and dcmi as determined by immunohistologically quantified cd3+ infiltrates. although it would be beyond the scope of this methodology paper, significantly different gene expression analyses as determined by preamp real-time pcr should be ideally confirmed by protein expression analyses in future investigations. further investigations on larger patient cohorts are warranted to explore associations with the various cardiotropic viruses, the impact of lvef, the acuity of the disease, the prognostic role of gene expression for the diverse natural course of the disease, as well as the changes of gene expression in dcmi patients under immunomodulatory treatment modalities. the application of the t-preamp procedure might be especially useful in light of the new emb guidelines. moreover, these promising results of t-preamp real-time rt-pcr in embs might be stimulating for researchers analyzing biopsies or comparably small sized tissue samples from other organs, and for preamp real-time rt-pcr analyses from limited cell amounts such as those obtained by laser capture microdissection. finally, we here provide first evidence that cdkn1b, in addition to its function as the suggested reference gene for the preamp uniformity, can be used as a relevant housekeeping gene for the relative quantification of gene expression in real-time rt-pcr in myocardial tissues, equivalent to hprt. whereas the tumor suppressor gene function of cdkn1b is well understood in other tissues and pathologic conditions, the physiological role and expression pattern of cdkn1b in myocardial tissues has not been investigated, yet. congruent with our observations, denning et al. have previously confirmed that cdkn1b can also serve as housekeeping gene in thyroid tissues.  in comparison to the ssrt-preamp, t-preamp enables a relatively simple workflow, and results in a robust preamp of multiple target genes by a mean ct improvement around cycles, and in a lower inter-assay variance in rna derived from embs. preliminary analyses comparing embs from dcm and dcmi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that t-preamp is a suitable tool to perform gene expression analyses in embs, expanding gene expression investigations with the limited rna/cdna amounts derived from embs. cdkn1b, in addition to its function as a reference gene for the calculation of preamp uniformity, might serve as a suitable housekeeping gene for real-time rt-pcr analyses of myocardial tissues. 
only two of the tested taqman® abi inventoried gene assays did not maintain preamp uniformity levels between - and +. preliminary analyses comparing embs from dcm and dcmi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that t-preamp is a suitable tool to perform gene expression analyses in embs, expanding gene expression investigations with the limited rna/cdna amounts derived from embs. in comparison to the ssrt-preamp, t-preamp enables a relatively simple workflow, and results in a robust preamp of multiple target genes by a mean ct improvement around cycles, and in a lower inter-assay variance in rna derived from embs. the preamp uniformity values related to the reference gene cdkn1b for of the investigated gene assays were - ±, without significant differences between self-designed and abi inventoried taqman® gene assays. comparing embs from each patients with dilated cardiomyopathy and inflammatory cardiomyopathy, t-preamp real-time rt-pcr analyses revealed differential regulation regarding of the investigated genes related to both hprt-ccm and cdkn1b. due to the limited rna amounts from endomyocardial biopsies and low expression levels of certain genes, gene expression analyses by conventional real-time rt-pcr are restrained in embs. we applied two preamplification techniques, the taqman® preamp master mix and a multiplex preamplification following a sequence specific reverse transcription. cdkn1b, in addition to its function as a reference gene for the calculation of preamp uniformity, might serve as a suitable housekeeping gene for real-time rt-pcr analyses of myocardial tissues. t-preamp encompassing gene assays with cycles resulted in a mean improvement of ± ct values. ct values of hprt and cdkn1b did not differ in equal rna amounts from explanted dcm and donor hearts. the coefficients for inter- and intra-assay variation were low for all gene assays tested. in comparison, the ssrt-preamp tested on self-designed gene assays yielded higher ct improvement, however was not as robust regarding the maintenance of preamp uniformity related to hprt-ccm, and demonstrated comparable intra-assay cvs, albeit higher inter-assay cvs.
 due to the limited rna amounts from endomyocardial biopsies and low expression levels of certain genes, gene expression analyses by conventional real-time rt-pcr are restrained in embs. we applied two preamplification techniques, the taqman® preamp master mix and a multiplex preamplification following a sequence specific reverse transcription.  t-preamp encompassing gene assays with cycles resulted in a mean improvement of ± ct values. the coefficients for inter- and intra-assay variation were low for all gene assays tested. the preamp uniformity values related to the reference gene cdkn1b for of the investigated gene assays were - ±, without significant differences between self-designed and abi inventoried taqman® gene assays. only two of the tested taqman® abi inventoried gene assays did not maintain preamp uniformity levels between - and +. in comparison, the ssrt-preamp tested on self-designed gene assays yielded higher ct improvement, however was not as robust regarding the maintenance of preamp uniformity related to hprt-ccm, and demonstrated comparable intra-assay cvs, albeit higher inter-assay cvs. comparing embs from each patients with dilated cardiomyopathy and inflammatory cardiomyopathy, t-preamp real-time rt-pcr analyses revealed differential regulation regarding of the investigated genes related to both hprt-ccm and cdkn1b. ct values of hprt and cdkn1b did not differ in equal rna amounts from explanted dcm and donor hearts.  in comparison to the ssrt-preamp, t-preamp enables a relatively simple workflow, and results in a robust preamp of multiple target genes by a mean ct improvement around cycles, and in a lower inter-assay variance in rna derived from embs. preliminary analyses comparing embs from dcm and dcmi patients, revealing differential regulation regarding 30% of the investigated genes, confirm that t-preamp is a suitable tool to perform gene expression analyses in embs, expanding gene expression investigations with the limited rna/cdna amounts derived from embs. cdkn1b, in addition to its function as a reference gene for the calculation of preamp uniformity, might serve as a suitable housekeeping gene for real-time rt-pcr analyses of myocardial tissues. 