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 [9, 19]. 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 [4, 11–13, 16]. 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 14 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 100 gene assays can be preamplified simultaneously in each T-PreAmp reaction, which is confirmed by our data on the simultaneously preamplified 92 gene assays both in PBMCs' and EMBs' cDNA. A further advantage of this technique is that out of 60 μl cDNA obtained from the herein applied RNA extraction and cDNA synthesis methods for EMBs, 8 different PreAmp reactions are feasible from one single EMB, which would hypothetically enable gene expression analysis of 784 different target genes (minus one housekeeping gene such as HPRT, and CDKN1B as the suggested reference gene for PreAmp uniformity). This is a substantially higher number of quantifiable target genes compared with the direct conventional real-time PCR, by which a maximum of 30 different target genes would be applicable in duplicate real-time RT-PCR analyses and 1 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 14 PreAmp cycles results in a substantial increase of sensitivity of the real-time RT-PCR by around 7 Ct values, with PreAmp uniformity values ranging between -1.5 to +1.5 both in PBMCs' and EMBs' cDNA (except for the gene assays HPRT-ABI and CD56). The low intra- and interassay CVs in EMBs cDNA (<4%, including CD56) 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 (Ct ≥ 35 in direct real-time RT-PCR) . 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 (TRBV primers) as well as to wobbled primer designs (TRBV5, 6 and 7 forward primers). 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 -1.5 to 1.5 with regard to CD3z, IFNg, T-bet and Perforin, albeit the same gene assays performed within the suggested PreAmp uniformity range of -1.5 to 1.5 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 (Ct ≥ 35 in direct real-time RT-PCR) , which implies a potential application in preclinical diagnostics especially for the T-PreAmp procedure due to its numerous advantages (i.e. robust PreAmp uniformity values for most gene assays, easier to use, expansion of possible real-time PCR analyses out of limited RNA/cDNA). 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 (i.e. PCR efficiency), 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 (data not shown). 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 (TRBV primers), 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 CD56 (both Taqman® ABI inventoried gene assays). 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 CD56, the low inter- and intra-assay variations of the PreAmp technique (CVs <4%) 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 -1.5 and +1.5. However, in the context of the relatively robust PreAmp uniformity performance of the T-PreAmp technique (especially compared to the SSRT-PreAmp procedure), 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 (122 bp) and 12 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 TRBV19 and TRBV29 gene assays yielding amplicons around 260 bp and 320 bp (depending on the respective diversity and joining regions) resulted in PreAmp uniformities in the range between -1.5 and +1.5, performing comparably to further self-designed gene assays (i.e. HPRT-CCM: 101 bp; CD3z: 108 bp; TRBC: 151 bp), and Taqman® ABI inventoried gene assays (i.e. CD3d: 92 bp; NFATC3: 74 bp; av5b1: 75 bp). 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 (100 bp) and CD56 (61 bp) 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 CD56 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 10 DCM patients with 10 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 27/90 (30%) 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 (CD3d, CD3z, TRBC and NFATC3). Up-regulation of distinct TRBV families (TRBV2, 4, 6, 10, 20, 23, 24 and 29) 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 IL6, TNFa and CX3CL1 infer that T-cell infiltration in DCMi is paralleled by differential increment of these cytokines, whereas a further chemokine, CXCL14, 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-R1 and APN-R2 were not differentially expressed, and an up-regulation of CYR61 . 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 (IL4, IL5, IL10, TGFb, GATA3, GRAIL, FoxP3) 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 (TLRs) 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 [14, 26], 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 2, 8, 9, TIMPs 1, 4 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 av5b1 and CD62E. GDF15 is a newly recognized marker of heart failure, and our data implicate an additional role of DCMi for increased GDF15 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 [28, 29]. We show here a significant association between expression of T-cellular markers (CD3d, CD3z, TRBC, NFATC3) 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 [2, 30], the impact of LVEF, the acuity of the disease (i.e. patients presenting with acute myocarditis versus DCM), the prognostic role of gene expression for the diverse natural course of the disease (improvement versus further deterioration under heart failure medication) , as well as the changes of gene expression in DCMi patients under immunomodulatory treatment modalities (i.e. immunosuppression, immunoadsorption, antiviral treatment; [4, 11–13, 16]). 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 [23, 32].
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 [33, 34], 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 .