Unknown,Transcriptomics,Genomics,Proteomics

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Transcription profiling of human cardiac muscle from patients with DCM or specific cardiomyopathy vs. controls to develop a diagnostic or prognostic tool for dilated cardiomyopathy

ABSTRACT: There is an emerging hypothesis that dilated cardiomyopathy (DCM) is a manifestation of end-stage heart failure (ESHF) resulting from âfinal common pathwayâ despite heterogeneous primary etiologies. We performed genome-wide expression profiling by means of high-density oligonucleotide microarrays using cardiac muscles from patients with DCM or specific cardiomyopathy as well as non-disease control hearts. Differentially expressed genes between ESHF and non-disease samples should include both genes reactive to heart failure (HF) and those responsible for ESHF. With the aid of samples with acute HF without DCM and those with DCM without HF (corrected with left ventricular assist device), we successfully distinguished ESHF genes from HF genes. Our findings implicate that transcriptional signature of cardiac muscle can be potentially applied as a diagnostic or prognostic tool for severe HF. Experiment Overall Design: The expression profiles of approximately 20,227 genes were analyzed using a microarray, Human 1A ver.2 (Agilent Technologies). A total of 30 cardiac RNA samples (21 clinical samples and 9 purchased samples) were used in the hybridizations. 200ng of total RNA was used for T7 RNA polymerase-based cRNA labeling. The microarray experiments were then carried out using competitive hybridization experiments with Cy5-labeled heart RNAs as a test RNA and with Cy3-labeled pooled heart RNA (Sample N) as a template control for normalization. The glass slides were scanned using an Agilent G2565BA microarray scanner. Scanned images were then analyzed using Feature Extraction software. The average signal intensities were corrected for median background intensity and transferred with GenBank descriptors to a Microsoft Excel data spreadsheet (Microsoft, Redmond, WA). Experiment Overall Design: Data analysis was performed using Genespring software version 6.1 (Silicon Genetics, Redwood City, CA). To avoid âfalse positiveâ signals, we excluded certain genes from the analysis for which the average reference signal level constraints were under 70. After intensity dependent normalization (Lowess), the expression levels relative to the control were calculated as a ratio, and the expression profiles were then compared between each disease or normal sample. Statistical analysis was done using non-parametric tests. To order the samples according to the correlation coefficient, we applied âFind Similar Samplesâ algorithm using Spearman Correlation.

ORGANISM(S): Homo sapiens

SUBMITTER: Masatsugu Ohtsuki

PROVIDER: E-GEOD-9800 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The clinical results obtained with organ allografts from marginal or extended donors are less satisfactory over both the short-term and long-term than the outcome of grafting from living donors. Early postoperative graft function depends on several pretransplant factors, with the major donor influences being brain death (BD) and ischemia/reperfusion(I/R) injury. The effects of BD and I/R injury not only reduce the number of functioning nephrons, but also trigger a host immune response to the grafted kidney. It has been hypothesized that allografts from marginal sources may not be biologically inert at the time of surgery, but may already be programmed to initiate or amplify a host response. To exclude the effects of allogenicity, we established a rat renal isograft model using dead donors and compared the results with those obtained after grafting from living donors. In this study, we performed an analysis of the changes of gene expression in rat kidney isografts using samples obtained at 0(pre-transplant) and 1 hour time points since transplantation procedure. The results enhanced our insight into the pathways and cascades that are activated or down-regulated by BD and/or I/R injury. Better knowledge of the key components of BD or I/R injury will provide clues to the factors triggering progression that leads to a decline of organ viability, as well as ideas on how to overcome such graft failures. Such data may also allow us to identify novel biomarkers as predictors of adverse outcomes. Experiment Overall Design: Inbred male Lewis rats were used for these experiments as recipients and donors. The left kidney was transplanted orthotopically by end-to-end anastomosis. The contralateral right kidney was removed at the time of transplantation. The time of operative ischemia for transplantation was 30 min, which did not vary between animals. Donor animals were divided into two groups. The experimental group received kidneys from BD rats, while kidneys from living donors were used in the control group. Brain death was produced by gradually increasing the intracranial pressure that led to brain stem herniation. The rats were mechanically ventilated for a period of 6 hours. We compared the gene expression profiles of renal isografts from BD donors and grafts from living donors using a high-density oligonucleotide microarray that contained approximately 20,500 genes. Experiment Overall Design: For DNA microarray experiments, 200 ng aliquots of total RNA were labeled using the Agilent Low RNA Input Fluorescent Linear Amplification Kit (Agilent Technologies Product) according to the manufacturerâ??s instructions. RNA purified from each kidney graft was used for microarray analysis (Cy3-labeled), with pooled RNA derived from normal kidneys as a template control (Cy5-labeled). After checking the labeling efficiency, 1ï?g aliquots of Cy5-labeled normal control RNA and Cy3-labeled RNA from individual grafts (control 0 hour, 1 hour, BD 0 hour, BD 1 hour, n=3/group) were mixed, and then hybridized to Agilent Rat Oligo Microarrays (Product No. G4130A). After washing, the microarray slides were analyzed with an Agilent Microarray scanner and software (scanner model G2565BA). Data analysis was performed using Agilent Feature Extraction software (Ver. A.7.1.1), and Excel 2003 (Microsoft). The data were imported into GeneSpring 7.0 (Silicon Genetics, Redwood City, CA), with per spot, per chip, and intensity dependent (lowess) normalization being applied for each array. The ratio of the normalized channels (Cy3/Cy5) was used to assess the level of expression.

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Transcription profiling of human cardiac muscle from patients with DCM or specific cardiomyopathy vs. controls to develop a diagnostic or prognostic tool for dilated cardiomyopathy

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