Unknown,Transcriptomics,Genomics,Proteomics

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Circulating miRNA microarray analysis from archival plasma samples

ABSTRACT: Using a microarray-based method we report on: i) adequate intra- and interarray reproducibility of miRNA profiling; ii) feasibility of using archival plasma samples stored for an extended period of time and available in limited amounts; and iii) good correlation between different batches. A total of 11 archived plasma samples collected between 1988 and 1999 were evaluated in this study. Firstly, RNA extracted from two samples (S1 and S2) were hybridized in triplicate. The same samples were also hybridized in triplicate after increasing the starting material volume from 2 to 2.5. Nine additional plasma samples were processed and RNA was hybridized on two different batches of arrays. Finally, two samples (S3 and S4) were re-hybridized on an array of the same batch of the first hybridization but close to the chip expiration date.

ORGANISM(S): Homo sapiens

SUBMITTER: Maurizio Callari

PROVIDER: E-GEOD-42654 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Feasibility of circulating miRNA microarray analysis from archival plasma samples.

Callari Maurizio M Tiberio Paola P De Cecco Loris L Cavadini Elena E Dugo Matteo M Ghimenti Chiara C Daidone Maria Grazia MG Canevari Silvana S Appierto Valentina V

Analytical biochemistry 20130313 2

MicroRNAs have been found to be deregulated in several diseases and, due to their high stability in body fluids, represent promising noninvasively detectable biomarkers. However, numerous technical variables can affect accurate measurement of circulating miRNAs. Using a microarray-based method we assessed the: (i) adequate intra- and inter-array reproducibility of miRNA profiling; (ii) feasibility of using archival plasma samples stored for an extended period of time and available in limited amo ...[more]

PMID: 23499963

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Project description:Background. Chronic renal failure is characterized by progressive renal scarring and accelerated cardiovascular disease. In animal models, this is thought to be due to non-dialyzable uremic toxins M-bM-^@M-^S small, protein-bound molecules normally secreted via Organic Anion Transporters (OATs) in the proximal renal tubule,rather than filtered at the glomerulus. The best studied of these is indoxyl sulfate (IS). Methods. We examined global gene expression responses in cultured normal human renal tubular cells incubated with control plasma (n=5) or pre- and post-dialysis uremic plasma (n=10). Results. GeneSpring analysis of microarrays revealed significantly altered expression of 2016 genes. The expression of 537 genes M-bM-^@M-^\normalizedM-bM-^@M-^] in post-dialysis plasma, suggesting removal of M-bM-^@M-^\low molecular weight uremic toxinsM-bM-^@M-^] by dialysis. The expression of the majority of dysregulated genes (1479) was not normalized in post-dialysis plasma. These likely represent the effects of substances not effectively removed by dialysis (M-bM-^@M-^\protein-bound uremic toxinsM-bM-^@M-^]). Addition of indoxyl sulfateto control plasma simulated most effects (81.1%) of uremic plasma, while the addition of probenecid, an OAT inhibitor, to uremic plasma reversed most changes in gene expression. Analysis of molecular programs with the Gene Set Enrichment Analysis and the DAVID database revealed patterns common to indoxyl sulfate-treated control plasma, pre-dialysis, and post-dialysis uremic plasma. These included increased cell cycle, pro-inflammatory, and pro-fibrotic molecular programs, particularly genes in the TGF-M-NM-2 pathway. Conclusion: These findings provide insight into the role of non-dialyzable, protein-bound uremic toxins in the pathogenesis of renal scarring and uremic vasculopathy. The GSEA patterns confirm that inflammatory and fibrotic programs are active. Transcriptomic remodeling in response to external stimulus RNA from non-immortalized cultured renal cortical cells incubated with plasma samples from 5 controls, 10 uremics pre-dialysis, 10 uremics post-dialysis, 5 controls with indoxyl sulfate added, 5 uremics pre-dialysis with probenecid added, 5 uremics post-dialysis with indoxyl sulfate added

Dataset Information

Circulating miRNA microarray analysis from archival plasma samples

Publications

Feasibility of circulating miRNA microarray analysis from archival plasma samples.

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