Unknown,Transcriptomics,Genomics,Proteomics

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Evaluation of a New High-Dimensional miRNA Profiling Platform

ABSTRACT: We evaluated a new miRNA profiling platform that utilizes Illuminaâs existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evaluated the reproducibility of miRNA expression levels across replicates and with varying amounts of input RNA. The beta test version was comprised of 735 miRNA targets of Illuminaâs miRNA profiling application. The study used four SAMs to assess variability due to separate total RNA extractions, technical replicates of each extraction and varying total RNA input. Total RNA extracted from five patient samples (IDs 45, 165, 565, 919, 133) and RNA from four cell lines (HeLa, PC3, 293 and MCF-7) supplied by Illumina, was evaluated on each of 4 SAMs. SAM 1 was hybridized in week one, SAMs 2 and 3 were hybridized in week two and SAM 4 (with cell line RNA only) was hybridized in week three of the study. Sample allocation on SAM 1 was designed to assess reproducibility between two separate total RNA extractions, between duplicate samples (technical replicates) and between two input RNA amounts. Sample allocation on SAM 2 was designed to assess technical reproducibility and the effect of six input levels of total RNA. Sample allocation on SAM 3 was designed to assess technical replication. The four cell lines, but none of the 5 patient RNA, were run on a fourth SAM . Reproducibility across all four SAMs was also assessed.

ORGANISM(S): Homo sapiens

SUBMITTER: Ann Oberg

PROVIDER: E-GEOD-17346 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Evaluation of a new high-dimensional miRNA profiling platform.

Cunningham Julie M JM Oberg Ann L AL Borralho Pedro M PM Kren Betsy T BT French Amy J AJ Wang Liang L Bot Brian M BM Morlan Bruce W BW Silverstein Kevin A T KA Staggs Rod R Zeng Yan Y Lamblin Anne-Francoise AF Hilker Christopher A CA Fan Jian-Bing JB Steer Clifford J CJ Thibodeau Stephen N SN

BMC medical genomics 20090827

<h4>Background</h4>MicroRNAs (miRNAs) are a class of approximately 22 nucleotide long, widely expressed RNA molecules that play important regulatory roles in eukaryotes. To investigate miRNA function, it is essential that methods to quantify their expression levels be available.<h4>Methods</h4>We evaluated a new miRNA profiling platform that utilizes Illumina's existing robust DASL chemistry as the basis for the assay. Using total RNA from five colon cancer patients and four cell lines, we evalu ...[more]

PMID: 19712457

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Project description:Runx2 and Axin2 regulate skeletal development. We recently determined that Axin2 and Runx2 molecularly interact in differentiating osteoblasts to regulate intramembranous bone formation, but the relationship between these factors in endochondral bone formation was unresolved. To address this, we examined the effects of Axin2 deficiency on the cleidocranial dysplasia (CCD) phenotype of Runx2+/-M-BM- mice, focusing on skeletal defects attributed to improper endochondral bone formation. Axin2 deficiency unexpectedly exacerbated calvarial components of the CCD phenotype in the Runx2+/-M-BM- mice; the endocranial layer of the frontal suture, which develops by endochondral bone formation, failed to mineralize in the Axin2-/-:Runx2+/-mice, resulting in a cartilaginous, fibrotic and larger fontanel than observed in Runx2+/-M-BM- mice. Transcripts associated with cartilage development (e.g., Acan, miR140) were expressed at higher levels, whereas blood vessel morphogenesis transcripts (e.g., Slit2) were suppressed in Axin2-/-:Runx2+/-calvaria. Cartilage maturation was impaired, as primary chondrocytes from double mutant mice demonstrated delayed differentiation and produced less calcified matrix in vitro. The genetic dominance of Runx2 was also reflected during endochondral fracture repair, as both Runx2+/-M-BM- and double mutant Axin2-/-:Runx2+/-M-BM- mice had enlarged fracture calluses at early stages of healing. However, by the end stages of fracture healing, double mutant animals diverged from the Runx2+/-M-BM- mice, showing smaller calluses and increased torsional strength indicative of more rapid end stage bone formation as seen in the Axin2-/-M-BM- mice. Taken together, our data demonstrate a dominant role for Runx2 in chondrocyte maturation, but implicate Axin2 as an important modulator of the terminal stages of endochondral bone formation. 4 mice per genotype X 4 genotypes: wildtype (WT), Runx2+/- (R-Het), Axin2-/- (A-KO), Axin2-/-:Runx2+/- (A-KO:R-Het). Total = 16 samples

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Evaluation of a New High-Dimensional miRNA Profiling Platform

Publications

Evaluation of a new high-dimensional miRNA profiling platform.

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