Unknown,Transcriptomics,Genomics,Proteomics

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Real-time quantitative PCR analysis of 88 microRNAs involved in human cell differentiation and development in normal fibroblasts stimulated with exogenous TGF-β1 and systemic sclerosis (SSc) dermal fibroblasts


ABSTRACT: Normal fibroblasts and SSc fibroblasts between the third and six subpassages were used for experiments. Normal and scleroderma fibroblasts were serum-starved for 24 hours and incubated in the presence or absence of TGF-β1 (2ng/ml) for 6 hours. Total RNA was extracted from culture cells with ISOGEN (Nippon Gene, Tokyo, Japan). MicroRNA isolation from total RNA was performed using RT2 qPCR-Grade miRNA Isolation Kit (SA Bioscience). For RT2 Profiler PCR Array (SABioscience), microRNAs were reverse-transcribed into first strand cDNA using RT2 miRNA First Strand Kit (SABiosciences). A mixture of equal amounts of cDNAs from 5 normal fibroblasts or 5 SSc fibroblasts was prepared. The cDNA was mixed with RT2 SYBR Green/ROX qPCR Master Mix and the mixture was added into a 96-well RT2 miRNA PCR Array (SABiosciences) that included primer pairs for 88 human microRNAs. Human dermal fibroblasts were obtained by skin biopsy from the affected areas (dorsal forearm) of 5 patients with diffuse cutaneous SSc and <2 years of skin thickening. Control fibroblasts were obtained by skin biopsies from 5 healthy donors. Control donors were each matched with a SSc patient for age, sex, and biopsy site.

ORGANISM(S): Homo sapiens

SUBMITTER: Noritoshi Honda 

PROVIDER: E-GEOD-34827 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

TGF-β-mediated downregulation of microRNA-196a contributes to the constitutive upregulated type I collagen expression in scleroderma dermal fibroblasts.

Honda Noritoshi N   Jinnin Masatoshi M   Kajihara Ikko I   Makino Takamitsu T   Makino Katsunari K   Masuguchi Shinichi S   Fukushima Satoshi S   Okamoto Yoshinobu Y   Hasegawa Minoru M   Fujimoto Manabu M   Ihn Hironobu H  

Journal of immunology (Baltimore, Md. : 1950) 20120229 7


Previous reports indicated the significance of the TGF-β signaling in the pathogenesis of systemic sclerosis. We tried to evaluate the possibility that microRNAs (miRNAs) play a part in the type I collagen upregulation seen in normal fibroblasts stimulated with exogenous TGF-β and systemic sclerosis (SSc) fibroblasts. miRNA expression profile was evaluated by miRNA PCR array and real-time PCR. The protein expression of type I collagen was determined by immunoblotting. In vivo detection of miRNA  ...[more]

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