Quantitative Analysis of Osteopontin Gene Expression Using a Real-Time Reverse Transcription-Polymerase Chain Reaction Assay
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概要
- 論文の詳細を見る
Osteopontin(OPN)is a non-collagenous matrix protein in bone. It is involved in bone formation, resorption, and remodeling. In this study, the expression of OPN gene was determined by real-time quantitative reverse-transcription polymerase chain reaction using SYBR Green dye and an ABI Prism 7700 Sequence Detection System. In this system, the amplified products are directly monitored by measuring the increase in fluorescence generated by the intercalation of SYBR Green I to amplified double-stranded DNA. To avoid non-specific increase in fluorescence due to the formation of primer dimer and amplification of contaminating genomic DNA, the primer concentrations were optimized and genomic DNA were eliminated by treating total RNA with ribonuclease-free deoxyribonuclease I. The RNA loads from various samples were normalized using the transcripts of glyceraldehyde-s-phosphate dehydrogenase. We determined quantitatively the OPN gene expression in mouse tissues and differentiating osteoblastic cells using the established protocol. The highest expression of OPN gene was observed in kidney, followed by liver, salivary glands and testis. In osteoblastic MC3T3-E1 cells, TGF-β1 induced the expression of OPN gene in a time-dependent manner.
- 硬組織再生生物学会の論文
著者
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Ohta Kazumasa
Department Of Biochemistry Tokyo Dental College
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Ohta Kazumasa
Departments Of Biochemistry Tokyo Dental College
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Kizaki Harutoshi
Department Of Biochemistry Tokyo Dental College
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Kizaki Harutoshi
Departments Of Biochemistry Tokyo Dental College
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Isshiki Yasushige
Department Of Orthodontics Tokyo Dental College
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Isshiki Yasushige
Departments Of Orthodontics Tokyo Dental College
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OTSUKA Waka
Departments of Orthodontics, Tokyo Dental College
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Otsuka Waka
Departments Of Orthodontics Tokyo Dental College
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