A Sensitive Method of Non-Radioactive In Situ Hybridization for mRNA Localization within Human Renal Biopsy Specimens: Use of Digoxigenin Labeled Oligonucleotides.
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概要
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A sensitive method of non-radioactive in situ hybridization using digoxigenin-labeled oligonucleotides is described for the detection of mRNA within human renal biopsy specimens. Although non-radioactive in situ hybridization typically has the drawback of low sensitivity, we increased the sensitivity of this method, providing a practical alternative to the use of radiolabelled probes. The four main points are: 1) assessment of the efficiency of labeling, 2) optimization of the probe concentration for hybridization, 3) requirement of deproteinization of tissues with HC1 and proteinase K, and 4) the use of a four-layer immunoperoxidase staining system. This technique was found to clearly localize individual mRNA positive cells within cryostat tissue sections. A variety of controls including sense probes, excess unlabeled anti-sense probes, and RNase-treatment demonstrated the specificity of the technique. This improved method is a powerful technique for detecting mRNA within human renal tissue and will be most useful in the study of gene expression in the pathogenesis of renal diseases.(Internal Medicine 33: 87-91, 1994)
- 社団法人 日本内科学会の論文
著者
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Nomoto Yasuo
The Department Of Internal Medicine School Of Medicine. Keio University
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Nikolic-paterson David
Department Of Nephrology Monash Medical Centre
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Sakai Hideto
The Division Of Nephrology And Metabolism Department Of Internal Medicine School Of Medicine Tokai U
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Koji Takehiko
Department Fo Histology And Cell Biology Nagasaki University School Of Medicine
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Atkins Robert
Department Of Nephrology Monash Medical Centre
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ENDOH Masayuki
the Department of Internal Medicine, School of Medicine, Tokai University
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MIYAZAKI Masanobu
The Department of Nephrology, Tokai University School of Medicine
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MIYAZAKI Masanobu
the Department of Internal Medicine, School of Medicine, Tokai University
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SAKAI Hideto
The Department of Nephrology, Tokai University School of Medicine
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SAKAI Hideto
the Department of Internal Medicine, School of Medicine, Tokai University
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NOMOTO Yasuo
The Department of Nephrology, Tokai University School of Medicine
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NIKOLIC-PATERSON David
Department of Nephrology, Monash Medical Center, Clayton
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