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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- MIYAZAKI Masanobu
- The Department of Nephrology, Tokai University School of Medicine
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- NIKOLIC-PATERSON David J.
- Department of Nephrology, Monash Medical Center, Clayton
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- ENDOH Masayuki
- The Department of Nephrology, Tokai University School of Medicine
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- NOMOTO Yasuo
- The Department of Nephrology, Tokai University School of Medicine
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- SAKAI Hideto
- The Department of Nephrology, Tokai University School of Medicine
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- ATKINS Robert C.
- Department of Nephrology, Monash Medical Center, Clayton
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- KOJI Takehiko
- Department of Anatomy, Nagasaki University School of Medicine
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抄録
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.<br>(Internal Medicine 33: 87-91, 1994)
収録刊行物
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- Internal Medicine
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Internal Medicine 33 (2), 87-91, 1994
一般社団法人 日本内科学会
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詳細情報 詳細情報について
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- CRID
- 1390001204869687296
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- NII論文ID
- 130000771047
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- NII書誌ID
- AA10827774
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- COI
- 1:STN:280:ByuB1cbovVE%3D
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- ISSN
- 13497235
- 09182918
- http://id.crossref.org/issn/09182918
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- PubMed
- 8019048
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- 本文言語コード
- en
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- データソース種別
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- JaLC
- Crossref
- PubMed
- CiNii Articles
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- 抄録ライセンスフラグ
- 使用不可