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使用碱性磷酸酶标记的寡脱氧核苷酸探针通过原位杂交和Northern印迹分析检测mRNA。

Detection of mRNA by in situ hybridisation and in northern blot analysis using oligodeoxynucleotide probes labelled with alkaline phosphatase.

作者信息

Farquharson M A, Harvie R, Kennedy A, McNicol A M

机构信息

University Department of Pathology, Royal Infirmary, Glasgow, Scotland.

出版信息

J Clin Pathol. 1992 Nov;45(11):999-1002. doi: 10.1136/jcp.45.11.999.

DOI:10.1136/jcp.45.11.999
PMID:1360478
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC495032/
Abstract

AIMS

To assess whether a reduction in intensity of signal observed using an alkaline phosphatase labelled oligodeoxynucleotide probe could be explained on the basis of procedural steps rather than reduced sensitivity.

METHOD

Signal intensity was assessed on in situ hybridisation for pro-opiomelanocortin (POMC) mRNA in rat pituitary and for somatostatin mRNA in human pancreas and in northern blot analysis for POMC mRNA in the presence and absence of formamide. The direct effects of formamide on the alkaline phosphatase detection step were assessed using histochemical enzyme detection in rat kidney.

RESULTS

All signals were reduced in systems containing formamide.

CONCLUSIONS

In the absence of formamide clear, strong signals for specific mRNAs can be obtained by in situ hybridisation and northern blot analysis using oligodeoxynucleotide probes directly labelled with alkaline phosphatase. Formamide seems to inhibit the activity of alkaline phosphatase.

摘要

目的

评估使用碱性磷酸酶标记的寡脱氧核苷酸探针观察到的信号强度降低是否可以基于操作步骤而非灵敏度降低来解释。

方法

在大鼠垂体中对促阿片黑素皮质激素原（POMC）mRNA进行原位杂交，在人胰腺中对生长抑素mRNA进行原位杂交，并在有或没有甲酰胺存在的情况下对POMC mRNA进行Northern印迹分析，以此评估信号强度。使用大鼠肾脏的组织化学酶检测来评估甲酰胺对碱性磷酸酶检测步骤的直接影响。

结果

在含有甲酰胺的系统中所有信号均降低。

结论

在没有甲酰胺的情况下，使用直接用碱性磷酸酶标记的寡脱氧核苷酸探针通过原位杂交和Northern印迹分析可以获得清晰、强烈的特定mRNA信号。甲酰胺似乎会抑制碱性磷酸酶的活性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/4e930adb4991/jclinpath00425-0064-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/29c5135fe87b/jclinpath00425-0062-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/05b68134abeb/jclinpath00425-0063-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/c703e83172da/jclinpath00425-0064-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/4e930adb4991/jclinpath00425-0064-b.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/29c5135fe87b/jclinpath00425-0062-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/05b68134abeb/jclinpath00425-0063-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/c703e83172da/jclinpath00425-0064-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1f10/495032/4e930adb4991/jclinpath00425-0064-b.jpg

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本文引用的文献

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Use of oligodeoxynucleotide probes for quantitative in situ hybridization to actin mRNA.
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Detection of proopiomelanocortin mRNA by in situ hybridization, using a biotinylated oligodeoxynucleotide probe and avidin-alkaline phosphatase histochemistry.使用生物素化寡脱氧核苷酸探针和抗生物素蛋白-碱性磷酸酶组织化学，通过原位杂交检测阿黑皮素原mRNA。
Histochemistry. 1988;89(2):109-16. doi: 10.1007/BF00489913.
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In situ hybridization: alkaline phosphatase visualization of biotinylated probes in cryostat and paraffin sections.原位杂交：在冷冻切片机切片和石蜡切片中对生物素化探针进行碱性磷酸酶显色。
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