一种用于脑组织中神经生长因子mRNA非放射性Northern印迹分析的可靠且灵敏的方法。

A reliable and sensitive method for non-radioactive northern blot analysis of nerve growth factor mRNA from brain tissues.

作者信息

Shifman M I, Stein D G

机构信息

Brain Research Laboratory, State University of New Jersey, Newark 07102, USA.

出版信息

J Neurosci Methods. 1995 Jul;59(2):205-8. doi: 10.1016/0165-0270(94)00184-i.

DOI:10.1016/0165-0270(94)00184-i

PMID:8531488

Abstract

This report describes a sensitive, rapid and reproducible protocol for non-isotopic Northern blotting analysis. Church buffer and probes labeled with digoxigenin (DIG) were used for studying the expression of nerve growth factor (NGF) in the rat brain. Using the described method, NGF cRNA probe was hybridized to blotted RNA and the results were compared to Northern blot obtained using the method recommended by the manufacturer (Boehringer Mannheim). Comparison revealed that the blot treated with Church buffer detected at least 10-fold more NGF mRNA as compared to blot hybridized with formamide buffer. In summary, we have developed an optimal hybridization protocol to perform non-radioactive Northern blot analysis using antisense RNA as a probe. This method allowed us to detect the specific low-abundant mRNA and analyze the expression of neurotrophic factors in the rat brain.

摘要

本报告描述了一种用于非同位素Northern印迹分析的灵敏、快速且可重复的方案。使用Church缓冲液和地高辛（DIG）标记的探针来研究大鼠脑中神经生长因子（NGF）的表达。采用所述方法，将NGF cRNA探针与印迹RNA杂交，并将结果与使用制造商（宝灵曼）推荐方法获得的Northern印迹结果进行比较。比较发现，与用甲酰胺缓冲液杂交的印迹相比，用Church缓冲液处理的印迹检测到的NGF mRNA至少多10倍。总之，我们开发了一种最佳杂交方案，以使用反义RNA作为探针进行非放射性Northern印迹分析。该方法使我们能够检测特定的低丰度mRNA，并分析大鼠脑中神经营养因子的表达。