Robbins E, Baldino F, Roberts-Lewis J M, Meyer S L, Grega D, Lewis M E
Cephalon, Inc., West Chester, Pennsylvania 19380.
Anat Rec. 1991 Dec;231(4):559-62. doi: 10.1002/ar.1092310417.
Non-radioactive detection of mRNA with in situ hybridization histochemistry has emerged as an important new technology for the study of gene expression. Quantitative in situ hybridization studies have generally relied upon counting of autoradiographic grains in the emulsion overlying cells containing hybridized, radioactively labeled probe. However, such high resolution studies require tedious grain counting over individual cells, frequently in addition to weeks of exposure to nuclear emulsion. The present report describes a quantitative, non-radioactive approach to the detection of a specific mRNA in the brain with the advantages of comparatively rapid tissue processing and computerized image analysis. The validity of this approach was tested by measuring the haloperidol-induced increase in the level of preproenkephalin mRNA in striatal sections of the rat brain using an RNA probe labeled with digoxigenin-11-UTP. Detection of probe hybridized to tissue sections was carried out enzymatically following complex formation with an antidigoxigenin-alkaline phosphatase conjugate. Using computerized image analysis, it was found that chronic treatment of rats with haloperidol resulted in a 50 +/- 6% increase in striatal neuronal optical density, a value in good agreement with previous studies using low-resolution radioactive methods, showing a 30-80% increase in striatal preproenkephalin mRNA hybridization signal.
原位杂交组织化学对mRNA进行非放射性检测已成为研究基因表达的一项重要新技术。定量原位杂交研究通常依赖于对含有杂交的、放射性标记探针的细胞上覆盖的乳胶中的放射自显影片颗粒进行计数。然而，此类高分辨率研究需要对单个细胞进行繁琐的颗粒计数，通常还需要数周时间暴露于核乳胶中。本报告描述了一种定量、非放射性方法，用于检测大脑中的特定mRNA，具有组织处理相对快速和计算机化图像分析的优点。通过使用地高辛配基-11-UTP标记的RNA探针，测量氟哌啶醇诱导的大鼠脑纹状体切片中前脑啡肽原mRNA水平的增加，对该方法的有效性进行了测试。在与抗地高辛配基-碱性磷酸酶偶联物形成复合物后，通过酶法对杂交到组织切片上的探针进行检测。使用计算机化图像分析发现，用氟哌啶醇长期治疗大鼠导致纹状体神经元光密度增加50±6%，该值与先前使用低分辨率放射性方法的研究结果高度一致，显示纹状体前脑啡肽原mRNA杂交信号增加30 - 80%。
