Quantitative non‐radioactive in situ hybridization of preproenkephalin mRNA with digoxigenin‐labeled cRNA probes

Abstract

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.