DOUBLE-LABEL IN-SITU HYBRIDIZATION USING BIOTIN AND DIGOXIGENIN-TAGGED RNA

We describe a double-label in situ hybridization protocol based on the optimized synthesis of biotin-labled RNA probes. Biotin-labeled probe s are used in conjunction with digoxigenin-labeled probes to simultane ously visualize two different transcripts. One transcript is hy bridiz ed with a biotin-labeled RNA probe and visualized as a brown peroxidas e reaction product, and the other transcript is hybridized with a digo xigenin-labeled RNA probe and visualized as a blue alkaline phosphatas e reaction product. We present several examples in which this double-l abeling method has proven useful in determining the spatial and tempor al relationships between various transcripts expressed during Drosophi la embryogenesis, indicating that this method should be of general use in establishing the relationship of two independent transcription pat terns.