Abstract
We have devised a novel protocol that optimizes three fluorescent dyes, Cy3 and Cy5, plus FluorX-dCTP/Fluorescein-dUTP for triple RNA labeling and hybridization, in order to identify differential transcriptional regulation in different compartments of the developing mouse brain. Simple triangle mathematics, based on the De Finetti diagram for the calculation of allele frequencies in a population, were tested for identification of expressed genes in particular brain compartments. Each significant result found at different stages of brain development was verified by whole mount in situ hybridization. The fidelity of the triple hybridization results, using three RNA inputs simultaneously, has valuable potential for implementation in many biological and medical contexts.