Gene-expression profiling has been successfully applied in various diagnostic applications, but its full capacity is yet to be realized. Samples are generally prepared from a mixture of different cells that are present in unknown proportions. Cells are, in many aspects, unique in their characteristics and this heterogeneity confounds the expression profile. The development of new and robust techniques to measure gene expression in single cells opens new avenues in molecular medicine. Today, gene-expression profiles of individual cells can be measured with high precision and accuracy, identifying different cell types as well as revealing heterogeneity among cells of the same kind. Here, we review practical aspects of single-cell gene-expression profiling using reverse transcription quantitative real-time PCR and its potential use in diagnostics.