In recent years, studies have shown that expression profiling of carefully chosen intermediary gene sets, comprising approximately 10 to 100 genes, can convey the most relevant information compared to much more complex whole-genome studies. In this paper, we present a novel method suitable for expression profiling of moderate gene sets in a large number of samples. The assay implements the parallel amplification features of the trinucleotide threading technique (TnT), which encompasses linear transcript-based DNA thread formation in conjunction with exponential multiplexed thread amplification. The amplifications bestow the method with high sensitivity. The TnT procedure together with thread detection, relying on thread-specific primer extension followed by hybridization to universal tag arrays, allows for three distinction levels, thus offering high specificity. Additionally, the assay is easily automated and flexible. A gene set, comprising 18 protein epitope signature tags from the Swedish Human Protein Atlas program, was analyzed with the TnT-based approach and the data were compared with those generated by both real-time PCR and genome-wide cDNA arrays, with the highest correlation observed between TnT and real-time PCR. Taken together, expression profiling with trinucleotide threading represents a reliable approach for studies of intermediary gene sets.