Comprehensive and systematic analysis of airway gene expression represents a strategy for addressing the multiple, complex, and largely untested hypotheses that exist for disease mechanisms, including asthma. Here, we report a novel real-time PCR-based method specifically designed for quantification of multiple low-abundance transcripts using as little as 2.5 fg of total RNA per gene. This method of gene expression profiling has the same specificity and sensitivity as RT-PCR and a throughput level comparable to low-density DNA microarray hybridization. In this two-step method, multiplex RT-PCR is successfully combined with individual gene quantification via real-time PCR on generated cDNA product. Using this method, we measured the expression of 75 genes in bronchial biopsies from asthmatic versus healthy subjects and found expected increases in expression levels of Th2 cytokines and their receptors in asthma. Surprisingly, we also found increased gene expression of NKCC1—a Na⁺-K⁺-Cl⁻ cotransporter. Using immunohistochemical method, we confirmed increased protein expression for NKCC1 in the asthmatic subject with restricted localization to goblet cells. These data validate the new transcriptional profiling method and implicate NKCC1 in the pathophysiology of mucus hypersecretion in asthma. Potential applications for this method include transcriptional profiling in limited numbers of laser captured cells and validation of DNA microarray data in clinical specimens.