A novel method of gene transcript profiling in airway biopsy homogenates reveals increased expression of a Na+-K+-Cl- cotransporter (NKCCl) in asthmatic subjects

Comprehensive and systematic analysis of airway gene expression represents a strategy for addressing the Multiple, complex, and largely untested hypot heses 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 specifi city and sensitivity as RT-PCR and a throughput level comparable to low-den sity DNA microarray hybridization. In this two-step method, multiplex RT-PC R is successfully combined with individual gene quantification via real-tim e PCR on generated cDNA product. Using this method, we measured the express ion of 75 genes in bronchial biopsies from asthmatic versus healthy subject s and found expected increases in expression levels of Th2 cytokines and th eir receptors in asthma. Surprisingly, we also found increased gene express ion of NKCCl-a Na+-K+-Cl- cotransporter. Using immunohistochemical method, we confirmed increased protein expression for NKCCl in the asthmatic subjec t with restricted localization to goblet cells. These data validate the new transcriptional profiling method and implicate NKCCl in the pathophysiolog y of mucus hypersecretion in asthma. Potential applications for this method include transcriptional profiling in limited numbers of laser captured cel ls and validation of DNA microarray data in clinical specimens.