Abstract
As part of a study of in vivo gene expression levels in the human airway epithelium in response to chronic cigarette smoking, we have identified a number of genes whose expression levels are altered in a time-dependent fashion resulting from the procedure used to sample epithelial cells. Fiberoptic bronchoscopy and airway epithelium brushing were used to obtain independent samples from a single individual, 1st from the right lung, followed by sampling of the left lung. We observed that a specific subset of early response genes encoding proteins involved in transcription, signal transduction, cell cycle/growth, and apoptosis were significantly up-regulated in the left lung samples (the 2nd region to be sampled) compared with the right lung samples (the 1st region to be sampled). This response was due to the temporal nature of the sampling procedure and not to inherent gene expression differences between airway epithelium of the right and left lungs. When the order of sampling was reversed, with the left airway epithelium sampled 1st, the same subset of genes were up-regulated in the samples obtained from the right airway epithelium. The time-dependent up-regulation of these genes was likely in response to the stress of the procedure and/or the anesthesia used. Sampling-dependent uncertainty of gene expression is likely a general phenomenon relevant to the procedures used for obtaining biological samples, particularly in humans where the sampling procedures are dependent on ensuring comfort and safety.
Highlights
The development of the technology of gene expression arrays has opened the possibility of assessment of the levels of mRNA for many genes from small samples [1,2,3]
Whereas our example is related to sampling of the airway epithelium in humans, the sampling dependent superimposition of “uncertainty” of gene expression is likely applicable to assessment of gene expression in general
The fiberoptic bronchoscopy procedure requires mild, systemic, conscious sedation and local anesthesia with a topical anesthetic. This is followed by the bronchoscopy procedure, in which a flexible fiberoptic bronchoscope is inserted through the mouth, passed through the vocal cords into the airways where the sampling is done by gliding a 1-mm brush along the epithelium to be sampled [10]
Summary
The development of the technology of gene expression arrays has opened the possibility of assessment of the levels of mRNA for many genes from small samples [1,2,3]. Experimental animal models can be used to evaluate some aspects of the pathogenesis of these disorders [6,7,8], the ultimate goal is to understand disease pathogenesis in humans In this context, we have adapted the methodology of sampling the airway epithelium using fiberoptic bronchoscopy and brushing to collect pure samples of airway epithelium for gene expression studies using microarrays [9,10,11]. The fiberoptic bronchoscopy procedure requires mild, systemic, conscious sedation and local (nose, pharynx, vocal cords, trachea, and bronchi) anesthesia with a topical anesthetic This is followed by the bronchoscopy procedure, in which a flexible fiberoptic bronchoscope is inserted through the mouth, passed through the vocal cords into the airways where the sampling is done by gliding a 1-mm brush along the epithelium to be sampled [10]. After an aliquot is taken for cell number and morphology assessment, the mRNA is extracted and handled as is typical for microarray studies [4,9,10,11]
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