Noninvasive method for obtaining RNA from buccal mucosa epithelial cells for gene expression profiling.

Swabs and scrapings from the buc-cal mucosa in the mouth have been used to obtain DNA from epithelial cells for genetic studies (1,2). RNA has been obtained from resected tissues and from biopsy samples of mouth epithelium in various disease states to measure gene expression (3,4). However , RNA has not been extracted from scrapings of buccal mucosa because ribonucleases that are present in saliva rapidly degrade epithelial cell RNA (5) during collection. To collect intact RNA from buc-cal mucosal epithelium for studies of the biologic effect of smoking on the airway epithelium, we developed a relatively noninvasive method for obtaining small amounts of RNA from the mouth. We measured the expression of selected genes in individual subjects using real-time PCR and used a recently described mass spectrometry method that requires only nanogram amounts of total RNA for analysis and lends itself to high-throughput analysis of hundreds of genes (6). Initially, we used a micropipet tip cut lengthwise to collect epithelial cells from the buccal mucosa in a relatively noninvasive fashion. We subsequently designed a standardized plastic tool that is concave with serrated edges. It is 5/16 inches wide and 1 6/16 inches long with a 3 inch handle that can be broken off when the scraping tool with the collected cells is inserted into a 2-mL mi-crofuge tube containing 1 mL RNAlater™ solution (Qiagen, Valencia, CA, USA). The tool has two features that allow the collection of a significant amount of good-quality RNA from the buccal mucosa: a finely serrated edge that can scrape off several layers of epithelial cells and a concave surface that collects the cells. Using gentle pressure, the serrated edge was scraped (10 times) against the buccal mucosa on the inside of the cheek, and the cells collected were immediately immersed in 1 cm 3 RNAlater solution. After storage at 4°C for up to 24 h, total RNA from buccal epithelial cells was isolated from the cell pellet using TRI ® reagent (In-vitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol. The integrity of the RNA was confirmed in select cases on a RNA denaturing gel (Figure 1). Epithelial cell content was quantified by cytocentrifugation at 700× g (Cytospin ® ; ThermoShan-don, Pittsburgh, PA, USA) of the cell pellet and staining with a cytokeratin antibody (Signet, Dedham, MA, USA) (Figure 2). Using this protocol, we were able to obtain 300–1500 ng RNA from each subject …