Isolation of Pure Human Mucosal Epithelium fo r RNA Analysi s

Techniques for isolating the desired cel l populations from complex tissues are essen tial for characterizing cells through mRNA analysis. We established a procedure fo r isolating pure mucosal epithelium from th e human alimentary tract. To do this, w e made rotating hooks that hold mucosa l strips and detach the epithelial sheets from the irregular mucosae surface in medium containing EDTA. An additional step using a cell strainer was required to reduce con tamination by lymphoid cells. Sheets of ep ithelial cells were detached successfull y from mucosal samples derived from five di f ferent parts of the human alimentary tract . Contamination by lymphoid cells or fibrob lasts was monitored by competitive RT-PCR and was no more than 0.5% of the tota l cells. Total RNA yields were 12.5–17 μ g fo r each separation, and the integrity of th e RNA was as good as that of RNAs extracted from mucosa immediately after resection. In conclusion, our method permits isolation o f RNAs from a pure population of epithelia l cells that can be used for mRNA-based gen e expression analyses .