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Gene expression analysis of specific cell types isolated from mouse inner ear following laser capture microdissection.
Alagramem K; Wang W; Davis R; Wright C
Abstr 28th Midwinter Res Meet 2005 Feb; 28:10
Laser Capture Microdissection (LCM) allows procurement of specific cells types from microscopic regions of tissue sections, which can then be used for gene expression analysis. Cryosections, rather than formalin-fixed paraffin embedded (FFPE) sections, are preferred because the recovery of quality RNA from FFPE sections can be quite challenging. However, the morphology of the various cells types within the inner ear is well preserved in FFPE sections (compared to cryosections) making it easier to identify the cell types and their boundaries. We explored the possibility of using FFPE for gene expression following LCM. Relatively pure populations of cells from the organ of Corti, spiral ganglion, and the maculae were isolated from eight-micron FFPE sections by LCM (Pixcell II, Arcturus, CA). RNA was extracted from captured cells, amplified and assessed for quality by gel electrophoresis. Expression of a select number of genes were tested by reverse transcription PCR and real-time PCR. We were able to detect the expression of several genes reproducibly. This included housekeeping gene Hprt, deafness genes (ex. Protocadherin 15) and other uncharacterized genes. While the house keeping genes were detected in all cell types, some of the other genes showed a restricted expression pattern. The method described here has potential use in many areas of hearing research. The sensitivity and accuracy of molecular profiling can be increased substantially by focusing the analysis on specific cell types within target tissues. For example, following noise exposure in mice, it would be highly desirable to perform gene expression analysis using RNA isolated from hair cells or spiral ganglion cells, instead of whole inner ear tissue or mixed populations of cells from surface preparations. Further, the method we describe here for mouse FFPE sections could be used for retrospective analysis of human archival ear tissue for investigation in search of disease mechanisms.
Noise-exposure; Exposure-levels; Noise-induced-hearing-loss; Noise-exposure; Noise; Hearing; Hearing-disorders; Hearing-loss; Laboratory-animals; Animals; Cell-damage; Cellular-reactions; Tissue-culture; Genes
Disease and Injury: Hearing Loss
Abstracts of the 28th Midwinter Research Meeting
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