Assessment of differential gene expression in vestibular epithelial cell types using microarray analysis.
Current global gene expression techniques allow the evaluation and comparison of the expression of thousands of genes in a single experiment, providing a tremendous amount of information. However, the data generated by these techniques are context-dependent, and minor differences in the individual biological samples, methodologies for RNA acquisition, amplification, hybridization protocol and gene chip preparation, as well as hardware and analysis software, lead to poor correlation between the results. One of the significant difficulties presently faced is the standardization of the protocols for the meaningful comparison of results. In the inner ear, the acquisition of RNA from individual cell populations remains a challenge due to the high density of the different cell types and the paucity of tissue. Consequently, laser capture microdissection was used to selectively collect individual cells and regions of cells from cristae ampullares followed by extraction of total RNA and amplification to amounts sufficient for high throughput analysis. To demonstrate hair cell-specific gene expression, myosin VIIA, calmodulin and alpha9 nicotinic acetylcholine receptor subunit mRNAs were amplified using reverse transcription-polymerase chain reaction (RT-PCR). To demonstrate supporting cell-specific gene expression, cyclin-dependent kinase inhibitor p27kip1 mRNA was amplified using RT-PCR. Subsequent experiments with alpha9 RT-PCR demonstrated phenotypic differences between type I and type II hair cells, with expression only in type II hair cells. Using the laser capture microdissection technique, microarray expression profiling demonstrated 408 genes with more than a five-fold difference in expression between the hair cells and supporting cells, of these 175 were well annotated. There were 97 annotated genes with greater than a five-fold expression difference in the hair cells relative to the supporting cells, and 78 annotated genes with greater than a five-fold expression difference in the supporting cells relative to the hair cells.
Cristobal
Ricardo
R
Department of Otolaryngology and Communication Sciences, Medical College of Wisconsin, 9200 W Wisconsin Avenue, Milwaukee, WI 53226-3596, USA.
Wackym
P Ashley
PA
Cioffi
Joseph A
JA
Erbe
Christy B
CB
Roche
Joseph P
JP
Popper
Paul
P
D008267:Acoustic Maculae / Q000166:cytology*; D000818:Animals; D015152:Blotting, Northern; D002147:Calmodulin / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D050760:Cyclin-Dependent Kinase Inhibitor p27; D004398:Dyneins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000502:physiology*; D020869:Gene Expression Profiling; D018069:Hair Cells, Vestibular / Q000378:metabolism*; D046228:Microarray Analysis / Q000379:methods*; D042282:Microdissection / Q000379:methods; D000081412:Myosin VIIa; D009218:Myosins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011978:Receptors, Nicotinic / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; C493962:Cdkn1b protein, rat; D018797:Cell Cycle Proteins; C000631457:Myo7a protein, rat; D000081412:Myosin VIIa; D012333:RNA, Messenger; D011978:Receptors, Nicotinic; D025521:Tumor Suppressor Proteins; D050760:Cyclin-Dependent Kinase Inhibitor p27; D009218:Myosins; D004398:Dyneins