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C. Jin, Y. Xia; Using Laser Capture Microdissection and cDNA Microarray to Elucidate the Molecular Signatures of Ocular Surface Development. Invest. Ophthalmol. Vis. Sci. 2010;51(13):1648. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
A critical developmental event of ocular surface in mammals is the formation of eyelid. Eyelid development is a dynamic process involving a transient "eyelid closure". Genetic mutant mice deficient in eyelid closure display an eye-open at birth (EOB) phenotype and severe ocular surface defects. We have shown that mice deficient in the MAP3K1, a cytoplasmic protein kinase, display EOB. Laser capture microdissection (LCM) is a method to procure subpopulations of cells under direct microscopic visualization to give histologically pure enriched cell populations. The purpose of the present study is to isolate pure cell from developing eyelid of wild type and Map3k1 (-/-) fetuses by LCM and to elucidate MAP3K1-dependent gene expression signatures by microarray.
The Map3k1 (+/-) mice were used for time mating. The fetuses were harvested at various embryonic days (ED), ranging from ED 13.5 -16.5, embedded in OCT and stored at -80°C. Before LCM, 8 µm sections were stained using HistoGene LCM Frozen Section Staining Kit, followed by microdissection (Arcturus XTTM Microdissection System.). For each section, pictures were taken before and after LCM procedures. The number of cells was estimated and the RNA isolated using Trizol reagent. The RNA quality was evaluated using Agilent bioanalyzer.
From an E13.5 fetus, approximately 200 cells were obtained from the "leading edge" eyelid epithelium, yielding 3-5 ng RNA, while 600 cells were obtained from the developing conjunctiva, giving rise 12-15 ng RNA. From an E15.5 fetus, approximately 400 cells were obtained from the "leading edge" eyelid epithelium, generating 7-10 ng RNA, while 1200 cells were obtained from the developing conjunctiva and 20-30 ng RNA.
It is estimated that 12 E13.5 and 6 E15.5 fetuses will yield sufficient RNA, assisted by two-round cDNA amplification, for triplicate microarray studies. The LCM technique can be used to isolate pure cell populations and to elucidate gene expression signatures in development.
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