Abstract
Laser capture microdissection (LCM) is a useful method to isolate specific cells or cell layers of interest from heterogeneous tissues, such as the retina. The collected cells can be used for DNA, RNA, or protein analysis. We have applied LCM technology to isolate cells from the outer nuclear, inner nuclear, and ganglion cell layers of the retina for mRNA and microRNA (miRNA) expression and epigenetic (DNA methylation) analysis. Here, we describe the methods we have employed for sample preparation, LCM-based isolation of retinal layers, RNA/DNA extraction, RNA quality check, microRNA analysis by quantitative PCR, and DNA methylation analysis by bisulfite sequencing.
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Acknowledgments
The work described in this chapter was supported by grants from the National Eye Institute, NIH (5R01EY009769, 5P30EY001765, and 5R01EY020406), and by generous gifts from the Guerrieri Family Foundation and from Mr. and Mrs. Robert and Clarice Smith.
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Hackler, L., Masuda, T., Oliver, V.F., Merbs, S.L., Zack, D.J. (2012). Use of Laser Capture Microdissection for Analysis of Retinal mRNA/miRNA Expression and DNA Methylation. In: Wang, SZ. (eds) Retinal Development. Methods in Molecular Biology, vol 884. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-848-1_21
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DOI: https://doi.org/10.1007/978-1-61779-848-1_21
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