Protocol

Retinal Development

Volume 884 of the series Methods in Molecular Biology pp 289-304

Date:

Use of Laser Capture Microdissection for Analysis of Retinal mRNA/miRNA Expression and DNA Methylation

  • Laszlo HacklerJrAffiliated withAvidin Ltd
  • , Tomohiro MasudaAffiliated withWilmer Eye Institute, Johns Hopkins University School of Medicine
  • , Verity F. OliverAffiliated withWilmer Eye Institute, Johns Hopkins University School of Medicine
  • , Shannath L. MerbsAffiliated withWilmer Eye Institute, Johns Hopkins University School of Medicine
  • , Donald J. ZackAffiliated withWilmer Eye Institute and Departments of Molecular Biology and Genetics, Neuroscience, and Institute of Genetic Medicine, Johns Hopkins University School of Medicine Email author 

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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.

Key words

Laser capture microdissection Retina Gene expression microRNA Epigenetics Quantitative PCR RNA extraction DNA methylation Bisulfate sequencing