Retinal Development pp 289-304

Part of the Methods in Molecular Biology book series (MIMB, volume 884)

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

  • Laszlo HacklerJr
  • Tomohiro Masuda
  • Verity F. Oliver
  • Shannath L. Merbs
  • Donald J. Zack
Protocol

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 

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Laszlo HacklerJr
    • 1
  • Tomohiro Masuda
    • 2
  • Verity F. Oliver
    • 2
  • Shannath L. Merbs
    • 2
  • Donald J. Zack
    • 3
  1. 1.Avidin LtdSzegedHungary
  2. 2.Wilmer Eye InstituteJohns Hopkins University School of MedicineBaltimoreUSA
  3. 3.Wilmer Eye Institute and Departments of Molecular Biology and Genetics, Neuroscience, and Institute of Genetic MedicineJohns Hopkins University School of MedicineBaltimoreUSA

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