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Laser Capture Microdissection for Protein and NanoString RNA Analysis

  • Yelena Golubeva
  • Rosalba Salcedo
  • Claudius Mueller
  • Lance A. Liotta
  • Virginia Espina
Part of the Methods in Molecular Biology book series (MIMB, volume 931)

Abstract

Laser capture microdissection (LCM) allows the precise procurement of enriched cell populations from a heterogeneous tissue, or live cell culture, under direct microscopic visualization. Histologically enriched cell populations can be procured by harvesting cells of interest directly or isolating specific cells by ablating unwanted cells. The basic components of laser microdissection technology are (a) visualization of cells via light microscopy, (b) transfer of laser energy to a thermolabile polymer with either the formation of a polymer-cell composite (capture method) or transfer of laser energy via an ultraviolet laser to photovolatize a region of tissue (cutting method), and (c) removal of cells of interest from the heterogeneous tissue section. The capture and cutting methods (instruments) for laser microdissection differ in the manner by which cells of interest are removed from the heterogeneous sample. Laser energy in the capture method is infrared (810 nm), while in the cutting mode the laser is ultraviolet (355 nm). Infrared lasers melt a thermolabile polymer that adheres to the cells of interest, whereas ultraviolet lasers ablate cells for either removal of unwanted cells or excision of a defined area of cells. LCM technology is applicable to an array of applications including mass spectrometry, DNA genotyping and loss-of-heterozygosity analysis, RNA transcript profiling, cDNA library generation, proteomics discovery, and signal kinase pathway profiling. This chapter describes LCM using an Arcturus XT instrument for downstream protein sample analysis and using an mmi CellCut Plus® instrument for RNA analysis via NanoString technology.

Key words

DNA Infrared laser Laser capture microdissection Molecular profiling NanoString Phopshoprotein Pre-analytical variability Protein RNA Tissue Tissue heterogeneity UV laser 

Notes

Acknowledgements

Thanks to the staff of Pathology-Histotechnology Laboratory, SAIC-NCI-Frederick. The work was funded in part by NCI Contract HHSN261200800001E.

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Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Yelena Golubeva
    • 1
  • Rosalba Salcedo
    • 1
  • Claudius Mueller
    • 2
  • Lance A. Liotta
    • 2
  • Virginia Espina
    • 2
  1. 1.National Cancer Institute-Frederick/SAICFrederickUSA
  2. 2.Center for Applied Proteomics and Molecular MedicineGeorge Mason UniversityManassasUSA

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