Advertisement

Laser Capture Microdissection of Embryonic Cells and Preparation of RNA for Microarray Assays

  • Latasha C. Redmond
  • Christopher J. Pang
  • Catherine Dumur
  • Jack L. Haar
  • Joyce A. Lloyd
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1092)

Abstract

In order to compare the global gene expression profiles of different embryonic cell types, it is first necessary to isolate the specific cells of interest. The purpose of this chapter is to provide a step-by-step protocol to perform laser capture microdissection (LCM) on embryo samples and obtain sufficient amounts of high-quality RNA for microarray hybridizations. Using the LCM/microarray strategy on mouse embryo samples has some challenges, because the cells of interest are available in limited quantities. The first step in the protocol is to obtain embryonic tissue, and immediately cryoprotect and freeze it in a cryomold containing Optimal Cutting Temperature freezing media (Sakura Finetek), using a dry ice–isopentane bath. The tissue is then cryosectioned, and the microscope slides are processed to fix, stain, and dehydrate the cells. LCM is employed to isolate specific cell types from the slides, identified under the microscope by virtue of their morphology. Detailed protocols are provided for using the currently available ArcturusXT LCM instrument and CapSure® LCM Caps, to which the selected cells adhere upon laser capture. To maintain RNA integrity, upon removing a slide from the final processing step, or attaching the first cells on the LCM cap, LCM is completed within 20 min. The cells are then immediately recovered from the LCM cap using a denaturing solution that stabilizes RNA integrity. RNA is prepared using standard methods, modified for working with small samples. To ensure the validity of the microarray data, the quality of the RNA is assessed using the Agilent bioanalyzer. Only RNA that is of sufficient integrity and quantity is used to perform microarray assays. This chapter provides guidance regarding troubleshooting and optimization to obtain high-quality RNA from cells of limited availability, obtained from embryo samples by LCM.

Key words

Laser capture microdissection RNA Microarray Infrared laser 

References

  1. 1.
    Chimge NO, Ruddle F, Bayarsaihan D (2007) Laser-assisted microdissection (LAM) in developmental biology. J Exp Zool B Mol Dev Evol 308:113–118PubMedCrossRefGoogle Scholar
  2. 2.
    Murray GI (2007) An overview of laser microdissection technologies. Acta Histochem 109:171–176PubMedCrossRefGoogle Scholar
  3. 3.
    Nawshad A, LaGamba D, Olsen BR, Hay ED (2004) Laser capture microdissection (LCM) for analysis of gene expression in specific tissues during embryonic epithelial-mesenchymal transformation. Dev Dyn 230:529–534PubMedCrossRefGoogle Scholar
  4. 4.
    Sainson RC, Johnston DA, Chu HC, Holderfield MT, Nakatsu MN, Crampton SP, Davis J, Conn E, Hughes CC (2008) TNF primes endothelial cells for angiogenic sprouting by inducing a tip cell phenotype. Blood 111:4997–5007PubMedCrossRefGoogle Scholar
  5. 5.
    Canto-Soler MV, Huang H, Romero MS, Adler R (2008) Transcription factors CTCF and Pax6 are segregated to different cell types during retinal cell differentiation. Dev Dyn 237:758–767PubMedCrossRefGoogle Scholar
  6. 6.
    Toba Y, Tiong JD, Ma Q, Wray S (2008) CXCR4/SDF-1 system modulates development of GnRH-1 neurons and the olfactory system. Dev Neurobiol 68:487–503PubMedCrossRefGoogle Scholar
  7. 7.
    Redmond LC, Dumur CI, Archer KJ, Haar JL, Lloyd JA (2008) Identification of erythroid-enriched gene expression in the mouse embryonic yolk sac using microdissected cells. Dev Dyn 237:436–446PubMedCrossRefGoogle Scholar
  8. 8.
    Spencer MW, Casson SA, Lindsey K (2007) Transcriptional profiling of the Arabidopsis embryo. Plant Physiol 143:924–940PubMedCrossRefGoogle Scholar
  9. 9.
    Bhattacherjee V, Mukhopadhyay P, Singh S, Johnson C, Philipose JT, Warner CP, Greene RM, Pisano MM (2007) Neural crest and mesoderm lineage-dependent gene expression in orofacial development. Differentiation 75:463–477PubMedCrossRefGoogle Scholar
  10. 10.
    Xiao W, Liu W, Li Z, Liang D, Li L, White LD, Fox DA, Overbeek PA, Chen Q (2006) Gene expression profiling in embryonic mouse lenses. Mol Vis 12:1692–1698PubMedGoogle Scholar
  11. 11.
    Plummer S, Sharpe RM, Hallmark N, Mahood IK, Elcombe C (2007) Time-dependent and compartment-specific effects of in utero exposure to Di(n-butyl) phthalate on gene/protein expression in the fetal rat testis as revealed by transcription profiling and laser capture microdissection. Toxicol Sci 97:520–532PubMedCrossRefGoogle Scholar
  12. 12.
    Williams EO, Xiao Y, Sickles HM, Shafer P, Yona G, Yang JY, Lin DM (2007) Novel subdomains of the mouse olfactory bulb defined by molecular heterogeneity in the nascent external plexiform and glomerular layers. BMC Dev Biol 7:48PubMedCrossRefGoogle Scholar
  13. 13.
    Brunskill EW, Aronow BJ, Georgas K, Rumballe B, Valerius MT, Aronow J, Kaimal V, Jegga AG, Yu J, Grimmond S, McMahon AP, Patterson LT, Little MH, Potter SS (2008) Atlas of gene expression in the developing kidney at microanatomic resolution. Dev Cell 15:781–791PubMedCrossRefGoogle Scholar
  14. 14.
    Redmond LC, Dumur CI, Archer KJ, Grayson DR, Haar JL, Lloyd JA (2011) Krüppel-like factor 2 regulated gene expression in mouse embryonic yolk sac erythroid cells. Blood Cell Mol Dis 47(1):1–11PubMedCrossRefGoogle Scholar
  15. 15.
    Pang CJ, Lemsaddek W, Alhashem YN, Bondzi C, Redmond LC, Ah-Son N, Dumur CI, Archer KJ, Haar JL, Lloyd JA, Trudel M (2012) Krüppel-like factor 1 (KLF1), KLF2, and myc control a regulatory network essential for embryonic erythropoiesis. Mol Cellular Biol 32(13):2628–2644PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, New York 2014

Authors and Affiliations

  • Latasha C. Redmond
    • 1
  • Christopher J. Pang
    • 1
  • Catherine Dumur
    • 2
  • Jack L. Haar
    • 3
  • Joyce A. Lloyd
    • 4
  1. 1.Departments of Human and Molecular GeneticsVirginia Commonwealth UniversityRichmondUSA
  2. 2.Departments of PathologyVirginia Commonwealth UniversityRichmondUSA
  3. 3.Departments of Anatomy and NeurobiologyVirginia Commonwealth UniversityRichmondUSA
  4. 4.Department of Human and Molecular GeneticsVirginia Commonwealth UniversityRichmondUSA

Personalised recommendations