ArticleMethods in molecular biology (Clifton, N.J.)2014
Laser capture microdissection of embryonic cells and preparation of RNA for microarray assays.
Article in Methods in molecular biology (Clifton, N.J.), 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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Who cites it
5 citing papers in PubMed.
- Multidimensional quantitative analysis of mRNA expression within intact vertebrate embryos.Development (Cambridge, England) · 2018Article
- Microdissection of lampbrush chromosomes as an approach for generation of locus-specific FISH-probes and samples for high-throughput sequencing.BMC genomics · 2016Article
- An efficient and sensitive method for preparing cDNA libraries from scarce biological samples.Nucleic acids research · 2015Article
- Ensuring good quality RNA for quantitative real-time PCR isolated from renal proximal tubular cells using laser capture microdissection.BMC research notes · 2014Article
- Kruppel-like factor 1 (KLF1), KLF2, and Myc control a regulatory network essential for embryonic erythropoiesis.Molecular and cellular biology · 2012Article
Corrections and comments
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Authors and funding
5 authors.
Funding
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.
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