Laser-Capture Microdissection of Developing Barley Seeds and cDNA Array Analysis of Selected Tissues
Laser microdissection provides a useful method for isolating specific cell types from complex biological samples for downstream applications. In contrast to the texture of mammalian cells, most plant tissues exhibit a cell organization with hard, cellulose-containing cell walls, large vacuoles, and air spaces, thus complicating tissue preparation and extraction of macromolecules such as DNA and RNA. Especially, barley seeds show cell types with enormous differences in osmolarity (degenerating and differentiating tissues) and contain high amounts of the main storage product starch, thus requiring specific procedures for morphological preservation and RNA extraction. In this study, we report about methods allowing tissue-specific gene expression profiling of developing barley seeds. Details on aspects of tissue preparation, including fixation and embedding procedures, laser-capture microdissection, RNA isolation, and linear mRNA amplification to produce high-quality labelled probes for large-scale expression analysis are provided. Particular emphasis is placed on the fidelity of transcript data obtained by the developed methods in relation to the in vivo transcriptome.
Key wordsLaser-capture microdissection mRNA amplification Gene expression analysis cDNA array Barley seeds
We are grateful to Uta Siebert for her excellent and expert assistance in tissue processing and operating of the PALM Laser Microbeam instrument. We also wish to thank Ursula Tiemann and Karin Lipfert for graphical artwork. This work was supported by the Deutsche Forschungsgemeinschaft (DFG, FKZ 39205123) and by the Federal Ministry of Education and Research (BMBF, FKZ 0313821A).
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