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Isolation of intact RNA from cytometrically sorted Saccharomyces cerevisiae for the analysis of intrapopulation diversity of gene expression

Nature Protocols volume 2, pages 2203–2211 (2007)Cite this article

Abstract

Characterizing and understanding the functional heterogeneity in a given population on the cellular and molecular level is a great challenge in microbiology. Each microorganism contributes differently to the overall performance of the community and responds differently to changing microenvironmental conditions. Here, we present a method for isolation of intact RNA out of small subpopulations of live Saccharomyces cerevisiae cells for differential gene expression analysis. The protocol includes fluorescence staining, flow cytometric analysis and sorting of live yeast cells, subsequent isolation of RNA from the resulting subpopulations and finally RNA quantification and integrity check. The isolated RNA can be transcribed into cDNA and successfully used for microarray analysis. This aids in relating molecular regulation processes within subpopulations with the dynamics and functioning of the entire population. The procedure can be accomplished in 2 d.

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Figure 1
Figure 2: Gate settings for the sorting procedure.
Figure 3: Microarray for the analysis of gene expression of a vital, stained and sorted subpopulation of S. cerevisiae with high affinity to 2-NBDglucose, harvested at a dilution rate of D = 0.160 h−1, and of cells from the whole population without further treatment grown at the same dilution rate.
Figure 4: Agarose gel showing RNA isolated from 5 × 107 freshly harvested (1–5) and frozen (with dry ice; 6–10) S. cerevisiae cells after treatment with lyticase (300 U in 100 μl) for various durations (2–30 min).
Figure 5: Agarose gel showing RNA isolated from 5 × 107 frozen (with dry ice) S. cerevisiae cells after treatment with lyticase (100 U in 100 μl (1–4) and 200 U in 100 μl (5–8)) at several time intervals (5–30 min).
Figure 6: Fractionation of RNA isolated from 5 × 107 S. cerevisiae cells harvested after triple-staining.
Figure 7: Agarose gel showing RNA isolated from 5 × 107 sorted S. cerevisiae cells, frozen with dry ice.

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Acknowledgements

We thank H. Engewald and C. Süring for technical assistance. We thank T. Hübschmann for technical assistance as well as for helpful discussions and A. Pernthaler for critical reading of the manuscript. We also thank C. Repenning and M. Pähler from the working group 'Chip Technology' at the Institute of Technical Chemistry of the University of Hannover. This work was supported by the Deutsche Forschungsgemeinschaft (MU 1089/5-3).

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  1. Department of Environmental Microbiology, UFZ, Helmholtz Centre for Environmental Research, Permoserstrasse 15, Leipzig, 04318, Germany

    Jeannette Achilles, Hauke Harms & Susann Müller

  2. Institute for Technical Chemistry, University of Hannover, Callinstrasse 3, Hannover, 30167, Germany

    Frank Stahl

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Correspondence to Susann Müller.

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Achilles, J., Stahl, F., Harms, H. et al. Isolation of intact RNA from cytometrically sorted Saccharomyces cerevisiae for the analysis of intrapopulation diversity of gene expression. Nat Protoc 2, 2203–2211 (2007). https://doi.org/10.1038/nprot.2007.322

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