Details
| Original language | English |
|---|---|
| Pages (from-to) | 2203-11 |
| Number of pages | 9 |
| Journal | Nature protocols |
| Volume | 2 |
| Issue number | 9 |
| Publication status | Published - 2007 |
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.
Keywords
- Flow Cytometry/methods, Gene Expression Profiling/methods, Genetic Variation, Oligonucleotide Array Sequence Analysis, RNA, Fungal/isolation & purification, Saccharomyces cerevisiae/genetics
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In: Nature protocols, Vol. 2, No. 9, 2007, p. 2203-11.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Isolation of intact RNA from cytometrically sorted Saccharomyces cerevisiae for the analysis of intrapopulation diversity of gene expression
AU - Achilles, Jeannette
AU - Stahl, Frank
AU - Harms, Hauke
AU - Müller, Susann
N1 - Funding information: ACKNOWLEDGMENTS 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).
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
KW - Flow Cytometry/methods
KW - Gene Expression Profiling/methods
KW - Genetic Variation
KW - Oligonucleotide Array Sequence Analysis
KW - RNA, Fungal/isolation & purification
KW - Saccharomyces cerevisiae/genetics
U2 - 10.1038/nprot.2007.322
DO - 10.1038/nprot.2007.322
M3 - Article
C2 - 17853877
VL - 2
SP - 2203
EP - 2211
JO - Nature protocols
JF - Nature protocols
SN - 1750-2799
IS - 9
ER -