Isolation of intact RNA from cytometrically sorted Saccharomyces cerevisiae for the analysis of intrapopulation diversity of gene expression.
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.
Achilles
Jeannette
J
Department of Environmental Microbiology, UFZ, Helmholtz Centre for Environmental Research, Leipzig, Germany.
Stahl
Frank
F
Harms
Hauke
H
Müller
Susann
S
D005434:Flow Cytometry / Q000379:methods*; D020869:Gene Expression Profiling / Q000379:methods*; D014644:Genetic Variation; D020411:Oligonucleotide Array Sequence Analysis; D012331:RNA, Fungal / Q000302:isolation & purification* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012331:RNA, Fungal