Linear amplification of limiting amounts of RNA for gene expression studies (Prot 20)
Expression profiling of cells is a valuable tool in understanding gene regulatory networks and in identifying genes necessary for normal developmental processes. In the haematopoietic system, cell surface markers are well defined allowing the isolation of cells from various developmental stages using flow cytometry. However gene expression studies of solid tissues are often hampered by the difficulty in obtaining a homogenous cell population. Often appropriate cell markers are not well defined for given cell stages therefore making gene expression studies difficult. In addition studies of rare cell populations mean that the amount of material obtained is insufficient for analysis. To overcome these problems we have employed a system where a given cell population is GFP-labeled and then sorted by flow cytometry in order to obtain a pure homogenous population. RNA was then prepared from this material and amplified by a linear amplification method allowing large scale microarray gene expression analysis. This approach was successfully applied to gene expression studies of the midbrain-hindbrain organizer region of the mouse but can be applied to many other systems to isolate cell populations. [...]
Research Institute for Molecular Pathology
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A1030, Vienna, Austria
RNAse A Treatment of Mouse Cells (Prot 10)
RNAse A treatment of permeabilized cells followed by immunostaining is a method which allows to show if the localization of a protein into the cell involves an RNAse-sensitive structure. This technique is highly dependent of the quality of the used RNAse A. In our lab, we used RNAse A treatment of mouse cells to demonstrate that the enrichment in HP1 (Heterochromatin Protein 1) proteins at pericentric heterochromatin depends on the presence of an RNA component.
UMR 218 Institute Curie - 26, rue d'Ulm - 75231 Paris Cedec 05, France