An ambitious EC-funded research initiative on epigenetics advancing towards systems biology

Introduction

The following protocol describes a procedure for the purification and cloning of miRNAs and other small RNAs in the 20-30 nucleotide size range from plant tissue.

PDF version

Kevin V. Morris

Introduction

The recent discovery of RNA interference and in particular the observation that siRNAs can modulate gene expression at the level of transcription, i.e. small-interfering RNA (siRNA) directed transcriptional gene silencing (TGS) in Human cells (Matzke and Birchler 2005; Morris 2005) has illustrated the fact that RNA may be far more intricately involved in epigenetics than was previously assumed. To determine more clearly how siRNAs are interacting with the homologous genomic regions in the nucleus in human cell cultures we designed several RNA-biotin based pulldown assays which can be used alone or in combination with other known assays such as ChIP and Flag-tagged pulldown assays. Three protocols are explained in detail here. The first protocol is essentially a dual-pulldown assay employing Flag-tagged DNMT3A or antibody of choice for an endogenous protein and 5' biotin linked antisense RNA, while the second protocol is a triple pulldown assay which essentially expands upon the dual pulldown to incorporate a third pulldown which is an iteration of the ChIP and is a pulldown for H3K27me3+. The third assay described here is the biotin-RNA pulldown of a low-copy RNA that spans the siRNA targeted promoter region. Data generated from this assay is currently in submission.

PDF version

Bryan K. Sun & Jeannie T. Lee

Introduction

RNA-protein interactions play important roles within the cell. Using a variation of the widely-used chromatin immunoprecipitation (ChIP) assay, the potential association of cellular RNAs and candidate proteins can be evaluated in a process named “RNA-ChIP”. This technique has been successfully used in mammalian cells, for example to examine the relationship of noncoding RNAs with histone proteins or to examine interactions between viral RNAs and proteins in the host mammalian cell. In RNA-ChIP, RNA-protein interactions are fixed by reversible chemical cross-linking with formaldehyde followed by immunoprecipitation with antibodies against the candidate protein(s). RNAs that are associated with the protein are detected by reverse transcriptase-PCR (RT-PCR). The following procedure was used to examine protein-RNA interactions in mouse embryonic stem cells, but can be modified for other cell types.

PDF version

Introduction

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. [...]

PDF version

Introduction

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.

PDF version