Advancing Epigenetics Towards Systems Biology

RNA-biotin based pulldown assays for the detection of siRNA targeted genomic regions and siRNA directed histone modifications (Prot 32)

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

Kevin V. Morris

The Scripps Research Institute
Department of Molecular and Experimental Medicine
The Scripps Research Institute - 10550 N. Torrey Pines Road
La Jolla, CA, 92037 , USA

Kevin V. Morris