Epigenetics
Figure 16
have been shown to be able to revert malignant cells to a more normal epigenetic state and have now entered the clinics following their FDA approval. Despite the field being still in its infancy, epigenetic modulators are expected to be an attrac- tive new class of chemotherapeutic agents. Consequently, there is currently great interest in the genome-wide identification and thorough analysis of the effects of novel modulators of the epigenetic machinery. To test their effects in cell line and in in vivo systems, reliable high-through- put technologies are required both for read-out as well as sample preparation. The advances in sec- ond generation sequencing technologies have enabled the analysis of large variety of epigenetic alterations at unprecedented resolution, sensitivity and speed. The use of dedicated robotics for sam- ple preparation (eg SX-8G IP-Star Robot), Diagenode (
www.diagenode.com) performing the liquid handling steps of the immunoprecipitation process and processing up to 16 samples in paral- lel, leads to a cost- and time-effective sample pro- cessing and a significant reduction of the inter- experimental variation inherent to experiments with multiple washing and incubation steps. The automated sample preparation of the IP-Star can be directly integrated into standard Next Generation Sequencing library preparation work- flow using the enriched and/or immunoprecipita- tion fraction of the genome obtained from the robot without the need for additional experimental steps. The IP-Star can automate ChIP assays for both abundant histone modifications as well as site specific transcription factor ensuring high quality and more reproducible ChIP-seq results. For MeDIP-seq or MBD-seq, ligation of the respective sequencing adaptors prior to the immunoprecipita-
Drug Discovery World Spring 2011
tion on the IP-Star assesses the efficiency of the enrichment using standard qPCR analysis with sample independent control oligonucleotides added to the automated sample preparation process. The automation of the immunoprecipita- tion and/or affinity enrichment increases signifi- cantly the signal-to-noise ratio permitting a more rapid and reliable genome-wide identification of enrichment as assessed by high-resolution quanti- tative technologies, tiling microarrays and second generation sequencing (Figure 16).
EMD Millipore (
www.millipore.com/epigenetics) offers a comprehensive range of products for the analysis of epigenetic regulation. EMD Millipore’s offering of kits, assays, proteins, peptides, antibod- ies and inhibitors enables multiple approaches for laboratories, validating potential hits, identifying potential targets, or evaluating the effects of candi- date compounds. For this purpose, EMD Millipore offers a variety of recombinant proteins, antibodies and inhibitors for laboratories creating customised assays. This is complemented by a variety of off-the- shelf kits for analysis of histone modifications, DNA methylation status and protein interactions utilising approaches such as ELISA, xMAP® and flow cytometry. Examples include HDAC Assay Kits that provide a simple, two-step procedure for colorimet- ric, fluorometric or radiometric detection of histone deacetylase activity in 96- or 384-well plates. For the measurement of acetylation, the HAT Assay Kit uses biotinylated histone peptides to measure his- tone acetyltransferase activity. For multiplex analy- sis utilising multiplex bead technology, the H2A.X Phosphorylation Multiplex Assay Kits enables bead- based multiplex measurement of phosphorylated histone H2A.X (Ser139). In labs performing cell
49
Genome-wide differential DNA methylation between wildtype MEFs and MEFs mutated for a protein associated with the MLL complex. The Methylated DNA immunoprecipitation (MeDIP) was performed manually (left) or with the automated MeDIP protocol performed on the Diagenode IP-Star system and analysed on NimbleGEN promoter and CpG island tiling arrays. Data is centred around the transcription start site (x-axis, red line). Positive values on the y axis indicate hypermethylation in the mutated cell lines. Data shows an improved signal-to-noise ratio in the automated MeDIP preparation
(Data courtesy of Dr JorgTost, Laboratory for Epigenetics. CEA Institut de Genomique, Evry, France)
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