Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
To study the interaction between proteins and DNA in cells, researchers often turn to chromatin immunoprecipitation (ChIP), using the technique to monitor the interactions of DNA with modified histones, transcription factors and other chromatin-associated proteins.
The basic idea of the ChIP assay is simple: Use protein as bait to capture any DNA sequences that bind to it, enrich for the protein-DNA complexes by immunoprecipitation and then isolate and analyze the DNA that was “caught” by your protein bait. The DNA is analyzed downstream, using PCR or microarray analysis, or more recently, DNA sequencing.
Sounds simple enough. But the not-so-simple fact is that ChIP assays can be tedious and time consuming—and difficult to scale to high-throughput. Newer ChIP assays and variants, though, overcome many of those issues. Here are some of your newer options.
High-throughput ChIP assays
Although numerous commercial ChIP kits exist, only recently have assays (and variations) designed for high-throughput studies become available. Earlier ChIP assays took several days to perform, required more cells per reaction well and involved labor-intensive protocols. But because of recent innovations, today’s ChIP assays can be accomplished in just a few hours with fewer cells and significantly less hands-on time.
With Epigentek’s microplate-based EpiQuik™ and ChromaFlash™ High Sensitivity ChIP kits, for example, users can run up to 96 reactions in a 96-well plate in two to five hours, with as few as 2,000 cells. “The maximum throughput of these assays is 96 reactions, and typically the 48-reaction option is used by our customers,” says William Lee, vice president of operations at Epigentek.
Epigentek’s ChromaFlash One-Step Magnetic ChIP Kit is the company’s fastest ChIP assay. Used as a stand-alone kit with your own sheared DNA, the assay takes about four hours. But when used in conjunction with Epigentek’s EpiSonic™ sonication device, which performs DNA shearing and immunoprecipitation simultaneously (and also accelerates the association of antibodies with protein-DNA complexes), the entire ChIP assay takes less than 70 minutes.
First introduced in 2008, the ChIP-IT® Express High Throughput (HT) Kit from the epigenetics specialists at Active Motif, likewise accelerates the ChIP process. “This kit is based on our widely popular ChIP-IT Express format that utilizes either sonication or enzymatic digestion to fragment chromatin, and our low-sample-volume, magnetic protein-G beads for fast washing and reduced nonspecific binding,” says Kyle Hondorp, technical product manager at Active Motif.
Naturally, smaller reaction sizes and microplate formats simplify reactions. But when it comes to epigenetics, less isn’t always more. “While the ChIP-IT Express HT works well evaluating multiple ChIP antibody targets in a sample, the kit is not designed to prepare chromatin from a 96-well plate, as the chromatin yield from such a small sample amount is not sufficient for detection,” says Hondorp. “As a result, larger chromatin samples should be prepared separately for use in the assay.” And preparation of your starting materials is worth paying attention to, she says. “Successful ChIP is dependent upon the quantity of input material, the binding affinity of the ChIP antibody and the abundance of the target protein of interest.”
ChIP assay variations
As vendors optimize their assays to address researchers’ needs, commercial ChIP variants have arisen. For example, the Thermo Scientific™ Pierce Magnetic ChIP Kits use magnetic beads that have been blocked to reduce background signals, “and the bead-blocking reagent does not contain nucleic acids, making them more amenable to [downstream sequencing] applications [such as ChIP-seq],” says Kay Opperman, technical product manager at Thermo Fisher Scientific.
To streamline the ChIP workflow and increase throughput, Thermo Fisher also validated the kits using the automated lab instruments of the KingFisher Flex System. Opperman says that although ChIP kits are still limited by process-sampling times, “ChIP assays are rapidly evolving to reduce [input] cell number, to increase throughput for automation and to reduce processing time.”
EMD Millipore also offers its own high-throughput variations on ChIP. The company’s Magna ChIP™ HT96 Chromatin Immunoprecipitation Kit is a magnetic plate-based version of its Magna ChIP HiSens Kit, which uses the same buffers and protocol but not in a 96-well format. This can be helpful in developing experiments because it enables “researcher[s] to move between a low-throughput and a high-throughput approach without significant changes to their approach,” explains Michael Sturges, senior product manager for epigenetics and assay technologies at EMD Millipore.
The Magna ChIP kits’ magnetic beads use a mixture of protein A and protein G antibody-capture reagents instead of using only one type. This combination offers generally lower background and “allows the use of a wider variety of antibody isotypes than reactions with protein A or G alone,” says Sturges. To streamline ChIP assays, these kits also use a single buffer for sonication, precipitation and wash steps, and they reduce handling to facilitate the adoption of lab automation, if desired.
Subsequent to the high-throughput ChIP assay workflow, Illumina offers a valuable tool for sequencing the assay’s results. Using the DNA resulting from a compatible ChIP assay as input, Illumina’s TruSeq® ChIP Sample Preparation kit (PDF) turns the DNA into a DNA library that is ready for sequencing. “Since the amount of DNA isolated from ChIP is often limited, these kits have been optimized to perform reliably with as little as 5 ng as input DNA,” says Dave Delano, senior product manager in gene expression and regulation applications at Illumina. The TruSeq ChIP Library Preparation kits are designed to be high-throughput friendly, running up to 96 samples in parallel.
RNA Variants
Also commercialized are ChIP variants for detecting interactions between RNA and protein. Many of these are not yet considered high-throughput, but change is coming.
In a recent paper in Nature Methods, Tome et al., presented the new “high-throughput sequencing-RNA affinity profiling” (HiTS-RAP) assay [1]. The team adapted Illumina’s high-throughput DNA sequencer to measure the binding of a fluorescent protein to millions of RNAs that are anchored to sequenced cDNA templates. Importantly, the method does not rely on antibodies, which can be expensive and time consuming to develop; instead, the authors use the sequencer to measure the affinity of specific RNA aptamers for their protein targets.
What is becoming clear, says Sturges, is that to fully understand chromatin, researchers may need to study not protein-DNA and protein-RNA interactions alone, but both together. “In some cases, chromatin-associated proteins that bind to DNA are found to also associate with RNA,” he says. “This triad of association suggests that these various nucleic acid-protein interactions work cooperatively to modulate gene expression.”
References
[1] Tome, JM, et al., “Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling,” Nature Methods, 11:683-8, 2014. [PubMed ID: 24809628]
Image: Screenshot of the UCSC Genome Browser.