: Chip qPCR Assays
Q: How do I choose ChIP-grade antibodies?
A: Many companies have so-claimed ChIP-grade antibodies available. However, they might not work in your experiment. In many cases, one has to experimentally test the antibodies of interest. We provide experimentally validated ChIP-Grade Antibody Kits, so you do not have to go through the antibody screening process. If your antibody of interest is not on the list of our ChIP-Grade Antibody Kits, our One-Day ChIP kit enables you a much easier and quicker screening for the antibodies that work on your ChIP assays.
Q. Should I use monoclonal or polyclonal antibodies in ChIP assay?
A. Both monoclonal and polyclonal antibodies have been used in ChIP. In general, monoclonal antibodies give lower background signals than polyclonal antibodies. However, monoclonal antibodies may not be suitable for X-ChIP. The epitope may have become masked during cross-linking thus preventing epitope recognition. We would suggest using polyclonal antibodies that will recognize multiple epitopes such that there is an increased chance of immunoprecipitating the protein of interest.
Q: Can I use your ChIP system on RNA study?
A: No. Our ChIP system is designed specifically for DNA/protein interaction. However, there are ChIP systems to target RNA in the literature. If the demand for RNA ChIP is high, our R&D group will develop ChIP products for RNA use.
Q: How do you design your primers? Do they work with SB GR?
A: ChampionChIP Real-Time PCR Primers are designed to detect specific genomic DNA sequences within a 30 kb promoter region surrounding every transcription start site (TSS) in the RefSeq database. They are optimized and extensively validated with SBGR master mix using both genomic DNA and real ChIP samples. ChIP PCR Primers meet all the essential requirements for accurate and reliable delta-delta Ct calculations for absolute fractional enrichment of a target nuclear factor at a specific promoter site as well as relative fold-change differences across multiple samples. For details on these calculations and on interpreting the results, see our ChIP-qPCR Assay Data Analysis Template.
Q: The primers I used show very high Ct. Are there any solutions?
A: It is not reliable to interpret your results when the primers show high Ct, especially over 34, in all three fractions: input, specific antibody and control IgG. That is because 1). Error occurs with high Ct; 2) The PCR with high Ct might not produce the real amplicon. It could be just the background noise. With our ChIP PCR Primers, you should see Ct at 24-30 at least in the input fraction with recommended ChIP starting material. In addition, our ChIP PCR Primers meet all the essential requirements for accurate and reliable delta-delta Ct calculations using real ChIP DNA samples. Also they are available within a 30 kb promoter region around every transcription start site (TSS) in the RefSeq database.
Q: Can I purchase only a few primers from SABiosciences?
A: Of course. You can purchase specific primers, or pathway-focused panel primers or custom-requested primers.
Q: How can I normalize my ChIP results?
A: There are many normalization methods available in the literature. SABiosciences adopt a very stringent normalization method to ensure the accurate analysis of your results. Our data analysis tool (ChampionChIP™ qPCR Data Analysis Excel File) can be found on the following website: http://www.sabiosciences.com/chipqpcr.php
Q: Can I use your One-Day kit with transcription factors?
A: Yes, but make sure that your antibodies are suitable for ChIP assay. And generally, you might want to increase the fixation time to insure the maximal cross-linking. Our system has been tested in house using at least two different transcription factors, p53 and NF-kappa B.
Q: How can I identify the binding sites of my transcription factor using ChIP?
A: First of all, the actual binding site for a specific TF might be known already in the literature. If so, the primer can be designed based on that information and ChIP-qPCR can be performed to detect the enrichment of the region by TF. If binding information for the TF is not known, ChIP-tiling assay allows sensitive analysis of up to 30kb around the promoter region if the TF of interest is known to bind to a specific promoter region. If nothing is known about the TF, ChIP-chip or ChIP-seq are recommended to discover unknown binding sites. When using these techniques, ChIP-qPCR is recommended to validate your results.
Q: At what point can I stop and freeze my samples?
A: There are several points at which the procedure may be put on hold. A) After pelleting cells following formaldehyde fixation and washes, you can freeze the cell pellet at -80° C. B) After making the lysate, you can freeze the lysate at -80° C. C) After sonication, you can store the chromatin in aliquot and store at -80° C. D) After DNA isolation prior to DNA purification, you may store DNA at -20° C or 4 ° C.
Q: How is the "150bp region" designed for EpiTect ChIP primer designs?
A: EpiTect ChIP 1-kb tiles are defined around each transcription start site from 20 kb upstream to 10 kb downstream of each site. A real-time PCR assay is designed for each tile. The size of the amplicon will vary, and they will be in a 150-bp range, which is optimal for real-time amplification.
Optimal real-time PCR designs at the center of the tile, but the final assay design may not necessarily be in the center depending on what if any sequences in the tile are optimal for real-time PCR. Neither the tile design nor the assay design is based on any knowledge of the precise location of any protein binding sites on the DNA, but on how PCR-based detection of chromatin immunoprecipitation works.
These binding sites have not all been determined and could be located anywhere in the promoter region. Therefore, it is not possible to make any assumptions about binding site locations in our genome-wide design strategy.
In the chromatin immunoprecipitation, the chromatin DNA is sheared into an average size range of 500-1000 bp, particularly in the protocol that is recommended for use with the EpiTect ChIP qPCR assays. For detection, the binding site and the real-time PCR amplicon simply need to be on the same piece of sheared DNA. The real-time PCR assay amplicon does not need to contain the binding site of interest. The assay system was designed to ensure that there is as an assay within 500 bp of any possible binding site of interest, maximizing the likelihood of co-existence on as many sheared DAN molecules as possible and maximizing sensitivity.