Why is there a special database for ChIP primers?
Designing primers for ChIP-qPCR is significantly more challenging as for other qPCR methods for multiple reasons: 1) Intron-spanning primers are used to enhance specificity in qRT-PCR reactions, but as the template in ChIP is genomic DNA, this is not possible. 2) The quality of the DNA is not as good. ChIP requires chemical crosslinking of the DNA and proteins, which is not 100% reversed downstream. Further, the commonly used mechanical shearing by ultrasound damages the DNA. 3) Quantities of available DNA are very low, frequently around 5 ng or below. 4) In contrast to qRT-PCR experiments, where many regions of the transcript may be used to amplify, ChIP primers have to target very specific regions, limiting the options for primer design. For broad marks like histones, primers specific for promoter regions close to the transcription start site (TSS) of genes are mostly suitable for other histone ChIP experiments too, but for proteins with narrow binding regions, like transcription factors, primers verified in one promoter region might not work efficiently in other ChIP experiments, as the enriched regions are too far apart.
How was the quality of these primers determined?
All primers listed are published in peer-reviewed manuscripts and a reported >5-fold change over a valid control. A direct link to the publication reporting the primer is provided for each entry and we encourage the user to check the presented result themselves to assess whether the primer fits their needs.
How do I perform searches in the database and download primer information?
Keywords can be entered in the search field and the primer list will update in real time. By default, the search will be conducted in all database fields. Refined searches, e.g. for antibodies or genes, can be performed by clicking on the "Advanced Search" button. More details on the search results can be displayed by clicking on the "Show primers" button on the entry of interest, which will display the primer sequences, the antibody used for the ChIP experiment, the Sample (which cell line or tissue), notes on the experiment where applicable, and the PMID of the publication listing the primers. The PMID is a direct link that can be clicked to open a new browser window for the respective publication. Further, primer entries can be downloaded by clicking the "Download" button, which will provide a .csv file that can be viewed in e.g. Microsoft Excel.
How do I use the primers listed?
We suggest the original publication for the primer in question is consulted first, to ensure that the primers are suitable for the specific need. Second, it is always recommended to verify primers in-house before use, as the melting temperature (Tm) varies depending on the type of polymerase, the amount of primers used, and the presence of salt in the PCR reaction. The database provides a Tm estimate for both primers, calculated for 50 nM primers and 50 mM Salt (Na+). Calculations were performed with OligoCalc, which can be accessed here http://biotools.nubic.northwestern.edu/OligoCalc.html. As a general starting point for optimizations an annealing temperature of 5°C below the Tm is recommended. To ensure target specificity we further suggest running an agarose gel to verify that only one product has been amplified, followed by Sanger sequencing.
How do I submit primers to the ChIPprimers database?
Primers may be submitted by clicking on the "Primer Submission" menu item. All submissions will be checked by the site admin before being listed. Primers have to be previously published in a peer-reviewed journal and show a >5-fold enrichment over a valid control.
Why submit primers to ChIPprimersDB?
Designing and verifying ChIP primers is more time intensive than developing primers for other applications. By listing your primers in this database you will not only help the research community, but we further encourage users to cite the publications that listed the primers they are using.
Who can I contact with additional questions or inquiries?
For any questions please contact the site administrator by email: Stefan.Kurtenbach@med.miami.edu