Abstract
Chromosome conformation capture (3C) technology is a pioneering methodology that allows in vivo genomic organization to be explored at a scale encompassing a few tens to a few hundred kilobase-pairs. Understanding the folding of the genome at this scale is particularly important in mammals where dispersed regulatory elements, like enhancers or insulators, are involved in gene regulation. 3C technology involves formaldehyde fixation of cells, followed by a polymerase chain reaction (PCR)-based analysis of the frequency with which pairs of selected DNA fragments are crosslinked in the population of cells. Accurate measurements of crosslinking frequencies require the best quantification techniques. We recently adapted the real-time TaqMan PCR technology to the analysis of 3C assays, resulting in a method that more accurately determines crosslinking frequencies than current semiquantitative 3C strategies that rely on measuring the intensity of ethidium bromide-stained PCR products separated by gel electrophoresis. Here, we provide a detailed protocol for this method, which we have named 3C-qPCR. Once preliminary controls and optimizations have been performed, the whole procedure (3C assays and quantitative analyses) can be completed in 7–9 days.
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Acknowledgements
We thank Robert Feil and Franck Court for discussion and comments on the manuscript. This work was supported by grants from the Association pour la Recherche contre le Cancer (ARC contract no. 3279), the “GIS Longévité” (contract no. GISLO401), the “Fond National de la Science” (ACI jeune chercheur) given to T. Forné and by funds from the “Centre National de la Recherche Scientifique” (CNRS). C.B. was supported by an ARC fellowship (JR/MLD/MDV—P05/2 and P06/2). W.L. was supported by grants from the Dutch Scientific Organization (NWO) (016-006-026) and (912-04-082). J.D. was supported by a grant from the NIH (HG003143).
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Hagège, H., Klous, P., Braem, C. et al. Quantitative analysis of chromosome conformation capture assays (3C-qPCR). Nat Protoc 2, 1722–1733 (2007). https://doi.org/10.1038/nprot.2007.243
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DOI: https://doi.org/10.1038/nprot.2007.243
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