Quantitative analysis of chromosome conformation capture assays (3C-qPCR) View Full Text


Ontology type: schema:ScholarlyArticle      Open Access: True


Article Info

DATE

2007-07

AUTHORS

Hélène Hagège, Petra Klous, Caroline Braem, Erik Splinter, Job Dekker, Guy Cathala, Wouter de Laat, Thierry Forné

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. More... »

PAGES

1722-1733

Identifiers

URI

http://scigraph.springernature.com/pub.10.1038/nprot.2007.243

DOI

http://dx.doi.org/10.1038/nprot.2007.243

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1021403754

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/17641637


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