3C-digital PCR for quantification of chromatin interactions. Issue 1 (December 2016)

• Record Type: Journal Article
• Authors: Du, Meijun; Wang, Liang
• Is Part Of: BMC molecular biology. Volume 17:Issue 1(2016)
• Page Start: 1
• Page End: 9
• Publication Date: 2016-12
• Subjects: Chromatin interaction -- Chromosome conformation capture -- Digital PCR -- Quantitative PCR; Molecular biology -- Periodicals; 572.805
• DOI: 10.1186/s12867-016-0076-6
• Languages: English
• ISSNs: 1471-2199

Abstract

Abstract Background Chromosome conformation capture (3C) is a powerful and widely used technique for detecting the physical interactions between chromatin regions in vivo. The principle of 3C is to convert physical chromatin interactions into specific DNA ligation products, which are then detected by quantitative polymerase chain reaction (qPCR). However, 3C-qPCR assays are often complicated by the necessity of normalization controls to correct for amplification biases. In addition, qPCR is often limited to a certain cycle number, making it difficult to detect fragment ligations with low frequency. Recently, digital PCR (dPCR) technology has become available, which allows for highly sensitive nucleic acid quantification. Main advantage of dPCR is its high precision of absolute nucleic acid quantification without requirement of normalization controls. Results To demonstrate the utility of dPCR in quantifying chromatin interactions, we examined two prostate cancer risk loci at 8q24 and 2p11.2 for their interaction target genesMYC andCAPG in LNCaP cell line. We designed anchor and testing primers at known regulatory element fragments and target gene regions, respectively. dPCR results showed that interaction frequency between the regulatory element andMYC gene promoter was 0.7 (95% CI 0.40–1.10) copies per 1000 genome copies while other regions showed relatively low ligation frequencies. The dPCR results also showed that the ligation frequencies between the regulatory element and twoEco RI fragments containingCAPG gene promoter were 1.9 copies (95% CI 1.41–2.47) and 1.3 copies per 1000 genome copies (95% CI 0.76–1.92), respectively, while the interaction signals were reduced on either side of the promoter region ofCAPG gene. Additionally, we observed comparable results from 3C-dPCR and 3C-qPCR at 2p11.2 in another cell line (DU145). Conclusions Compared to traditional 3C-qPCR, our results show that 3C-dPCR is much simpler and more sensitive to detect weak chromatin interactions. It may eliminate multiple and complex normalization controls and provide accurate calculation of proximity-based fragment ligation frequency. Therefore, we recommend 3C-dPCR as a preferred method for sensitive detection of low frequency chromatin interactions.