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Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR

IFOM—The FIRC Institute of Molecular Oncology, 20139 Milan, Italy
CNR—Consiglio Nazionale delle Ricerche, Istituto di Genetica Molecolare, 27100 Pavia, Italy
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2020, 21(24), 9774;
Received: 30 November 2020 / Accepted: 17 December 2020 / Published: 21 December 2020
Because of their intrinsic characteristics, telomeres are genomic loci that pose significant problems during the replication of the genome. In particular, it has been observed that telomeres that are maintained in cancer cells by the alternative mechanism of the lengthening of telomeres (ALT) harbor higher levels of replicative stress compared with telomerase-positive cancer cells. R-loops are three-stranded structures formed by a DNA:RNA hybrid and a displaced ssDNA. Emerging evidence suggests that controlling the levels of R-loops at ALT telomeres is critical for telomere maintenance. In fact, on the one hand, they favor telomere recombination, but on the other, they are a source of detrimental replicative stress. DRIP (DNA:RNA immunoprecipitation) is the main technique used for the detection of R-loops, and it is based on the use of the S9.6 antibody, which recognizes preferentially DNA:RNA hybrids in a sequence-independent manner. The detection of DNA:RNA hybrids in repetitive sequences such as telomeres requires some additional precautions as a result of their repetitive nature. Here, we share an optimized protocol for the detection of telomeric DNA:RNA hybrids, and we demonstrate its application in an ALT and in a telomerase-positive cell line. We demonstrate that ALT telomeres bear higher levels of DNA:RNA hybrids, and we propose this method as a reliable way to detect them in telomeres. View Full-Text
Keywords: telomeres; DNA:RNA hybrids; DNA:RNA immunoprecipitation telomeres; DNA:RNA hybrids; DNA:RNA immunoprecipitation
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MDPI and ACS Style

Rosso, I.; d’Adda di Fagagna, F. Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR. Int. J. Mol. Sci. 2020, 21, 9774.

AMA Style

Rosso I, d’Adda di Fagagna F. Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR. International Journal of Molecular Sciences. 2020; 21(24):9774.

Chicago/Turabian Style

Rosso, Ilaria, and Fabrizio d’Adda di Fagagna. 2020. "Detection of Telomeric DNA:RNA Hybrids Using TeloDRIP-qPCR" International Journal of Molecular Sciences 21, no. 24: 9774.

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