In Vitro Detection of Long Noncoding RNA Generated from DNA Double-Strand Breaks
DNA damage response (DDR) is essential for the maintenance of genomic integrity. We have recently discovered the generation of noncoding RNA from a DNA double-strand break (DSB) in an MRE11-RAD50-NBS1 complex-dependent manner, which are necessary for full DDR activation. The low abundance of these noncoding RNA makes them difficult to identify and study. In this chapter, we describe an in vitro biochemical assay to study the generation of damage-induced long noncoding RNA (dilncRNA) from a DNA DSB. In this assay, transcriptionally competent cell-free extracts upon incubation with a linear DNA support RNA synthesis from DNA ends, as monitored by incorporation of 32P[UTP] in discrete products resolved on a denaturing polyacrylamide gel. This approach can be used to identify the role of different DDR proteins in generating dilncRNA.
Key wordsCell-free extracts In vitro transcription Double-strand break (DSB) DNA damage response (DDR) Damage-induced long noncoding RNA (dilncRNA) Plasmid DNA
S.S., a Structured International Postdoctoral Fellow, received funding from the People Programme, (Marie Curie Actions) of the European Union’s Seventh Framework Programme FP7 under grant agreement n.600399. F.d’A.d.F. was supported by the Associazione Italiana per la Ricerca sul Cancro, AIRC (application 12971), Human Frontier Science Program (contract RGP 0014/2012), Cariplo Foundation (grant 2010.0818 and 2014-0812), Marie Curie Initial Training Networks [FP7 PEOPLE 2012 ITN (CodAge)], Fondazione Telethon (GGP12059), Association for International Cancer Research (AICR-Worldwide Cancer Research Rif. N. 14-1331), Progetti di Ricerca di Interesse Nazionale (PRIN) 2010–2011, the Italian Ministry of Education Universities and Research EPIGEN Project, European Research Council advanced grant (322726), and AriSLA (project “DDRNA and ALS”).