Estimation of Telomeric Repeat-containing RNA from DNA/RNA Hybrid Complexes.

Telomeric repeat-containing RNA (TERRA) is a long non-coding RNA transcribed from all telomeres in higher eukaryotes, and only a subset of these molecules forms stable DNA/RNA hybrids at telomeres. We have outlined a detailed molecular protocol to identify and purify these hybrid estimates. This streamlined method enables the direct extraction of DNA/RNA hybrid interactions naturally formed in vivo and can be applied to detect all such regions. By minimizing procedural steps, this technique is highly efficient in isolating RNA attached to DNA, estimating the reliability and reproducibility of downstream sequencing. Guanidine isothiocyanate reagent is used for total cell lysis and homogenization, preserving the integrity of nucleic acids (DNA and RNA), particularly DNA/RNA hybrids. Adding chloroform initiates phase separation via centrifugation (three distinct layers): a lower phenol-chloroform phase, an interphase, and an upper aqueous phase. RNA, released from both the nucleus and cytoplasm, is retained in the aqueous phase, while high-molecular-weight DNA, including the DNA/RNA hybrid regions, is confined to the interphase. With isopropyl alcohol, followed by centrifugation, the aqueous phase (free RNA), interphase, and DNA, particularly DNA/RNA hybrids, are recovered. To remove the remaining traces of proteins, the sample is incubated with Proteinase K. Treatment with DNase, phenol/chloroform extraction, isopropyl alcohol, and centrifugation is used to release RNA hybridized to DNA.