Telomeres, the heterochromatic structures that protect the ends of the chromosomes, are transcribed into a class of long non-coding RNAs, telomeric repeat-containing RNAs (TERRA), whose transcriptional regulation and functions are not well understood. The identification of TERRA adds a novel level of structural and functional complexity at telomeres, opening up a new field of research. TERRA molecules are expressed at several chromosome ends with transcription starting from the subtelomeric DNA proceeding into the telomeric tracts. TERRA is heterogeneous in length and its expression is regulated during the cell cycle and upon telomere damage. Little is known about the mechanisms of regulation at the level of transcription and post transcription by RNA stability. Furthermore, it remains to be determined to what extent the regulation at different chromosome ends may differ.
Researchers from the Swiss Institute for Experimental Cancer Research present an overview on the methodology of how RT-qPCR and primer pairs that are specific for different subtelomeric sequences can be used to detect and quantify TERRA expressed from different chromosome ends.
Schematic representation of TERRA RT-qPCR assay
TERRA synthesis, which is mainly mediated by RNA polymerase II, derives from the subtelomeric region (purple) and extends towards the end of the chromosome into the telomeric repeats (red). A TERRA-specific oligonucleotide comprised of telomeric sequence will anneal and reverse transcribe TERRA from numerous positions in the telomeric tract (red), generating a pool of cDNA molecules with diverse subtelomeric sequences (purple) and length. In quantitative PCR specific primer pairs (purple arrows) amplify the desired subtelomeric sequence from the pool of transcribed TERRA molecules. Fluorescence intercalating dyes bind the double-stranded amplicon, which is detected and analyzed by the RT-qPCR system.