DdTRAP: A method for sensitive and precise quantification of telomerase activity

Abstract

Telomerase is a cellular RNA template-dependent reverse transcriptase that adds telomere repeats to the 3′ ends of chromosomes. Telomerase is expressed almost universally in tumor cells (>85%) to maintain telomere length, thus providing the ability of tumor cells to avoid senescence and to have unlimited replication ability, one of the key hallmarks of cancer. ddTRAP (droplet digital Telomere Repeat Amplification Protocol) is a two-step assay with whole cell lysates that utilizes a telomerase-mediated primer extension followed by droplet digital PCR (ddPCR) detection of extended products. The adoptation of the TRAP assay to ddPCR has resulted in improved throughput, increased sensitivity and better repeatability of the TRAP assay. The protocol described below details our procedures for ddTRAP.