Telomere length measurement by a novel monochrome multiplex quantitative PCR method

TLDR

Current QPCR telomere length assays use separate wells for telomere and single‑copy gene signals, making the measurement sensitive to DNA pipetting variation, while multiplexing would reduce this variability, increase throughput, and lower costs. This study introduces the first multiplexed QPCR method for telomere length measurement. The method employs a single fluorescent DNA‑intercalating dye, capturing telomere signals in early cycles before single‑copy gene signals rise, and then collecting single‑copy gene signals at a high temperature that melts the telomere product to baseline. The monochrome multiplex QPCR method shows a stronger correlation with Southern blot TRF lengths (R² = 0.844 versus 0.677 for the single‑plex assay) and yields highly reproducible results across days (R² = 0.91).

Abstract

The current quantitative polymerase chain reaction (QPCR) assay of telomere length measures telomere (T) signals in experimental DNA samples in one set of reaction wells, and single copy gene (S) signals in separate wells, in comparison to a reference DNA, to yield relative T/S ratios that are proportional to average telomere length. Multiplexing this assay is desirable, because variation in the amount of DNA pipetted would no longer contribute to variation in T/S, since T and S would be collected within each reaction, from the same input DNA. Multiplexing also increases throughput and lowers costs, since half as many reactions are needed. Here, we present the first multiplexed QPCR method for telomere length measurement. Remarkably, a single fluorescent DNA-intercalating dye is sufficient in this system, because T signals can be collected in early cycles, before S signals rise above baseline, and S signals can be collected at a temperature that fully melts the telomere product, sending its signal to baseline. The correlation of T/S ratios with Terminal Restriction Fragment (TRF) lengths measured by Southern blot was stronger with this monochrome multiplex QPCR method (R2 = 0.844) than with our original singleplex method (R2 = 0.677). Multiplex T/S results from independent runs on different days were highly reproducible (R2 = 0.91).