A novel pooled-sample multiplex luminex assay for high-throughput measurement of relative telomere length.

ABSTRACT: OBJECTIVES:Relative telomere length (RTL) is a potential biomarker of aging and risk for chronic disease. Previously, we developed a probe-based RTL assay on Luminex platform, where probes for Telomere (T) and reference gene (R) for a given DNA sample were tested in a single well. Here, we describe a method of pooling multiple samples in one well to increase the throughput and cost-effectiveness. METHODS:We used four different microbeads for the same T-probe and four different microbeads for the same R-probe. Each pair of probe sets were hybridized to DNA in separate plates and then pooled in a single plate for all the subsequent steps. We used DNA samples from 60 independent individuals and repeated in multiple batches to test the precision. RESULTS:The precision was good to excellent with Intraclass correlation coefficient (ICC) of 0.908 (95% CI 0.856-0.942). More than 67% of the variation in the RTL could be explained by sample-to-sample variation; less than 0.1% variation was due to batch-to-batch variation and 0.3% variation was explained by bead-to-bead variation. We increased the throughput of RTL Luminex assay from 60 to 240 samples per run. The new assay was validated against the original Luminex assay without pooling (r?=?0.79, P?=?1.44 × 10-15 ). In an independent set of samples (n?=?550), the new assay showed a negative correlation of RTL with age (r?=?-0.41), a result providing external validation for the method. CONCLUSION:We describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies.

SUBMITTER: Jasmine F 

PROVIDER: S-EPMC6105449 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature