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

作者：Jasmine Farzana; Shinkle Justin; Sabarinathan Mekala; Ahsan Habibul; Pierce Brandon L; Kibriya Muhammad G^*

来源：American Journal of Human Biology, 2018, 30(4): e23118.

DOI：10.1002/ajhb.23118

ObjectivesRelative 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.
MethodsWe 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.
ResultsThe 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 x 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.
ConclusionWe describe a novel high throughput pooled-sample multiplex Luminex assay for RTL with good to excellent precision suitable for large-scale studies.

• 出版日期2018-8