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Keywords: Telomere structure/function; Genomics; Methodology

Authors:
M.G. Kibriya; F. Jasmine; A. Shaikh; H. Ahsan; B.L. Pierce

Affiliation: Institute for Population and Precision Health, Public Health Sciences, Univ Chicago, Chicago, Illinois.


Background: Relative Telomere Length (RTL) is a potential biomarker of aging and chronic disease. Previously we developed a non-PCR, probe-based high throughput RTL assay on Luminex platform suitable for large-scale studies. This multiplexed assay (measuring the telomere and the reference gene from the same DNA sample in a single well) requires ~50ng of DNA. We use branched DNA technology of QuantiGene chemistry for signal amplification. The “telomere length” is measured against a standard or reference DNA sample (hence the term “relative”). In this study we examined if different source of DNA from same individual’s blood sample could affect the RTL measurement in this Luminex based assay.
Material Methods: Blood from 11 individuals were collected in different tubes - (a) K2-EDTA tube, (b) Trace element tube with EDTA, (c) Heparinized tube, and (d) serum separator SST tube with gel and clot activator. The blood in K2-EDTA tube was used to separate (1) Peripheral Blood Mononuclear Cell (PBMC) and (2) granulocyte cell population using standard Ficoll gradient for DNA extraction. DNA from Whole blood (WB) was derived from (i) trace element EDTA tube, (ii) heparinized tube and (iii) the serum separating tube (SST) with clot activator. DNA was extracted using Flexigene DNA kit (5 samples/person). Quantification was done by Nanodrop spectrophotometry. We tested a total of 55 different DNA samples from the 11 individuals. RTL assay was performed using Luminex method – each was assayed in quadruplicate in four different plates simultaneously in a single batch by same individual.
Result: The RTL measurement in DNA from PBMC or Granulocyte was not different (1.039 SD 0.169 vs 1.047 SD0.167, ANOVA p=0.911). RTL measurements of Whole Blood DNA from EDTA, Heparin or SST tubes did not show any difference (0.976 SD 0.125, 0.999 SD 0.188 and 1.011 SD 0.136; p=0.857). Regression using General Linear Model (GLM) showed that the variation in RTL measures was mainly attributed to “person-to-person” variation (69%) and the “tube-to-tube” variation contributed only 3.3% of the total variation. The precision of the assay was good to excellent with the Intraclass correlation coefficient (ICC) of 0.896 (95%CI 0.842 – 0.935) among the replicates.
Conclusion: The Luminex based multiplex assay for RTL yields similar RTL measures from an individual whole blood sample irrespective of blood collection tube (EDTA, Heparin or SST) or white blood cell type (PBMC or Granulocyte).