Repeatability of a terminal restriction fragment telomere length assay on red drum fin tissue

Abstract

Telomere length measurements are becoming increasingly popular in biology as a non-lethal tool for estimating organisms’ biological conditions. One particularly appealing application of telomere length measurements for conservation biologists is genetic age estimation. Most age estimation methods require animals to be sacrificed followed by the dissection and sectioning of calcified structures. Because telomeres degrade over time, telomere lengths may provide non-lethal and minimally invasive tool for estimating age. Furthermore, telomere length may also be useful for predicting longevity and fitness. Traditionally, telomere lengths have been measured using terminal restriction fragment (TRF) methodology; however, there is no one standard laboratory procedure to estimate the size of these fragments, and few studies supply repeatability data for the respective methods. While TRF telomere length assays are applicable to all organisms with known telomere repeat motif sequences, inconsistencies in TRF methodology include template DNA treatment, restriction enzymes used, gel electrophoresis methods, and probe/detection methods. Here we present a study documenting repeatability data for a specific laboratory procedure for estimating telomere length using two common restriction enzymes, denaturing pulsed field gel electrophoresis, and a chemiluminescent detection system for three replicates of 16 individual red drum collected in South Carolina. The data were analyzed following traditional methods using an average across the entire TRF distribution, which represents all TRF lengths found in the sample; and following non-traditional methods, where only a subsample of the TRF distributions are analyzed. Our results demonstrate a strong correlation among average TRF lengths between replicates while analyzing the entire TRF distribution; however, our results show a decreasing trend of consistency between replicates as smaller portions of the TRF distribution were analyzed. These results suggest our method provides a repeatable estimate of average TRF length in a sample, and we urge that caution should be exercised when subsampling TRF distributions for telomere length analysis. Repeatability data not only validate the assay producing the data, but also may allow researchers to develop standard optimal criteria to balance cost and extent of their study design.