Abstract C27: Evaluation of the telomere QPCR assay for use in gauging the effect of storage conditions on telomere length
Authors: Edelman Daniel C. , Tran Paul , Petersen David , Du Meng Meng , Wong Jason , De Vivo Immaculata 
Published: June 2012

Telomere length has been reported as potentially biomarker and prognostic indicator in cancer. Our goal was to evaluate whether DNA storage/handling or sample source would result in fragmented telomere DNA using an established QPCR-based telomere assay that quantifies the length of telomeres (Cawthon 2002) Because the effect of storage conditions on telomere length has not been thoroughly evaluated, we were concerned that the QPCR could misclassify telomere length and lead to misleading epidemiological results.

Genomic DNA samples for this preliminary study were gathered from either The Nurses Health Study, healthy volunteers, the MDA-MB-231 cell line (human breast cancer), and a commercial source (Promega). To create a model system of degraded DNA, the DNA were either subjected to multiple freeze thaws (FT, &lt;20), Covaris S2 sonication (&lt;6 Cycles; intensity 5/8, 200/500 bursts), or DNase I treatment (&lt;1 U/ 1 ug). The QPCR assay modified the reactants, parameters, and the thermocycler. By examining the distribution of telomeric PCR amplicons on the Agilent Bioanalyzer 2100 based upon their relative quantity and size, we sought to analyze the effects of damaging measures. Ct measurements provided data as to the relative number of telomeric targets present in the respective samples and melt peak analyses (MPA) identified the unique amplicon Tm compositions.

Due to the repetitive nature of the telomeric sequence, we expected to see discrete amplicon sizes starting at 76 Bp reaching over hundreds of Bps. Bioanalyzer electropherograms indicated that eight to twelve major products of ∼8 Bp iterations are commonly produced up to 150 Bp with some minor products &lt;200 Bp. A primer generated amplicon at ∼84 Bp, although not always present, confounded the analyses. Interestingly, MPA showed a single melt peak at ∼79C for all telomeric amplicons; the aberrant peak had a lesser Tm of ∼78C. Ct data do suggest that although the telomeres are sensitive to enzymatic assault (two–three log reduction), as many as 20 FTs and extensive sonication seem not to degrade the telomere (no Ct change). Ct values indicated that differences in telomere length exist between persons (range: 16–19); the MDA-MB-231 cell line had the shortest telomere length (Ct 22). However, neither individual samples nor samples subjected to DNase I digestion were significantly differentiated via end-point analysis on the Bioanalyzer.

In summary, under the current experimental conditions, the QPCR assay does not generate sufficient variable length amplicons as to present a measurable metric using the Bioanalyzer. Elevated Ct values do seem to correlate with the presence of degraded DNA, and therefore suggest under the conditions used multiple FTs and sonication do not damage the telomeric sequences. However this assay design cannot currently determine the relative contribution of full length vs. degraded telomeric sequence to the Ct value. Further investigation is warranted before this assay becomes clinically diagnostic or prognostic.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C27.