Jodczyk Sarah, Pearson John F, Aitchison Alan, Miller Allison L, Hampton Mark B, Kennedy Martin A
1 Gene Structure and Function Laboratory, Department of Pathology, University of Otago , Christchurch, Christchurch, New Zealand .
Genet Test Mol Biomarkers. 2015 Feb;19(2):63-8. doi: 10.1089/gtmb.2014.0208. Epub 2014 Dec 23.
The average length of telomeres as measured in genomic DNA from human peripheral blood leukocytes is proving to be a potential biomarker of great interest, particularly with respect to studies of aging, specific diseases, and the effects of various stresses on overall health.
The aim of this study was to establish an effective real-time quantitative polymerase chain reaction (qPCR) method for telomere length measurement on the Roche LightCycler® 480 (LC480) real-time PCR platform.
Measurement of relative average telomere length was achieved by comparing products amplified from telomere-specific primers and single copy reference gene primers in a ratio (T/S).
Extensive testing led us to conclude that a modification of the original two-plate T/S assay was more compatible with this platform than the recently developed single-plate assay, and that choice of hot-start Taq polymerase and intercalating dye were critical factors.
This modified assay generates reliable measurements as judged by correlation with data derived by the telomeric restriction fragment Southern blot-based method.
事实证明，人类外周血白细胞基因组DNA中测得的端粒平均长度是一个极具潜在价值的生物标志物，特别是在衰老、特定疾病以及各种应激对整体健康影响的研究方面。
本研究的目的是在罗氏LightCycler® 480（LC480）实时PCR平台上建立一种有效的实时定量聚合酶链反应（qPCR）方法来测量端粒长度。
通过比较从端粒特异性引物和单拷贝参考基因引物扩增的产物的比例（T/S）来实现相对平均端粒长度的测量。
大量测试使我们得出结论，与最近开发的单板检测法相比，对原始双板T/S检测法进行改进后更适合该平台，并且热启动Taq聚合酶和嵌入染料的选择是关键因素。
通过与基于端粒限制性片段Southern印迹法获得的数据进行相关性判断，这种改进后的检测法能产生可靠的测量结果。
