Impartial comparative analysis of measurement of leukocyte telomere length/DNA content by Southern blots and qPCR

Abstract

Telomere length/DNA content has been measured in epidemiological/clinical settings with the goal of testing a host of hypotheses related to the biology of human aging, but often the conclusions of these studies have been inconsistent. These inconsistencies may stem from various reasons, including the use of different telomere length measurement techniques. Here, we report the first impartial evaluation of measurements of leukocyte telomere length by Southern blot of the terminal restriction fragments and quantitative PCR (qPCR) of telomere DNA content, expressed as the ratio of telomeric product (T)/single copy gene (S) product. Blind measurements on the same samples from 50 donors were performed in two independent laboratories on two different occasions. Both the qPCR and Southern blots displayed highly reproducible results as shown by r values > 0.9 for the correlations between results obtained by either method on two occasions. The inter-assay CV measurement for the qPCR was 6.45%, while that of the Southern blots was 1.74%. The relation between the results generated by Southern blots versus those generated by qPCR deviated from linearity. We discuss the ramifications of these findings with regard to measurements of telomere length/DNA content in epidemiological/clinical circumstances.

Genetic and Environmental Effects on Telomere Length and Lung Function: A Twin Study

“…A comprehensive sample of subjects from the Finnish twin cohort study was recruited and all the measurements were carefully performed according to internationally standardized practices at the same university laboratory. Some criticism has been leveled at the qPCR method used in this study, although it is widely used and has good repeatability (40). The study sample is representative of Finnish older women aged 65 to 70 year old, the majority of whom were non-smokers, and therefore the results cannot be generalized to other populations.…”

Assessing the scholarly impact of health psychology: A citation analysis of articles published from 1993 to 2003.

“…Telomere length is presented in terms of base pairs. This method of LTL measurement is reliable and comparable to alternative methods, such as southern blot [31].…”

Leukocyte telomere length and age at menopause

“…In contrast, we assessed telomere length by using the Southern blot method, which provides a direct estimate of the average telomere length within a population of cells. 40,41 It is unknown whether a difference in the two methods of measuring leukocyte telomere length might explain the discrepancy. The Southern blot method is considered the gold standard, 42 and qPCR has a coefficient of variation nearly four times that of the Southern blot method, 41 which may partially explain the discrepant results.…”

“…40,41 It is unknown whether a difference in the two methods of measuring leukocyte telomere length might explain the discrepancy. The Southern blot method is considered the gold standard, 42 and qPCR has a coefficient of variation nearly four times that of the Southern blot method, 41 which may partially explain the discrepant results. In one of the two studies (a breast cancer case-control study 39 ), the inclusion of subjects having had previous hysterectomy and oophorectomy could also have biased the results toward the null.…”