An Optimised Step-by-Step Protocol for Measuring Relative Telomere Length

Abstract

Telomeres represent the nucleotide repeat sequences at the ends of chromosomes and are essential for chromosome stability. They can shorten at each round of DNA replication mainly because of incomplete DNA synthesis of the lagging strand. Reduced relative telomere length is associated with aging and a range of disease states. Different methods such as terminal restriction fragment analysis, real-time quantitative PCR (qPCR) and fluorescence in situ hybridization are available to measure telomere length; however, the qPCR-based method is commonly used for large population-based studies. There are multiple variations across qPCR-based methods, including the choice of the single-copy gene, primer sequences, reagents, and data analysis methods in the different reported studies so far. Here, we provide a detailed step-by-step protocol that we have optimized and successfully tested in the hands of other users. This protocol will help researchers interested in measuring relative telomere lengths in cells or across larger clinical cohort/study samples to determine associations of telomere length with health and disease.

Keywords: DNA; Telomeres; cells; real-time PCR; relative telomere length.

Figure 1

PCR plate map. A template of 96-well plate for telomere PCR that accommodates triplicates of no template control (NTC), 30 samples, and Control DNA (C) in different positions as indicated. Exact same PCR is setup for single-copy gene (human β-globin) with identical well positions for NTC, samples, and Control DNA.

Figure 2

Details of the PCR setup. Detailed information of the PCR setup on the ViiA7 instrument with telomere primers (A; annealing at 56 °C) and human β-globin primers (B; annealing at 58 °C) is provided. Cycling temperatures with time, number of cycles, as well as ramping rates are clearly indicated here. A melt curve analysis is added at the end of 40 cycles of PCR to ensure the absence of primer-dimers.

Figure 3

Expected PCR results. (A) representative amplification plot for three DNA concentrations performed in triplicates (100 ng/μL, green curves; 25 ng/μL, cyan curves and 6.25 ng/μL, blue curves). All replicates have almost identical cycle threshold value, and for each four-fold dilution, the cycle threshold values increase by two cycles. Representative images of the melt curve for telomere PCR products (B) and human β-globin PCR products (C).

Figure 4

Reproducibility of data: the actual cycle threshold (Ct) values for the three different dilutions (100 ng/μL, 25 ng/μL, 6.25 ng/μL) used in the qPCR reactions for telomere and human β-globin expression from three technical repeats and three biological samples (A). PCR data from the same set of samples were obtained by two different users from the same lab, and the correlation between their results are plotted for telomere (B) and the human β-globin gene (C). Reproducibility of the method across two different labs (Sydney; Blue circles and Hong Kong; Red squares) is presented as mean Ct values of the triplicates obtained after telomere qPCR (D) and human β-globin qPCR (F). Correlation plots for telomere qPCR data (E) and human β-globin qPCR data (G) performed in each lab on the same samples. B,C,E, and G show Pearson (r) correlation coefficient and p-values.