DNA extraction and measurement of telomere length by
quantitative real-time PCR
After isolating naive and memory T cells, the DNA was extracted using a
QIAamp RNA Mini Kit (Qiagen) according to the manufacturers’
instructions. The concentration and purity of the DNA was quantified
using a Nanodrop Spectrophotometer (ThermoFisher Scientific, Waltham,
Massachusetts, USA). The telomere length was determined by quantitative
real-time PCR adapted from Cawthon [20] . The relative telomere
length was measured as the ratio of standard DNA quantities for telomere
template (T) over single copy gene, 36B4 (S). The telomere and 36B4 gene
primers were as follows:
(Tel-forward,5’-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3’; Tel reverse,
5’-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3’) for Telomere PCR and
(36B4-forward, 5’-CAGCAAGTGGGAAGGTGTAATCC-3’; 36B4-reverse,
5’-CCCATTCTATCATCAACGGGTACAA-3’) for the 36B4 PCR. All samples were
blindly and consecutively run in triplicate together with reference
samples. The intra-assay coefficient of variation was 5%.