Effect of Long-Term HT on Telomere Length 36B4 did. Whole blood samples were collected by veni- puncture and stored in EDTA-containing tubes. Genomic DNA was extracted from white blood cells by phenol/chloroform extraction using a standard protocol. All DNA samples were di- luted to the same concentration (based on ab- sorbance) and stored at -80 until time of use. Real time PCR was performed using a Light Cycler (Roche, Mannheim, Germany). The rate of accumulation of amplified DNA was measured by continuous monitoring of SYBR Green I (Mole- cular Probes, Eugene, Oregon, USA) as fluorescent dye. Quantitative values were obtained from the threshold cycle value (CT) at which a signal in- crease associated with exponential growth of PCR products was detected using Light Cycler analysis software. Twelve microliters of the genomic DNA were used to obtain the quantitative PCR in a final volume of 20 μL. The concentrations of re- agents for telomere PCRs were 20 mM Tris-HCl pH 8.4, 50 mM KCl, 200 μM each dNTP, 1% DMSO, 2.5 mM DTT, 0.4×SYBR Green I, 0.08 U Platinum Taq Polymerase (Invitrogen, Carlsbad, California, USA), 1.5 mM MgCl2, and 300 nM of each telomere primer. The concentrations of reagents for β-globin PCRs were 20 mM Tris-HCl pH 8.4, 50 mM KCl, 200 μM each dNTP, 1% DMSO, 2.5 mM DTT, 0.4×SYBR Green I, 0.05 U Platinum Taq Polymerase, 3.5 mM MgCl2, 300 nM β-globin primer 1, and 500 nM β-globin primer 2. Telomere primer sequences were 5'-CGGTTTGT TTGGGTTTGGGTTTGGGTTT-GGGTTTGGGTT-3' and 5'-GGCTTGCCTTACCCTTACCCTTACCCTT ACCCTTA-CCCT-3'. β-globin primer sequences were 5'-GCTTCTGACACAACTGTGTTCACTAGC-3' and 5'-CACCAACTTCATCCACGTTCACC-3'. The thermal cycling conditions for both amplicons be- gan with two-minute incubation at 94 to activate the Platinum Taq DNA polymerase and denature the DNA. For telomere PCRs, 30 cycles were con- ducted for 15 sec at 95 and for one minute at 56 . Thirty-five cycles of 15 sec at 95 , 20 sec at 56 , and 20 sec at 72 were conducted for β-glo- bin PCR. At the end of the amplification cycles, a melting temperature analysis was performed using a slow increase in temperature (0.1 /sec, up to 95 ). Each sample was analyzed using dup- licates, and mean values were used for the study. For the inter-assay coefficient of variation (CV), nine DNA samples with the same concentration were tested on three different days: inter-assay CV for telomere and β-globin PCR were 1.5 ± 0.7% and 1.3 ± 0.9%, respectively. Nine different allo- cations for intra-assay CV were tested in triplicate with one DNA sample in the same plate: intra- assay CV for telomere and β-globin PCR were 1.0 ± 0.7% and 1.0 ± 1.1%, respectively. Calculation of relative T/ S ratio In order to reduce the inter-assay variation, the same number of HT and non HT DNA were allocated with reference DNA in each plate. Then the CT values of both telomere and β-globin were measured using quantitative PCR in each plate, and the T/S ratio and relative T/S ratio were calculated as follows: Total amount of telomere in a DNA sample= 2-CT (telomere) Total amount of β globulin in a DNA sample =2-CT (β-globin) T/S ratio in a DNA sample={2 CT (telomere)/2CT (β- globin)}-1=2-Δ CT The relative T/S ratio is the T/S ratio of one sample relative to the T/S ratio of a reference gene from a healthy young female, and this ratio was calculated as follows: Relative T/S ratio of study subjects={2Δ CT (telo- mere)/2Δ CT (reference gene)}-1=2-ΔΔCT. Mean value of Δ CT for a reference gene was 12.73 ± 0.50. Agarose gel electrophoresis of the telomere PCR product After 30 cycles of telomere PCR, each 20 μL of the amplified product was analyzed by electro- phoresis using 4% metaphor agarose gels running for one hr at 50 V in 45 mM tris borate and one mM EDTA with ethidium bromide. Gels were visualized under ultra-violet (UV) illumination. The PCR products were observed as a smear beginning with great intensity at 78 bp, which was the shortest possible product size, and progres- sively fading to background with larger products. This has been previously described by Cawthon19 (Fig. 1). Yonsei Med J Vol. 46, No. 4, 2005