Supplemental materials and methods
We amplified 36b4 as an endogenous control and telomere in one 384-well plate: 95 °C for 10 min, followed by 35 cycles of 15 s at 95 °C and 1 min 15 s at 58 °C. The quantitative real-time
PCR was performed using the ABI Prism 7900HT Sequence Detection System and ABI Prism 384well Clear Optical Reaction Plates. Each locus was amplified individually in a total volume of 10
μl, which contained 300 nM of each primer, 5 μl of ABI POWER SYBR Green Master Mix and 4 μl of DNA dilution. Fifty-six samples and two standard curves (telomere and 36b4) and nontemplate controls (each well in triplicate) were processed per plate. We measured mTL in 24 randomly chosen DNA samples in different time, the data were highly reproducible with
R 2 =0.865 (see Fig. 1).
During optimization of the assays, a dilution series (50ng/μl~0.78ng/μl) was run for both the telomere and 36b4 PCRs. We found that the linearity was poor for telomere amplification
(Telomere: R 2 =0.882, 36b4: R 2 =0.976; see Fig. 2 A&B). The first three high concentration plots were shifted from the trend line. So, we made another standard curve with a series of dilutions from 6.25ng/ul to 0.19ng/ul (R2=0.994 for both telomere and 36b4 amplifications; see Fig. 2
C&D). Samples with threshold cycle numbers that fell outside the range defined by the standard curve were redone by a different concentration to ensure the amplifications were within the linear range.
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Supplementary Figure 1. Reproducibility of measurement of mean telomere length with RT-
PCR.
24 samples were assayed in triplicate on consecutive days. They were highly correlated
(R 2 =0.865).
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C
A
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D
B

Supplementary Figure. 2. Standard curves of a series of dilution (left: telomere, right: 36b4).
For A and B, seven concentrations range from 50 ng/μl to 0.78 ng/μl were produced by several dilutions of two-fold change. The first three high concentration plots deviated from the line for telomere amplification. For C and D, standard curve was generated by amplification of DNA dilutions (6.25 ng/μl ~ 0.19 ng/μl). The R 2 of both telomere and 36b4 was 0.994.
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