A Synthetic Single-Stranded Dual-Template Oligonucleotide as a Reference Standard for Monochrome Multiplex qPCR Measurements of Average Telomere Length

Quantitative PCR is frequently used to measure average telomere length (TL) relative to the TL of a reference DNA sample chosen by the investigator. This makes comparisons of TLs across studies and laboratories difficult. Here we demonstrate that a single synthetic single-stranded dual-template oligonucleotide (DTO) containing both a telomere repeat sequence (T) and a segment of the human beta-globin (HBB) single copy gene (S) can be used as a universal reference standard for monochrome multiplex quantitative PCR (MMqPCR) measurements of average TL using SYBR Green I as the only fluorescent reporter dye. A set of twelve concentrations of the DTO is prepared by serial 3-fold dilutions, to a lowest concentration of approximately 20 copies per microliter. The 5 highest concentrations are used for the T standard curve, and the 5 lowest concentrations are used for the S standard curve. For each reaction 5 microliters containing approximately 3 ng of genomic DNA (or one of the DTO dilutions) is mixed with 5 microliters of a 2x MasterMix containing the primers for T and S amplification, and MMqPCR is performed. The design of the primers and thermal cycling profile allows all T amplification signals to be collected before exponential amplification of the S signal begins. Exponential amplification from S is then carried out in a temperature range that keeps the telomere product fully melted and therefore unable to influence the S amplification signal. The T value for each DNA sample is the Standard Curve DTO dilution that contains the same number of copies of the telomere sequence as the experimental sample, and the S value is the DTO dilution that contains the same number of copies of the single copy gene sequence as the experimental sample. Dividing the first dilution by the second dilution yields an absolute T/S ratio, since it is expressed relative to the fixed 1:1 T/S ratio that is built into the DTO by design. Absolute T/S ratios for average TL in 48 human DNA samples determined by this method correlated strongly with mean Terminal Restriction Fragment (mTRF) lengths for the same DNA samples determined by the Southern Blot method (R-squared = 0.801). This DTO and the accompanying protocol may facilitate the standardization of average telomere length measurements and analyses across laboratories.