Telomere Length Determination by qPCR

Telomere length was determined using real-time PCR (Cawthon, 2002 (link); O'Callaghan et al, 2008 ) with minor modifications. Two PCRs were performed for each sample, one to determine the cycle threshold (Ct) value for telomere (T) amplification and the other to determine the Ct value for the amplification of a single-copy (S) control gene (acidic ribosomal protein P0, RPLP0). The primer sequences for telomere amplification were TEL1B 5′-CGGTTTGTTTGGGTTTGGGTTTGGGTTTGGGTTTGGGTT-3′ and TEL2B 5′-GGCTTGCCTTACCCTTACCCTTACCCTTACCCTTACCCT-3′ (O'Callaghan et al, 2008 ) and those for RPLP0 amplification were RPLP01 5′-CAGCAAGTGGGAAGGTGTAATCC-3′ and RPLP02 5′-CCCATTCTATCATCAACGGGTACAA-3′ (Boulay et al, 1999 (link)). Each PCR reaction was performed using a 10 μl sample (1 ng of DNA per μl) and a 40 μl mixture containing 1.25 U AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 150 nM 6-ROX, 0.2 × SYBRGreen I nucleic acid stain 10 000 × (Invitrogen, Milan, Italy), 50 mM KCl, 2 mM MgCl₂, 0.2 mM of each deoxynucleoside triphosphate (Applied Biosystems), 5 mM dithiothreitol, 1% dimethyl sulphoxide, and 15 mM Tris–HCl pH 8.0, as well as primer pair TEL1B (300 nM) and TEL2B (900 nM) or primer pair RPL01 (300 nM) and RPL02 (500 nM). A reference curve was generated at each PCR run, consisting of reference DNA from the RAJI cell line (Nishikura et al, 1985 (link)) serially diluted from 10 to 0.41 ng μl⁻¹. All real-time PCR reactions were carried out using the ABI Prism 7900 HT Sequence Detection System (Applied Biosystems). Telomere and RPLP0 sequences were amplified using the following conditions: 95°C for 10 min to activate the AmpliTaq Gold DNA polymerase, and then 25 cycles each at 95°C for 15 s and 54°C for 2 min for telomere; 40 cycles each at 95°C for 15 s and 58°C for 1 min for RPLP0. ABI Prism software version 2.3 was used for analysis. Intra- and inter-assay reproducibility of both telomere and RPL0 PCR results was evaluated initially in a series of experiments using dilutions of the reference curve. The s.d. of Ct values was ⩽0.189 (% coefficient of variation ⩽1.13) in six replicates of samples amplified in the same PCR run, and ⩽0.251 (% coefficient of variation ⩽1.58) among mean values of triplicates in different PCR runs. Both reference and sample DNAs were analysed in duplicate. Variation of Ct values in the sample was ⩽0.3 Ct (s.d. ⩽0.212; % coefficient of variation ⩽1.25) in both telomere and RPL0 PCR runs. Mean Ct values were used to calculate the relative telomere length using the telomere/single-copy-gene ratio (T/S) according to the formula: ΔCt_sample=Ct_telomere−Ct_control, ΔΔCt=ΔCt_sample−ΔCt_{reference curve} (where ΔCt_{reference curve}=Ct_telomere−Ct_control ) and then T/S=2^−ΔΔCt (Cawthon 2002 (link); O'Callaghan et al, 2008 ).