Method for single cell analysis of telomere length using pre-amplification and multiple-copy reference sequence

A method for determining relative telomere length in a biological sample, which sample comprises less than 20 ng of genomic DNA, said method comprising:

• (a) performing a multiplex pre-amplification of DNA contained in the sample for 12-20 cycles using, in a single reaction mixture, a first primer pair specific for a telomere sequence and a second primer pair specific for a reference sequence, wherein the reference sequence is present in multiple copies in the genome,
• (b) performing a qPCR reaction on the products of step (a) using the first and the second primer pairs, wherein such primer pairs are the same as in step (a) and are present in a single or separate qPCR reaction mixtures,
• (c) calculating a T/R ratio as the ratio of (i) the amount of qPCR product obtained in step (b) using the first primer pair specific for the telomere sequence (T) and (ii) the amount of qPCR product obtained in step (b) using the second primer pair specific for the reference sequence (R), and
• (d) determining the relative telomere length for the sample based on the T/R ratio calculated in step (c), wherein the T/R ratio of more than a standard T/R ratio indicates the relative telomere length greater than normal and the T/R ratio of less than the standard T/R ratio indicates the relative telomere length shorter than normal, wherein the standard T/R ratio is (i) a predetermined value, or (ii) an average T/R ratio for multiple corresponding normal cells, tissues or individuals of the same species, or (iii) a standard curve of T/R ratios for multiple corresponding normal cells, tissues or individuals of the same species.