A New Accurate and Reproducible Method to Assess Telomere Length by Flow-FISH.

Telomeres have been related to many diseases including cancer, medullar insufficiency and to understand aging. To measure the average length of telomere repeats have been described at least three methods as Southern blot and quantitative fluorescence in situ hybridization using either digital fluorescence microscopy (Q-FISH) or flow cytometry (Flow-FISH). After two independents groups (Hultdin et al., 1998 and Rufer et al., 1999) have described different protocols using Flow-FISH to measure telomere lengths, the Southern blot and Q-FISH methods have been less used. However, when we were performed Hultdin et al., 1998 or Rufer et al., 1999 protocols were found some facts that could cause mistakes in the final results. Afterward, many steps of these protocols were explored to become the Flow-FISH results more accurate and reproducible. In our laboratory we developed a unlike protocol to assess the average length of telomere repeats by Flow-FISH taking advantage of the both Hultdin et al., 1998 and Rufer et al., 1999 techniques that was named Modified Hultdin and Rufer protocol.

We compare the telomere length repeats measure by three different methods of Flow-FISH with or lacking standard beads for calibrate fluorescence intensity, control cell line 1301 and compensation by DNA index of G0/G1 cells.

We demonstrated a linear relationship between the average length of telomere repeats by Hultdin et al., 1998 protocol and Modified Hultdin and Rufer protocol but showing that standard beads to quantify the fluorescence intensity was important to determine telomere length by Flow-FISH. However, the coefficient correlation (r) between the Rufer et al., 1999 protocol with Hultdin et al., 1998 and Modified Hultdin and Rufer protocols in our laboratory was poor, justifying of the internal control cell and compensation of the telomere length by DNA index of G0/G1 of unknown cells use.

We conclude that use of appropriate internal control cells with knowledge telomere length and DNA index is essential to improve accuracy and reproducibility of the telomere length measurement by Flow-FISH. Indeed the standard beads for fluorescence intensity quantification can allow intercenters comparison of the results.

No relevant conflicts of interest to declare.