A simplified method to calculate telomere length from Southern blot images of terminal restriction fragment lengths

Abstract

Southern blotting of DNA terminal restriction fragment lengths is the gold standard for measuring mean telomere length. Analysis of the final image is a crucial step in this process, however, current techniques are cumbersome and prone to error. Here we present a simple and accurate method for analyzing telomere smears. Basic 2D gel imaging software was used to automatically subtract background, generate standard curves and calculate net intensity and MW at each position (i) along the telomere smear. Our method required no statistical software or major data manipulation and correctly classified >80% of 18 samples as having short, medium or long telomeres compared with 33–72% using other methods.

METHOD SUMMARY

Here we demonstrate a new method of analysis to calculate mean telomere length from Southern blot images of DNA terminal restriction fragment lengths using basic imaging software. ImageQuant was used to automatically subtract background, generate standard curves and set MW across the width of the image. A series of 31–60 small (5 pixel) boxes were manually inserted along the length of each telomere smear to enable the software to calculate net intensity and MW at each position (i). Then, the data were batch exported into Microsoft Excel for final calculation of mean telomere length.