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Fig. 1 | Acta Neuropathologica Communications

Fig. 1

From: The level of activity of the alternative lengthening of telomeres correlates with patient age in IDH-mutant ATRX-loss-of-expression anaplastic astrocytomas

Fig. 1

Measurement of the level of ALT activity levels in human diffuse gliomas. Top panels illustrate the principle of the ALT C-circle assay [17] and describe its general steps. ALT cells have very long telomeres that have been amplified mainly by homologous recombination that generates partially single-stranded extra-chromosomal circles. Genomic DNA prepared from tumor samples is then incubated with the Phi29 DNA polymerase that specifically amplifies this telomeric DNA. Middle panel illustrates ALT-specific signals measured in tumor DNA samples using this assay, which were detected here on dot blots hybridized with a telomeric 32P-labeled probe. Genomic DNAs from HeLa (telomerase positive) and U2OS (ALT positive) cells were also probed, representing negative and posititve controls for the C-circle assay, respectively. These assays were systematically performed in duplicates and here dot blot 2, on the right, was loaded with the same tumor samples as dot blot 1, on the left, to insure for reproducibility. Bottom table illustrates examples of duplicate numbers obtained for each of the indicated tumors, real signals of which are represented in the middle panel above. The C-circle score was determined after calculating the intensity of the signal relative to that of the ALT positive U2OS cell line, designated to be 100 arbitrary units (AU). Note that the C-circle assays were performed on representative samples, including those from the two patient groups analyzed in the present study

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