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Fig. 2 | Acta Neuropathologica Communications

Fig. 2

From: Detection of Histone H3 mutations in cerebrospinal fluid-derived tumor DNA from children with diffuse midline glioma

Fig. 2

Selection of Precipitation Carriers and Mutation-Specific Primers. a The quantity and quality of DNA extracted from CSF using carrier RNA (yRNA) or linear polyacrylamide (LPA) were compared using matched CSF specimens (n = 4). PCR-amplification of H3F3A in CSF-derived DNA using yRNA and LPA yielded 300 bp bands at equivalent intensity (yRNA mean intensity normalized to 1; LPA mean relative intensity = 0.99; Mann-Whitney U test, p > 0.99, band intensities analyzed with ImageJ) with gel results from two specimens shown (PID 2 and 11). No significant difference was detected in the amount of DNA recovered per microliter CSF between the two carriers (yRNA mean = 1.74 ng DNA/μL CSF; LPA mean = 1.47 ng/μL CSF; Mann-Whitney U test, p = 0.97). b Prior to primer testing, H3F3A c.83 A > T mutation status of a DIPG cell line SF8628 (mutant) and pediatric glioblastoma (high-grade glioma, HGG) cell line SF9427 (wild type) was confirmed by Sanger Sequencing. Selective amplification of the mutant H3F3A allele in SF8628 was achieved using all three H3.3K27M primer pairs (Table 1)

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