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Page 4 of 15 Zhou et al. J Cancer Metastasis Treat 2018;4:41 I http://dx.doi.org/10.20517/2394-4722.2018.16

A B

C

Figure 1. Increase of trisomy-7 cell percentage and Chr7-defined cell diversity in higher grade of glioma. (A) Chr7-subpopulations in
gliomas of WHO grades II (Oligodendroglioma, OG), III (anaplastic oligodendroglioma, AO), and IV (glioblatoma multiforme, GBM),
determined by FISH. Cells with 4 copies of Chr7 were considered as tetraploid cells with 2-Chr7; (B) shannon diversity index (H)
calculated based on the percentage of cells in four groups shown in A; (C) comparison of copy number variation (CNV) of EGFR and
PTEN in gliomas by CQ-PCR analysis of DNA samples. Bar height and error bar are mean and SEM of individual tumors

Real-time comparative quantitative polymerase chain reaction
DNA samples from frozen glioma specimens were isolated using a DNeasy kit (QIAGEN, Valencia, CA).
Comparative quantitative polymerase chain reaction (CQ-PCR) standards (CQ101 for EGFR and CQ102 for
PTEN) and PCR primers of EGFR, PTEN, and three reference genes on 2q34 (SPAG16), 3p14.3 (ERC2), and 5q31.2
(SPOCK1) were from Ziren Research LLC (Irvine, CA). It is a recombinant DNA containing PCR fragments
of EGFR or PTEN and reference genes in one piece to determine copy number variation (CNV) as described
previously . Real-time PCR was carried out using FAST-START SYBR-Green I Master Mix (Roche).
[33]

Statistical analysis
Two tailed T-tests with equal sample variation were performed to measure significance on pairwise
comparisons, with P < 0.05, 0.01, and 0.001 are shown in presented figures.

RESULTS
Increase of trisomy-7 and Chr7-defined cell diversity in higher grades of glioma
Using FISH analysis with dual probes for centromere 7 and EGFR, our prior studies showed common
alterations of Chr7, both in number and structure in established glioblastoma multiforme (GBM) derived
cell lines of U251, A172, LN229, and T98G, and an increasing Chr7 score (average copy of Chr7 per cell) along
[13]
with increase in the grade of human gliomas . Here we re-analyzed the FISH data on these gliomas, and
showed a significantly higher percentage of cells with 3 copies of Chr7 in GBM (grade 4, n = 16) compared
to oligodendroglioma (OG, grade 2, n = 12) [Figure 1A]. The Shannon diversity index (H) was calculated