A blood-based gene expression and signaling pathway analysis to differentiate between high and low grade gliomas

Ponnampalam,Stephen N.; Kamaluddin,Nor Rizan; Zakaria,Zubaidah; Matheneswaran,Vickneswaran; Ganesan,Dharmendra; Haspani,Mohammed Saffari; Ryten,Mina; Hardy,John A.

doi:10.3892/or.2016.5285

A blood-based gene expression and signaling pathway analysis to differentiate between high and low grade gliomas

Authors:
- Stephen N. Ponnampalam
- Nor Rizan Kamaluddin
- Zubaidah Zakaria
- Vickneswaran Matheneswaran
- Dharmendra Ganesan
- Mohammed Saffari Haspani
- Mina Ryten
- John A. Hardy
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Affiliations: Cancer Research Center, Institute for Medical Research, Jalan Pahang, 50588 Kuala Lumpur, Malaysia, Department of Neurosurgery, University Malaya Medical Centre, Jalan Universiti, 50603 Kuala Lumpur, Malaysia, Department of Neurosurgery, Hospital Kuala Lumpur, Jalan Pahang, 50586 Kuala Lumpur, Malaysia, Department of Molecular Neuroscience, Institute of Neurology, University College London, Queen Square, London WC1N 3BG, UK
Published online on: November 29, 2016 https://doi.org/10.3892/or.2016.5285
Pages: 10-22
Copyright: © Ponnampalam et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

The aims of the present study were to undertake gene expression profiling of the blood of glioma patients to determine key genetic components of signaling pathways and to develop a panel of genes that could be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and control samples. In this study, blood samples were obtained from glioma patients, non-glioma and control subjects. Ten samples each were obtained from patients with high and low grade tumours, respectively, ten samples from non-glioma patients and twenty samples from control subjects. Total RNA was isolated from each sample after which first and second strand synthesis was performed. The resulting cRNA was then hybridized with the Agilent Whole Human Genome (4x44K) microarray chip according to the manufacturer's instructions. Universal Human Reference RNA and samples were labeled with Cy3 CTP and Cy5 CTP, respectively. Microarray data were analyzed by the Agilent Gene Spring 12.1V software using stringent criteria which included at least a 2-fold difference in gene expression between samples. Statistical analysis was performed using the unpaired Student's t-test with a p<0.01. Pathway enrichment was also performed, with key genes selected for validation using droplet digital polymerase chain reaction (ddPCR). The gene expression profiling indicated that were a substantial number of genes that were differentially expressed with more than a 2-fold change (p<0.01) between each of the four different conditions. We selected key genes within significant pathways that were analyzed through pathway enrichment. These key genes included regulators of cell proliferation, transcription factors, cytokines and tumour suppressor genes. In the present study, we showed that key genes involved in significant and well established pathways, could possibly be used as a potential blood-based biomarker to differentiate between high and low grade gliomas, non-gliomas and control samples.

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