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A novel amplification-based approach to enable gene expression profiling from small clinical tumor specimens
- Source :
- Journal of Neuro-Oncology. 126:69-75
- Publication Year :
- 2015
- Publisher :
- Springer Science and Business Media LLC, 2015.
-
Abstract
- Glioblastoma is the most common and deadly type of brain cancer. Over the past decade, several divergent genetic pathways have been implicated in the initiation, progression and clinical outcome of this disease. As our understanding of GBM expands and identifies actionable targets specific to individual tumors, there will be a pressing need for the development of new tools that will maximize the use of limited clinical samples to enable the employment of personalized care paradigms. We used PrimePCR validated assays to generate a custom real-time PCR screening panel, containing 74 previously published mRNA targets showing gene expression changes in glioblastoma, and five house-keeping genes. A cohort of 19 frozen brain specimens were analyzed, including WHO Grade II oligodendroglioma (n = 3), WHO Grade II astrocytoma (n = 2), WHO Grade III astrocytoma (n = 1), and glioblastoma (n = 13). Four normal brain samples were also analyzed. We performed RNA extraction, followed by cDNA synthesis, multiplexed pre-amplification and SYBR-based qPCR, to generate expression profiles on all samples. We demonstrated that the workflow shows high tolerance to variation in RNA quality (RIN 8.5-4) and high sensitivity in detection. cDNA input that is equivalent to 3 ng of starting RNA was sufficient to conduct accurate semiquantitative analysis of the panel of 79 assays. Using principal component analysis, we were able to accurately separate glioblastoma from low-grade glioma. The two WHO Grade III tumors analyzed clustered with glioblastoma, but showed more similarity to Grade II gliomas. In this study, we have shown the feasibility of consolidating high-throughput data into a single functional panel capable of accurately classifying glioma specimens based solely on semiquantitative gene expression profiling.
- Subjects :
- Male
0301 basic medicine
Cancer Research
Computational biology
Biology
Polymerase Chain Reaction
03 medical and health sciences
Glioma
Complementary DNA
Gene expression
Biomarkers, Tumor
medicine
Humans
RNA, Messenger
Gene
Principal Component Analysis
Brain Neoplasms
Gene Expression Profiling
Astrocytoma
medicine.disease
Molecular biology
nervous system diseases
Gene Expression Regulation, Neoplastic
Gene expression profiling
Logistic Models
030104 developmental biology
Neurology
Oncology
Female
Neurology (clinical)
Oligodendroglioma
RNA extraction
Glioblastoma
Subjects
Details
- ISSN :
- 15737373 and 0167594X
- Volume :
- 126
- Database :
- OpenAIRE
- Journal :
- Journal of Neuro-Oncology
- Accession number :
- edsair.doi.dedup.....bcafb150ae1e6abcc1b18e24a1beacbc