Validation of Reference Genes for Reliable Gene Expression Study in Human Glioblastoma Multiforme and Neural Stem Cell Lines

Abstract

Glioblastoma multiforme (GBM) is the most deadly malignant brain tumor in adults. GBM has a histologically heterogeneous group of neoplastic cells which are postulated to be the mediator of anti-cancer therapy resistance and tumor relapse. Therefore, it is important to design precise therapies targeting the GBM. The tumor homing of human neural stem cells (NSC) allows selective drug delivery to kill GBM. The therapeutic effects of NSC for GBM can be assessed by reverse transcription real-time polymerase chain reaction (RT-qPCR) using a validated internal control across GBM and NSC. This study is the first evaluation on the suitability of nine reference genes as the internal control in human glioblastoma cell line (DBTRG-05MG) and H9-derived human NSC (H9-hNSC) using RT-qPCR. The results were analyzed using geNorm, NormFinder and BestKeeper algorithms. The analyses revealed PPIA as the most stably expressed gene across DBTRG-05MG and H9-hNSC. Both geNorm and NormFinder suggested PPIA and RPL13A as the best combination of reference genes for RT-qPCR normalization across DBTRG-05MG and H9-hNSC. HPRT1 was ranked as the least stably expressed gene between DBTRG-05MG and H9-hNSC by all three analyses. In conclusion, PPIA and RPL13A were proposed as the best reference genes for these two cell types.

Keywords: Glioblastoma multiforme; Neural stem cell; Reverse transcription quantitative real-time polymerase chain reaction; reference genes