Brain Tumor Res Treat.  2015 Apr;3(1):24-29. 10.14791/btrt.2015.3.1.24.

Validation of Housekeeping Genes for Gene Expression Analysis in Glioblastoma Using Quantitative Real-Time Polymerase Chain Reaction

• Aithal MG
• Rajeswari

• KMID: 1882006
• DOI: http://doi.org/10.14791/btrt.2015.3.1.24

Abstract

BACKGROUND
Quantitative real-time polymerase chain reaction (qPCR) is the most reliable tool for gene expression studies. Selection of housekeeping genes (HKGs) that are having most stable expression is critical to carry out accurate gene expression profiling. There is no 'universal' HKG having stable expression in all kinds of tissues under all experimental conditions.
METHODS
The present study aims to identify most appropriate HKGs for gene expression analysis in glioblastoma (GBM) samples. Based on literature survey, six most commonly used HKGs that are invariant in GBM were chosen. We performed qPCR using RNA from formalin fixed paraffin embedded GBM samples and normal brain samples to investigate the expression pattern of HPRT, GAPDH, TBP, B2M, B2M, RPL13A, and RN18S1 with different abundance. A simple Deltacycle threshold approach was employed to calculate the fold change.
RESULTS
Our study shows that the expression of RPL13A and TBP were found to be most stable across all the samples and are thus suitable for gene expression analysis in human GBM. Except for TBP, none of the other conventionally used HKGs in GBM studies e.g., HPRT and GAPDH were found to be suitable as they showed variation in RNA expression.
CONCLUSION
Validation of HKGs is therefore immensely specific for a particular experimental setup and is crucial in assessing any new setup.

Keyword

Gene expression; Glioblastoma; Housekeeping genes; Quantitative real-time PCR

MeSH Terms

Brain
Formaldehyde
Gene Expression Profiling
Gene Expression*
Genes, Essential*
Glioblastoma*
Humans
Hypoxanthine Phosphoribosyltransferase
Paraffin
Real-Time Polymerase Chain Reaction*
RNA
Formaldehyde
Hypoxanthine Phosphoribosyltransferase
Paraffin
RNA

Figure

• 

  Fig. 1 Expression of candidate HKGs in GBM (filled circles) compared with normal brain samples used as control (open circles) on the basis of raw Ct values. Ct, cycle threshold; GBM, glioblastoma; HKGs, housekeeping genes.

• 

  Fig. 2 Fold change in gene expression. Scatter plots of HKGs in terms of fold change derived from qPCR analysis across GBM samples. Each dot represents data obtained from one patient sample and the bar represents average fold change. GBM, glioblastoma; HKGs, housekeeping genes; qPCR, quantitative real-time polymerase chain reaction.

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