Quantitative realtime reversetranscription PCR (qPCR) is a frequently used research tool in human glioma. So far, there has been no systematic approach to analysis of suitable reference genes for reliable gene expression analysis. In a recent study by Kreth et al. (S. Kreth, J. Heyn, S. Grau, H. A. Kretzschmar, R. Egensperger, F. W. Kreth, Identification of valid endogenous control genes for determining gene expression in human glioma. Neuro-Oncology Advance Access published online on February 5, 2010), 19 commonly used reference genes were tested for their expression stability in human astrocytoma WHO Grade II, astrocytoma WHO Grade III, and glioblastoma (WHO Grade IV) both alone and compared with normal brain. Equivalence tests for equal expression of candidate genes were applied. Genes showing differential expression were ruled out from further analyses. The authors used the RealTime ready Human Reference Gene Panel from Roche Applied Science (SIX: RO, ROG; OTCQX: RHHBY), which permits simultaneous expression analysis of the 19 reference genes. Realtime qPCR was performed in duplicate with the Roche LightCycler® 480 Instrument, 96well plate. The researchers used the NormFinder software to determine expression stability of the remaining candidate genes.
The authors found that in general glioblastoma exhibited the highest expression levels and largest variability of the candidate genes, whereas normal brain showed the lowest level. In each of the tumor subgroups and across these groups, the NormFinder analyses identified a large number of genes suitable for normalization. However, this number was drastically reduced after inclusion of normal brain into the analyses: it was expected that only GAPDH, IPO8, RPL13A, SDHA, and TBP would not be differentially expressed; statistical analysis indicated favorable stability values for all of these genes, TBP and IPO8 being the most stable. These 5 genes represent different physiological pathways and may be regarded as universal reference genes applicable for accurate normalization of gene expression in human astrocytomas of different grades (WHO Grades II-IV) alone and compared with normal brain, thereby enabling longitudinally designed studies (e.g., in astrocytoma before and after malignant transformation). The results show that the Roche RealTime ready Human Reference Gene Panel is a perfect tool for analyzing a wide variety of different genes in order to identify suitable reference genes for further relative quantification experiments.
The authors found that in general glioblastoma exhibited the highest expression levels and largest variability of the candidate genes, whereas normal brain showed the lowest level. In each of the tumor subgroups and across these groups, the NormFinder analyses identified a large number of genes suitable for normalization. However, this number was drastically reduced after inclusion of normal brain into the analyses: it was expected that only GAPDH, IPO8, RPL13A, SDHA, and TBP would not be differentially expressed; statistical analysis indicated favorable stability values for all of these genes, TBP and IPO8 being the most stable. These 5 genes represent different physiological pathways and may be regarded as universal reference genes applicable for accurate normalization of gene expression in human astrocytomas of different grades (WHO Grades II-IV) alone and compared with normal brain, thereby enabling longitudinally designed studies (e.g., in astrocytoma before and after malignant transformation). The results show that the Roche RealTime ready Human Reference Gene Panel is a perfect tool for analyzing a wide variety of different genes in order to identify suitable reference genes for further relative quantification experiments.
