In a recent study by Kreth et al, the Realtime ready Human Reference Gene Panel from Roche was used to identify valid endogenous control genes for determining gene expression in human glioma.
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 a normal brain.
Equivalence tests for equal expression of candidate genes were applied and genes showing differential expression were ruled out from further analyses.
Real-time qPCR was performed in duplicate with the Roche Lightcycler 480 Instrument, 96-well 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 tumour subgroups and across these groups, the Normfinder analyses identified a large number of genes suitable for normalisation.
However, this number was reduced after inclusion of the normal brain into the analyses.
It was expected that only GAPDH, IPO8, RPL13A, SDHA and TBP would not be differentially expressed, and statistical analysis indicated favourable stability values for all of these genes - TBP and IPO8 being the most stable.
These five genes represent different physiological pathways and may be regarded as universal reference genes applicable for accurate normalisation of gene expression in human astrocytomas of different grades (WHO Grades II - IV) alone and compared with normal brain, thereby enabling longitudinally designed studies (for example, in astrocytoma before and after malignant transformation).
The results show that the Roche Realtime ready Human Reference Gene Panel enables the analysis of a variety of different genes, in order to identify suitable reference genes for further relative quantification experiments.
