Evaluation of reference genes for normalization of quantitative real time PCR in non-small cell lung cancer

To compare gene expression levels of lung cancer by real time quantitative reverse transcription polymerase chain reaction (qRT-PCR), choosing appropriate control genes for normalization of RNA input is important. We examined whether frequently used control genes are really appropriate. Eighteen lung cancer tissue samples were used in this study. Gene expression levels were measured by qRT-PCR and compared to the data generated by a DNA microarray experiment. The expression levels of four housekeeping genes that are most frequently used as control genes for qRT-PCR published reports in lung cancer were exammed. Four test genes (TP53, laminin γ2, TGFα and cathepsin L2) were also examined. These genes had significantly higher expression in the high aggressive disease group. When taken as single genes, the expression levels of these so called control genes (glyceraldehydes-3phosphate dehydrogenase, TATA box binding protein, β actin and β2 microgloblin) were not consistent among the tested samples. However, the geometric mean of the threshold cycle of the TATA box binding protein and β2 microglobulin showed less variation than that of the single control gene. These results indicate that normalization by multiple control genes is more appropriate for comparison of gene expression levels of lung cancer when using qRT-PCR analysis.