GeneChip, geNorm, and gastrointestinal tumors: novel reference genes for real-time PCR

Accurate quantitation of target genes depends on correct normalization. Use of genes with variable tissue transcription ( GAPDH) is problematic, particularly in clinical samples, which are derived from different tissue sources. Using a large- scale gene database ( Affymetrix U133A) data set of 36 gastrointestinal ( GI) tumors and normal tissues, we identified 8 candidate reference genes and established expression levels by real-time RT- PCR in an independent data set ( n = 42). A geometric averaging method ( geNorm) identified ALG9, TFCP2, and ZNF410 as the most robustly expressed control genes. Examination of raw CT values demonstrated that these genes were tightly correlated between themselves ( R-2 > 0.86, P < 0.0001), with low variability [ coefficient of variation ( CV) < 12.7%] and high interassay reproducibility ( r = 0.93, P = 0.001). In comparison, the alternative control gene, GAPDH, exhibited the highest variability ( CV = 18.1%), was significantly differently expressed between tissue types ( P = 0.05), was poorly correlated with the three reference genes ( R2 < 0.4), and was considered the least stable gene. To illustrate the importance of correct normalization, the target gene, MTA1, was significantly overexpressed ( P = 0.0006) in primary GI neuroendocrine tumor ( NET) samples ( vs. normal GI samples) when normalized by geNorm(ATZ) but not when normalized using GAPDH. The geNormATZ approach was, in addition, applicable to adenocarcinomas; MTA1 was overexpressed ( P < 0.04) in malignant colon, pancreas, and breast tumors compared with normal tissues. We provide a robust basis for the establishment of a reference gene set using GeneChip data and provide evidence for the utility of normalizing a malignancy- associated gene ( MTA1) using novel reference genes and the geNorm approach in GI NETs as well as in adenocarcinomas and breast tumors.