Real-Time PCR Data Processing Shown by the Analysis of Colorectal Specific Candidate Genes, ERCC1, RRM1 and TS in Relation to β2M as Endogenous Control

Resistance to chemotherapy is a major cause of mortality in advanced cancer patients. In this study, digital karyotyping was used to search for genomic alterations in liver metastases that were clinically resistant to 5-fluorouracil (5-FU). In two of four patients, we identified amplification of an approximately 100-kb region on 18p11.32 that was of particular interest because it contained the gene encoding thymidylate synthase (TYMS), a molecular target of 5-FU. Analysis of TYMS by fluorescence in situ hybridization identified TYMS gene amplification in 23% of 31 5-FU-treated cancers, whereas no amplification was observed in metastases of patients that had not been treated with 5-FU. Patients with metastases containing TYMS amplification had a substantially shorter median survival (329 days) than those without amplification (1,021 days, P <0.01). These data suggest that genetic amplification of TYMS is a major mechanism of 5-FU resistance in vivo and have important implications for the management of colorectal cancer patients with recurrent disease.

Elevated levels of glycoprotein:sialyltransferase activity (EC 2.4.99.1; CMP-N-acetylneuraminate: D-galactosyl-glycoprotein N-acetylneuraminyltransferase) were found in human malignant neoplastic tissues compared to normal, benign, and "preneoplastic" tissues. This increase was not due to the cell density of the tissue. Elevated levels of certain proteases and glycosidases were also found. The increase in transferase activity may be associated with altered membrane synthesis in the neoplastic state; changes in the activity of degradative enzymes may be associated with tumor invasiveness and maintenance of the neoplastic state. Measurements on human tumors are possibly more directly relevant to cancer than those described for transformed fibroblastic cells in vitro.

Background Quantitative real-time PCR gene expression results are generally normalised using endogenous control genes. These reference genes should be expressed at a constant level across all sample groups in a study, and should not be influenced by study treatments or conditions. There has been no systematic investigation of endogenous control genes for bovine endometrium to date. The suitability of both commonly used and novel endogenous control genes was evaluated in this study, with the latter being selected from stably expressed transcripts identified through microarray analysis of bovine endometrium. Fifteen candidate endogenous control genes were assessed across different tissue subtypes in pregnant and cycling Holstein-Friesian dairy cows from two divergent genetic backgrounds. Results The expression profiles of five commonly used endogenous control genes (GAPDH, PPIA, RPS9, RPS15A, and UXT) and 10 experimentally derived candidate endogenous control genes (SUZ12, C2ORF29, ZNF131, ACTR1A, HDAC1, SLC30A6, CNOT7, DNAJC17, BBS2, and RANBP10) were analysed across 44 samples to determine the most stably expressed gene. Gene stability was assessed using the statistical algorithms GeNorm and Normfinder. All genes presented with low overall variability (0.87 to 1.48% CV of Cq). However, when used to normalise a differentially expressed gene (oxytocin receptor - OXTR) in the samples, the reported relative gene expression levels were significantly affected by the control gene chosen. Based on the results of this analysis, SUZ12 is proposed as the most appropriate control gene for use in bovine endometrium during early pregnancy or the oestrus cycle. Conclusion This study establishes the suitability of novel endogenous control genes for comparing expression levels in endometrial tissues of pregnant and cycling bovines, and demonstrates the utility of microarray analysis as a method for identifying endogenous control gene candidates.