For interpretation of quantitative gene expression measurements in clinical tumor samples, a normalizer is necessary to correct expression data for differences in cellular input, RNA quality, and RT efficiency between samples. In many studies, a single housekeeping gene is used for normalization. However, no unequivocal single reference gene (with proven invariable expression between cells) has been identified yet. As the best alternative, the mean expression of multiple housekeeping genes can be used for normalization. In this study, no attempt was made to determine the gold-standard gene for normalization, but to identify the best single housekeeping gene that could accurately replace the measurement of multiple genes. Expression patterns of 13 frequently used housekeeping genes were determined in 80 normal and tumor samples from colorectal, breast, prostate, skin, and bladder tissues with real-time quantitative RT-PCR. These genes included, large ribosomal protein, b-actin, cyclophilin A, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerokinase 1, b-2-microglobin, b-glucuronidase, hypoxanthine ribosyltransferase (HPRT), TATA-box-binding protein, transferrin receptor, porphobilinogen deaminase, ATP synthase 6, and 18S ribosomal RNA. Principal component analysis was used to analyze these expression patterns, independent of the level of expression. Our approach identified HPRT as the single best reference gene that could be used as an accurate and economic alternative for the measurement of multiple housekeeping genes. We recommend this gene for future studies to standardize gene expression measurements in cancer research and tumor diagnostics until a definite gold standard has been determined.

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Novel fluorescent oligonucleotides that contain a 3¢ minor groove binding group (MGB) hybridize to single-stranded targets with increased sequence-specificity compared to ordinary DNA probes. This reduces non-specific probe hybridization and results in low background fluorescence during the 5¢ nuclease PCR assay (TaqMan, Applied Biosystems, Foster City, CA). We developed a method for closed-tube genotyping using two allele-specific MGB probes labeled with different fluorophores in one reaction. After PCR, tubes were transported to a fluorescence plate-reader for analysis of fluorescence. Common spreadsheet software was used for automated genotype assignment. As an example, DNA samples from 172 hemochromatosis patients were selected and tested for molecular defects in the HFE gene, i.e., mutations in codon 63 and 282. Tight genotype clusters were observed for both codons and results with MGB probes were identical to conventional genotyping (PCR + restriction-fragment-length-polymorphism). We show that this fast and easy method can be used for large-scale (high-throughput) genetic studies but also for routine molecular diagnostics without post-PCR manipulation of amplicons or the need for real-time quantitative PCR machines. Hum Mutat 19:554 559, 2002.

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Background: A total of 4 additional splice variants (survivin-⌬Ex3, survivin 2␣, survivin-2B, and survivin-3B) have been described for survivin [baculoviral IAP repeat-containing protein (BIRC-5), approved gene symbol BIRC5], which has been implicated in both inhibition of apoptosis and regulation in mitosis in many tumor types. In this study, we assessed whether the survivin splice variants modulate or add to the prognostic value of total survivin in breast cancer. Methods: With quantitative reverse transcription-PCR, we measured mRNA concentrations of survivin and all variants in tumor tissue from 275 patients with breast cancer and associated these with clinicopathologic characteristics and relapse-free survival. Results: Total survivin, survivin-⌬Ex3, and survivin 2␣ mRNA levels were associated with young age and ductal histology. Total survivin and survivin-⌬Ex3 were highest in samples with advanced histological grade, whereas patients with 4 -9 involved lymph nodes expressed less survivin-2B mRNA than those with 1-3 involved nodes. All variants were higher in tumors negative for steroid hormone receptors. Total survivin, survivin 2␣, and survivin-3B were associated with poor relapse-free survival in univariate analyses. Survivin 2␣ and survivin-3B added to the prognostic value of total survivin in multivariate analyses. In addition, the prog-