No abstract

Objective: Human chorionic gonadotropin (hCG) is a marker of trophoblastic tumors, and the serum concentration of the free β-subunit (hCGβ) is an independent prognostic marker in several nontrophoblastic cancers. hCGβ is encoded by six genes, of which the type II genes (hCGβ 3/9, 5 and 8) are thought to be upregulated in relation to type I genes (hCGβ 6/7) in cancer. Method: We developed a novel quantitative RT-PCR method for the quantification of the relative expression levels of the two groups of hCGβ genes and analyzed 28 bladder tumors and 15 urine samples. Results: We found a higher relative expression level of type II genes in malignant compared with benign urothelia (p = 0.016) and in exfoliated urinary cells from cancer patients compared with those from benign controls (p = 0.026). The expression level was increasing with higher stage (p = 0.014) and grade (p = 0.001) and tended to be higher in relapsing tumors (p = 0.059). Conclusion: The increased hCGβ concentrations in body fluids of patients with aggressive bladder cancer may be due to overexpression of type II genes. Quantification of the relative mRNA expression levels of the hCGβ type I and II genes in urine cells should be further studied as a potential noninvasive tool for the diagnosis and follow-up of bladder cancer.

The new quantitative RT-PCR technique facilitates very accurate quantitation of the relative mRNA levels of homologous genes. Using this method we have shown that the ratio of hK2/PSA mRNA is higher in cancerous than in benign prostatic tissue.

Specific somatic MED12 mutations in prostate cancer and uterine leiomyomas accumulate in two separate regions of the gene and promote tumorigenesis through clearly distinct mechanisms.

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%-57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis.