Aberrant DNA methylation occurs early in oncogenesis, is stable, and can be assayed in tissues and body fluids. Therefore, genes with aberrant methylation can provide clues for understanding tumor pathways and are attractive candidates for detection of early neoplastic events. Identification of sequences that optimally discriminate cancer from other diseased and healthy tissues is needed to advance both approaches. Using well-characterized specimens, genome-wide methylation techniques were used to identify candidate markers specific for colorectal neoplasia. To further validate 30 of these candidates from genome-wide analysis and 13 literature-derived genes, including genes involved in cancer and others with unknown functions, a high-throughput methylation-specific oligonucleotide microarray was used. The arrays were probed with bisulfite-converted DNA from 89 colorectal adenocarcinomas, 55 colorectal polyps, 31 inflammatory bowel disease, 115 extracolonic cancers, and 67 healthy tissues. The 20 most discriminating markers were highly methylated in colorectal neoplasia (area under the receiver operating characteristic curve > 0.8; P < 0.0001). Normal epithelium and extracolonic cancers revealed significantly lower methylation. Real-time PCR assays developed for 11 markers were tested on an independent set of 149 samples from colorectal adenocarcinomas, other diseases, and healthy tissues. Microarray results could be reproduced for 10 of 11 marker assays, including eight of the most discriminating markers (area under the receiver operating characteristic curve > 0.72; P < 0.009). The markers with high specificity for colorectal cancer have potential as blood-based screening markers whereas markers that are specific for multiple cancers could potentially be used as prognostic indicators, as biomarkers for therapeutic response monitoring or other diagnostic applications, compelling further investigation into their use in clinical testing and overall roles in tumorigenesis. (Mol Cancer Res 2007;5(2):153 -63)
We developed a homogeneous format reverse transcription-polymerase chain reaction assay for quantitating hepatitis C virus (HCV) RNA based on the TaqMan principle, in which signal is generated by cleaving a target-specific probe during amplification. The test uses two probes, one specific for HCV and one specific for an internal control, containing fluorophores with different emission spectra. Titers are calculated in international units (IU)/ml by comparing the HCV signal generated by test samples to that generated by a set of external standards. Endpoint titration experiments demonstrated that samples containing 28 IU/ml give positive results 95% of the time. Based on these data, the limit of detection was set conservatively at 40 IU/ml. All HCV genotypes were amplified with equal efficiency and accurately quantitated: when equal quantities of RNA were tested, each genotype produced virtually identical fluorescent signals. The test exhibited a linear range extending from 64 to 4,180,000 IU/ml and excellent reproducibility, with coefficients of variation ranging from 21.6 to 30.4%, which implies that titers that differ by a factor of twofold (0.3 log10) are statistically significant (P = 0.005). The test did not react with other organisms likely to co-infect patients with hepatitis C and exhibited a specificity of 99% when evaluated on a set of samples from HCV seronegative blood donors. In interferon-treated patients, the patterns of viral load changes revealed by the TaqMan HCV quantitative test distinguished responders from nonresponders and responder-relapsers. These data indicate that the TaqMan quantitative HCV test provides an attractive alternative for measuring HCV viral load and should prove useful for prognosis and for monitoring the efficacy of antiviral treatments.
A key issue in nucleic acid-based assays related to infectious disease testing and genetic testing is the sample preparation. This has to be convenient, efficient and suitable for automation. We describe a new method based on the use of glass magnetic particles. The steps involved are the lysis of bacterial cells and viral particles by chaotropic salts, binding of released nucleic acids to the magnetic glass particles, the removal of inhibitors and the release of purified nucleic acids ready for PCR amplification. The method fulfills most of the requirements for sample preparation such as sensitivity, universality and suitability for automation.
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