U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

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The association of a particular mitochondrial DNA (mtDNA) mutation with different clinical phenotypes is a well-known feature of mitochondrial diseases. A simple genotype-phenotype correlation has not been found between mutation load and disease expression. Tissue and intercellular mosaicism as well as mtDNA copy number are thought to be responsible for the different clinical phenotypes. As disease expression of mitochondrial tRNA mutations is mostly in postmitotic tissues, studies to elucidate disease mechanisms need to be performed on patient material. Heteroplasmy quantitation and copy number estimation using small patient biopsy samples has not been reported before, mainly due to technical restrictions. In order to resolve this problem, we have developed a robust assay that utilizes Molecular Beacons to accurately quantify heteroplasmy levels and determine mtDNA copy number in small samples carrying the A8344G tRNA(Lys) mutation. It provides the methodological basis to investigate the role of heteroplasmy and mtDNA copy number in determining the clinical phenotypes.

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The association of human papillomaviruses (HPVs) with several human malignancies is well established.1 HPV has been found in benign, premalignant and malignant lesions of the skin 2 and of the anogenital 3 and aerodigestive system. 4,5 World-wide epidemiological studies have shown that cancer of the uterine cervix is the second most common malignant disease in women.6 Virtually every cervical cancer (99.7%) is HPV-positive, indicating that the presence of HPV is an obligatory element in the development of cervical cancer. 7Until now more than 80 types of HPV have been identified. The types associated with diseases of the anogenital tract can be classified on the basis of phylogenetic relationship 8 and of association frequencies with benign or malignant cervical lesions as high-risk types and low-risk types . The most common HR types are 9 The most commonly found LR types are HPV-6 and -11.Because the detection and type-specific classification of HPV infection by an in vitro viral culture test is not possible and serological tests are still ineffective, a molecular DNA diagnosis is needed. Direct hybridizationbased assays, such as Southern blotting and in situ hybridization have been described, but these methods lack sensitivity and specificity. Signal amplification assays such as used in the hybrid capture assays 10 for HPV are often applied. After recent improvements in this technique, it was shown that at the 1 pg/ml cut-off point (equivalent to 100.000 viral copies/ml) results agreed well with those of a polymerase chain reaction (PCR)-based assay. 11Because of the high-type specificity and sensitivity provided by target DNA amplification, the most widely