cyanine-dye-3 and Uterine-Cervical-Neoplasms

Development of novel approaches for quantitative analysis of gene expression in intact tumour cells could provide new methods for the detection of cervical cancer. Molecular beacons (MBs) are single-stranded oligonucleotide probes that form stem-and-loop structures. Survivin, a member of the 'inhibitor of apoptosis' family of proteins, is highly expressed in cervical cancer. The aim of this study was to determine the feasibility of MBs targeting wild-type survivin in the diagnosis of cervical cancer.. MBs with oligonucleotide sequences complementing survivin mRNA and covalently linked with FITC or Cy3 were designed and synthesized. The specificity and sensitivity of survivin MBs were examined in human cervical cancer cell lines and smears from cervical cancer patients, and confirmed by reverse transcriptase polymerase chain reaction (RT-PCR), immunocytochemistry, Western blotting and the thinprep cytological test (TCT).. Both survivin MB-FITC and MB-Cy3 produced a strong fluorescent signal in cervical cancer cells, and the intensity was consistent with the results from RT-PCR, Western blotting and immunocytochemical staining. In the initial clinical cohort study, the sensitivity of survivin MBs was 61.37% (27/44) and the specificity was 72.72% (34/44); the sensitivities of Western blotting and TCT were 76.1% (32/42) and 68.19% (30/44), respectively. No significant difference in sensitivity was observed between MBs, Western blotting and TCT for different tumour grades and International Federation of Gynecology and Obstetrics (FIGO) stages.. Survivin MBs are specific and sensitive molecular probes for the detection of cervical cancer cells. They have great potential in the early detection and follow-up of patients with cervical cancer.

Topics: Adult; Carbocyanines; Cell Line, Tumor; Cohort Studies; DNA, Single-Stranded; Feasibility Studies; Female; Fluorescein-5-isothiocyanate; Humans; Inhibitor of Apoptosis Proteins; Inverted Repeat Sequences; Middle Aged; Molecular Imaging; Neoplasm Grading; Neoplasm Proteins; Neoplasm Staging; Oligonucleotide Probes; Sensitivity and Specificity; Survivin; Uterine Cervical Neoplasms; Vaginal Smears

High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure.. All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range.. It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.

Topics: Algorithms; Carbocyanines; DNA, Complementary; Female; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Nucleic Acid Hybridization; Oligonucleotide Array Sequence Analysis; Reproducibility of Results; Uterine Cervical Neoplasms