Study On Distributed Localization Schemes For Wireless Sensor Networks (the Detection Of Circulating Tumor Cells By Quantitative Real-time PCR)

PartⅠDetection of hMAM mRNA by using quantitative real-time PCRObjectivesTo establish a quantitative real-time PCR method for detecting hMAM mRNA.MethodsThe hMAM fragment in pure form from classical RT-PCR was cloned to pGEM-Tvector, and recombinant plasmid pGEM- hMAM was purified and quantified. Standardcurve was established using a series dilution of quantified plasmids to measure hMAM bySYBR Green I quantitative real-time PCR and the characteristic of specific hMAMamplicon was analyzed by melting curve.ResultsThe method can detect as low as 2 copies. A good linearity was found from2～2×10~6copies/reaction and the correlation coefficient was -0.99. The intraassay and interassayvariation of 2×10~4copies/μ1 was 1.50 % and 3.26 % ,respectively. Melting curve analysisshowed a single peak.ConclusionsSYBR Green I quantitative real-time PCR is specific, sensitive and accurate can beused to further research on human hMAM.PartⅡThe detection of circulating tumor cells of breast cancer patients by usingmultimarker (Survivin, hTERT and hMAM) quantitative real-time PCRObjectives To develop a specific, reliable assay for detecting circulating tumor cells (CTCs) inperipheral blood of breast cancer patients and explore the correlation between theexpression of molecular markers (Survivin, hTERT and hMAM ) and a variety ofclinicopathological features.Methods94 breast cancer patients, 35 patients with benign breast tumor, 40 healthy individuals,and 25 patients with other solid tumors were evaluated by quantitative real-time reversetranscription-PCR (qRT-PCR) for detecting Survivin, hTERT, and hMAM mRNA inperipheral blood (PB) of breast cancer patients. In addition, analyses were carried out fortheir correlation with patients' clinicopathologic features.ResultsThe positive rate of Survivin, hTERT, and hMAM mRNA in the PB of breast cancerpatients was 36.2%, 59.6% and 33.0%, respectively. Survivin and hTERT were detected inthe PB patients with solid tumors other than breast cancer, but hMAM mRNA was onlydetected in breast cancer patients. The three combined markers in the parallel test had apositive rate (70.2%). Compared to that of single marker detection, the three combinedmarkers' percentage was significantly higher. However, the specificity of three combinedmarkers of serial test was 100%. The expression of the three mRNAs significantlycorrelated with TNM stage, and lymph node metastasis.ConclusionsSurvivin, hTERT and hMAM mRNA assays are powerful methods for detection ofCTCs of breast cancer patients. With combination of the three markers fordetection of CTCs of breast cancer, the parallel test increases the sensitivity. This analysiscan offer a simple, noninvasive, and promising adjuvant tool for the early detection ofmicrometastatic tumor cells in breast cancer patients. PartⅢQuantitative real-time RT-PCR detection for Survivin, CK20 and CEA inperipheral blood of colorectal cancer patientsObjectiveTo establish a sensitive method for early detection of circulating tumor cells (CTCs) inperipheral blood (PB) of colorectal cancer (CRC) patients, improve the sensitivity for earlydetection of CTCs of CRC diagnosis in PB, and explore the correlation between theexpression of these molecular markers and a variety of clinicopathological features.MethodsPB samples were collected from 156 CRC, 40 benign colorectal disease patients, 40healthy individuals and 45 patients with other solid tumors. The combination negative andpositive immunomagnetic bead method was used to enrich cancer cells. Then,cytokeratin-20 (CK-20), Survivin, and carcinoembryonic antigen (CEA) mRNA weredetected by quantitative real-time reverse transcription-polymerase chain reaction(qRT-PCR). In addition, analyses were carried out for their correlation with patients'clinicopathologic features.ResultsThe positive rates of Survivin, CK20 and CEA mRNA in the PB of CRC patients were57.7%, 47.4%, and 39.1%, respectively, and the sensitivity increased from 39.1% of CEAmRNA alone to 60.9% of the combined panel. The expression of the three mRNAs in CRCpatients was significantly higher than that in benign control and healthy volunteers, and theexpression of Survivin and CK20 were not significantly higher than that of patients withother solid tumors. However, the expression of CEA mRNA was significantly higher thanthat of patients with other solid tumors. The expression of Survivin, CK20 and CEA mRNAsignificantly correlated with Dukes stages and lymph node metastasis. ConclusionsThe combined use of negative and positive immunomagnetic beads followed byamplification of Survivin, CK20 and CEA mRNA by means of qRT-PCR is a non-invasiveand sensitive assay for the detection of circulating CRC cells. The combined panelimproved the sensitivity of detection CRC patients.PartⅣDetection of telomerase gene expression in the peripheral blood mononuclear cells inpatients with colorectal carcinoma by a RTQ-TRAPObjective To establish real-time quantitative telomeric-repeat amplification protocolassay (RTQ-TRAP) method for detection of the gene expression of telomerase, andinvestigate the gene expression of telomerase in the peripheral blood mononuclear cells(PBMC) of colorectal carcinoma patients and the relationship between the telomerase geneexpression in PBMC and clinicopathological features.MethodsPeripheral blood samples were collected from 71 colorectal carcinoma patients, 20benign colorectal disease patients and 25 normal controls. The telomerase geneexpression in PBMC was measured by RTQ-TRAP, and the CEA of the colorectalcarcinoma patients was measured by chemiluminescence immunoassay.ResultsThe RTQ-TRAP method was highly both accurate and sensitive in measuring telomerasegene expression. Regarding the gene expression of telomerase in PBMC of patients, 50 outof 71 (70.4%) were found to be positive, The difference to the telomerase gene expressionof PBMC in benign colorectal diseases and cancer patients was significant (χ~2=24.521, P＜0.001). When the gender, age, Dukes stage, and tumor site of telomerase gene expression in patients with colorectal carcinoma were evaluated, there are not significant differences.The positive rate of CEA in colorectal carcinoma patients was not significantly higher thanthat of telomerase gene expression (χ~2=2.286, P=0.125).ConclusionsThe RTQ-TRAP method is a useful tool to rapidly and precisely quantify telomerasegene expression, the detection of telomerase gene expression in PBMC of colorectalcarcinoma patients is a simple and useful molecular marker for the diagnosis of colorectalcarcinoma.