Establishment Of A DNA-binding Dye Real-time Fluorescence Quantitative RT-PCR Assay For Quantification Of Human Telomerase Reverse Transcriptase (hTERT) MRNA And Evaluation Of Clinical Application

Objective: To establish a real-time fluorescence quantitative reverse transcriptase- polymerase chain reaction (RT-PCR) assay using intercalation dye SYBR Green Ⅰ for quantification of human telomerase reverse transcriptase (hTERT) mRNA and to detect hTERT mRNA in exfoliated cells in urine taken from patients with bladder transitional cell carcinoma(TCC) in order to demonstrate the diagnostic validity for TCC.Methods:1. T24 human bladder cancer cells, known to be positive telomerase activity, and IMR90 human fibroblast cells, a cell line that is negative for telomerase activity, were cultured.2. A real-time fluorescence quantitative RT-PCR assay using intercalation dye SYBR Green ⅠDye was established and the hTERT mRNA expression in T24 and IMR90 cells was quantified.3. Semi quantitative noncompetitive RT-PCR was also used to quantify hTERT mRNA expression in T24 and IMR90 cells and compared with real-time fluorescence quantitative RT-PCR.4. We measured hTERT mRNA expression in exfoliated cells in urine taken from 26 patients with TCC and 18 nonmalignant controls using real-time fluorescence quantitative RT-PCR.Results: 1. As the concentration of intercalation dye SYBR Green Ⅰwasincreased, the melting curve shifted to higher temperature. Rapid temperature transition also shifted melting curve to higher Tm. The melting curve of PCR amplification product was easily identified as low Tm peak and high Tm peak. Non-specific amplification product tended to be at much lower temperature than specific amplification product. The Tms of GAPDH and hTERT non-specific amplification products were 72.8℃ and 71.6℃, respectively. The Tms of GAPDH and hTERT specific amplification products were 87.2℃ and 90.5℃, respectively. The Tm peak of hot start PCR amplification product was flatter than the Tm peak of non-hot start PCR amplification product.2. The functions of the calibration curves for GAPDH and hTERT mRNA quantification were Ct=25.77－2.27log[GAPDH (copies)] (r=－1.000) and Ct=25.77－2.27log[hTERT(copies)] (r=－1.000), respectively. The functions of the dynamic ranges for GAPDH and hTERT mRNA quantification were Ct=10.46－3.98log[cDNA(μl)] (r=－1.000) and Ct=26.00－2.08log [cDNA(μl)] (r=－1.000), respectively. The mean intra-assay (3 replicates) CVs of GAPDH and hTERT PCR amplification were 2.85% and 1.59%, respectively. The efficiency of PCR between GAPDH and hTERT PCR amplification was almost equal with slope of 0.0068.3. With real-time fluorescence quantitative RT-PCR assay, the hTERT mRNA expression in IMR90 cells was negative and the normalized hTERT mRNA expression in T24 cells was 25.06[100×hTERT (copies)／GAPDH(copies)]. 4. With semi quantitative noncompetitive RT-PCR assay, the hTERT mRNA expression in IMR90 cells was negative and the normalized hTERT mRNA expression in T24 cells was 0.06[hTERT(IOD)／GAPDH(IOD)].5. The overall specificity, sensitivity and diagnostic validity for quantification of hTERT mRNA expression in exfoliated cells in urine using real-time fluorescence quantitative RT-PCR were 100% (26/26), 76.9% (20/26) and 86.4% (38/44), respectively. Quantitative result of hTERT mRNAexpression was significantly correlated with TCC tumor grade (P<0.01) and clinical stage (P<0.01). Quantitative result of hTERT mRNA expression could discriminate TCC tumor grade and clinical stage with predictive classification results of 100% (Grade Ⅰ),72.7% (Grade Ⅱ),83.3% (Grade Ⅲ), 100% (Ta-T1) and 56.3% (T2-T3).Conclusion: 1. With melting curve analysis, the real-time fluorescence quantitative RT-PCR using intercalation SYBR Green ⅠDye can easily discriminate specific product from non-specific product.2. The real-time fluorescence quantitative RT-PCR assay using intercalation SYBR Green ⅠDye is sensitive and has a broad dynamic range. With real-time fluorescence monitoring, it can ensure PCR amplification running among the exponential period. It is rapid, accuracy and need not perform gel electrophoresis.3. The semi quantitative noncompetitive R...