比较单管和双管SYBR Green荧光定量PCR方法在端粒长度测量中的应用
Comparison of the Applications of SYBR Green Single Tube and Two Tubes Fluorescent Quantitative PCR Method in Measuring the Length of Telomere

目的：探讨单色多重定量PCR方法在人类端粒长度测量中的临床应用价值。方法：我们设计了针对端粒基因多次重复序列的能产生固定长度产物的引物，同时设计能扩增单拷贝基因的引物作为内参；两对引物在同一管中进行SYBR green QPCR反应，数据进行分析获得端粒相对长度。结果：1) 两对引物在单管中反应没有改变单独反应时的扩增效率和溶解温度；2) 同一样本在单管内和双管内反应T/S的差异很大；3)通过对同一样本进行10次重复发现，单管的值很均一，而双管的值相差很大，CV%分别是15.25%和53%。结论：我们的结果提示，单色单管方法准确性和重复性都大大优于单色双管，能够准确测量端粒的相对长度，有利于在生物医疗研究领域中的应用。
Objective: To investigate the clinical value of the monochrome multiplex quantitative PCR method in measuring the length of the human telomere. Methods: We designed primers targeting the multiple repeating sequences producing fixed-length products in the gene encoding telomere. Primers were also designed to amplify single-copied genes which served as the internal control. The two primers were loaded into the same tube for the SYBR green QPCR, and data were analyzed to get the information of the relative length of the telomere. Results: 1) The amplification efficiency and the melting temperature were not changed in reactions with two pairs of primers in the single tube versus two separate tubes; 2) There was a significant difference in the T/S of the same samples with reactions occurring in single or separate tubes; 3) After repeating the test 10 times of the same sample, we found that the results from single tube were stable while there were big variations for the results obtained for reactions in two separate tubes (the CV% are 15.25%, 53%, respectively). Conclusions: Our results indicate that the accuracy and reproducibility of monochrome single tube reactions in measuring the relative length of telomeres are significantly better than those of monochrome two tubes reactions. These data are helpful for the use of the qPCR in biomedicine.