Park S, Lee B, Kim I, Choi I, Hong K, Ryu Y, Rhim J, Shin J, Park S C, Chung H, Chung J
Department of Biochemistry, Dongguk University School of Medicine, Kyung-Buk, Korea.
J Cancer Res Clin Oncol. 2001 Aug;127(8):489-94. doi: 10.1007/s004320100239.
The reverse transcription polymerase chain reaction (RT-PCR) amplification of tumor-specific mRNA has been used for the detection of cancer cells in peripheral blood. More recently, an immunomagnetic isolation and reverse transcription polymerase chain reaction (immunobead RT-PCR) was developed which has reportedly significant advantages over the previous RT-PCR analysis. In our study, we compared these two methods using a model set of peripheral blood containing tumor cells under standardized conditions.
In order to compare the false positive rate, normal peripheral blood samples from five volunteers were analyzed by both methods. A model set of peripheral blood containing tumor cells was established by adding SNUC4 human colon cancer cells to peripheral blood collected from normal volunteers not showing any nonspecific bands upon electrophoresis of the PCR products. RT-PCR amplification of carcinoembryonic antigen (CEA) mRNA was done with total RNA and mRNA prepared from this model sample. In immunobead RT-PCR analysis, mRNA was prepared from the cells isolated with anti-CEA antibody-coated magnetic beads or anti-Ber-EP4 antibody-coated magnetic beads before the RT-PCR analysis.
The immunobead RT-PCR yielded no non-specific band, while the regular RT-PCR using total RNA did show non-specific band formation in all five samples. When mRNA rather than total RNA was used, nonspecific bands were formed in three of the five samples. Immunobead RT-PCR allowed the detection of 10(1) tumor cells in 1 ml of peripheral blood. The regular RT-PCR analysis had a detection limit of 10(2) tumor cells in 1 ml of peripheral blood.
The immunobead RT-PCR proved to be more sensitive and specific than the regular RT-PCR at least in our model system.
肿瘤特异性mRNA的逆转录聚合酶链反应（RT-PCR）扩增已用于检测外周血中的癌细胞。最近，开发了一种免疫磁珠分离和逆转录聚合酶链反应（免疫磁珠RT-PCR），据报道它比以前的RT-PCR分析具有显著优势。在我们的研究中，我们在标准化条件下使用一组含有肿瘤细胞的外周血模型比较了这两种方法。
为了比较假阳性率，用两种方法分析了五名志愿者的正常外周血样本。通过将SNUC4人结肠癌细胞添加到从正常志愿者采集的外周血中，建立了一组含有肿瘤细胞的外周血模型，这些志愿者的PCR产物电泳后未显示任何非特异性条带。用该模型样本制备的总RNA和mRNA进行癌胚抗原（CEA）mRNA的RT-PCR扩增。在免疫磁珠RT-PCR分析中，在RT-PCR分析之前，从用抗CEA抗体包被的磁珠或抗Ber-EP4抗体包被的磁珠分离的细胞中制备mRNA。
免疫磁珠RT-PCR未产生非特异性条带，而使用总RNA的常规RT-PCR在所有五个样本中均显示有非特异性条带形成。当使用mRNA而非总RNA时，五个样本中有三个形成了非特异性条带。免疫磁珠RT-PCR能够检测出1 ml外周血中的10(1)个肿瘤细胞。常规RT-PCR分析在1 ml外周血中的检测限为10(2)个肿瘤细胞。
至少在我们的模型系统中，免疫磁珠RT-PCR比常规RT-PCR更敏感、更特异。
