Detection of tyrosinase mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence.
Detection of tyrosinase mRNA in melanoma by reverse transcription-PCR and electrochemiluminescence.
复制标题
DOI：
10.1093/clinchem/44.6.1161
复制
发表时间：
1998-06
影响因子：
9.3
通讯作者：
C. O’Connell;A. Juhász;C. Kuo;D. Reeder;D. Hoon
中科院分区：
文献类型：
--
作者：
C. O’Connell;A. Juhász;C. Kuo;D. Reeder;D. Hoon
生物技术和临床化学中使用的分析测试的增加的灵敏度和改进的定量是许多实验室的目标。我们已经使用酪氨酸酶引物，通过RT-PCR特异性地扩增由M12黑色素瘤细胞系在来自乳腺癌细胞的mRNA的背景下表达的酪氨酸酶mRNA。电化学发光检测程序被用作本研究的读出系统。将生物素化的PCR后cDNA样品与三（2，2 '-联吡啶）钌（II）（TBR）螯合物标记的寡核苷酸探针杂交，随后通过链霉亲和素包被的Dynabeads捕获杂交体。当使用QPCR系统5000或Origen 1分析仪系统时，评估捕获的特异性PCR后产物的TLR螯合物发出的发光。在10（7）个细胞的背景中，可检测到从约1-10个黑素瘤细胞中分离的酪氨酸酶特异性mRNA。我们通过使用rTth进行逆转录和扩增，提高了检测的灵敏度和物流。酪氨酸酶mRNA在16例黑色素瘤患者中的7例血液中检测到，而5例健康供体血液中无一阳性（P = 0.01; Wilcoxon检验）。
Increased sensitivity and improved quantitation of analytical tests used in biotechnology and clinical chemistry are goals of many laboratories. We have used tyrosinase primers to specifically amplify by RT-PCR the tyrosinase mRNA expressed by the M12 melanoma cell line in a background of mRNA from breast cancer cells. An electrochemiluminescence detection procedure was used as a readout system for this study. Biotinylated post-PCR cDNA samples were hybridized to a tris(2,2'-bipyridine)ruthenium(II) (TBR) chelate-labeled oligonucleotide probe, and the hybrid was subsequently captured by streptavidin-coated Dynabeads. When either the QPCR System 5000 or the Origen 1 Analyzer System were used, the luminescence emitted by the TBR-chelate of the captured specific post-PCR product was assessed. Tyrosinase-specific mRNA isolated from approximately 1-10 melanoma cells in a background of 10(7) cells could be detected. We improved the sensitivity and logistics of the assay through the use of rTth for reverse transcription and amplification. Tyrosinase mRNA was detected in blood from 7 of 16 melanoma patients, whereas none of the 5 healthy donor bloods were positive (P = 0.01; Wilcoxon test).