Kopreski M S, Benko F A, Kwak L W, Gocke C D
OncoMEDx, Inc., Columbia, Maryland, USA.
Clin Cancer Res. 1999 Aug;5(8):1961-5.
Serum RNases are known to be elevated in patients with cancer. Consequently, it is not clear whether human mRNA with sufficient integrity as to permit reverse transcription-PCR (RT-PCR) amplification is detectable in serum. We examined serum from six patients with malignant melanoma for human tyrosinase mRNA using RT-PCR. Serum from 20 normal volunteers served as controls. Tyrosinase mRNA could be demonstrated in serum from four of the six melanoma patients with detection by gel electrophoresis and confirmation by blotting amplified product to a tyrosinase-specific probe. The serum remained tyrosinase mRNA positive, even if passed through a 0.45 microm filter prior to RNA extraction, indicating that the mRNA was extracellular at the time of extraction. Tyrosinase mRNA could not be detected in any control serum (0 of 20 individuals). The presence and integrity of amplifiable RNA was confirmed in all serum specimens (patients and controls) by RT-PCR amplification of c-abl mRNA. Amplifiable RNA could be demonstrated regardless of whether serum was freshly drawn or stored frozen for several years. We conclude that human mRNA can be extracted and amplified from serum. The ability to amplify tumor mRNA from serum may have important utility in cancer diagnostics and monitoring.
已知癌症患者血清中的核糖核酸酶水平会升高。因此,目前尚不清楚血清中是否能检测到具有足够完整性以允许进行逆转录聚合酶链反应(RT-PCR)扩增的人类信使核糖核酸(mRNA)。我们使用RT-PCR检测了6例恶性黑色素瘤患者血清中的人类酪氨酸酶mRNA。来自20名正常志愿者的血清作为对照。通过凝胶电泳检测并将扩增产物与酪氨酸酶特异性探针杂交确认,6例黑色素瘤患者中有4例血清中可检测到酪氨酸酶mRNA。即使在RNA提取前血清经过0.45微米滤膜过滤,血清中酪氨酸酶mRNA仍为阳性,这表明提取时mRNA处于细胞外。在任何对照血清中均未检测到酪氨酸酶mRNA(20例个体中0例)。通过对c-abl mRNA进行RT-PCR扩增,证实了所有血清标本(患者和对照)中均可扩增的RNA的存在及其完整性。无论血清是新鲜采集的还是冷冻保存数年的,均可检测到可扩增的RNA。我们得出结论,人类mRNA可以从血清中提取并扩增。从血清中扩增肿瘤mRNA的能力在癌症诊断和监测中可能具有重要用途。