Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics
Characterization of amplifiable, circulating RNA in plasma and its potential as a tool for cancer diagnostics
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DOI：
10.1373/clinchem.2003.028506
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发表时间：
2004-03-01
影响因子：
9.3
通讯作者：
Godfrey, TE
中科院分区：
文献类型：
--
作者：
El-Hefnawy, T;Raja, S;Godfrey, TE
背景：最近的一些报道描述了在癌症患者的血清或血浆中检测到循环的癌症相关RNA分子，但对这种细胞外RNA的生物学知之甚少。我们的目的是确定RNA在血清中是如何防止降解的，优化从大量血清中分离RNA的方法，并测试我们优化的血清癌症检测方法。方法：采用定量逆转录pcr （QRT-PCR）技术对细胞外血浆RNA的分离及生物学特性进行研究。然后，我们检测了来自对照组和食管癌和恶性黑色素瘤患者的血浆和血清中可扩增RNA转录物的存在。结果：我们发现血浆中的细胞外RNA是高度降解的，采用胍-苯酚萃取-沉淀的方法可以最有效地分离胞外RNA。细胞外RNA在血清中稳定达3小时，但立即被添加洗涤剂破坏。细胞外RNA可以在0.2 μ m的过滤器上捕获，允许从几毫升血浆中提取RNA浓度。当我们从高达4ml的血清中浓缩RNA时，在癌症患者和对照组的血清中检测到癌症相关转录物的频率很低，而且不一致。结论：细胞外RNA很可能在蛋白质或脂质囊泡中受到保护，可能是凋亡小体，可被洗涤剂破坏。尽管优化了血浆RNA检测的许多方面，但我们无法重复检测癌症相关转录物。我们的数据表明，循环RNA的测量可能不是早期癌症诊断的好方法。(C) 2004美国临床化学学会。
Background: Several recent reports have described the detection of circulating, cancer-related RNA molecules in serum or plasma from cancer patients, but little is known about the biology of this extracellular RNA. We aimed to determine how RNA is protected against degradation in serum, to optimize RNA isolation from large volumes of serum, and to test our optimized assays for serum-based cancer detection.Methods: We used quantitative reverse transcription-PCR (QRT-PCR) analysis to investigate the isolation and biology of extracellular plasma RNA. We then examined the presence of amplifiable RNA transcripts in plasma and serum from controls and from patients with esophageal cancer and malignant melanoma.Results: We found that extracellular RNA in plasma is highly degraded and can be isolated most efficiently by guanidinium-phenol extraction followed by precipitation. Extracellular RNA is stable in serum for up to 3 h but is destroyed immediately by addition of detergents. Extracellular RNA can be captured on 0.2 mum filters, allowing concentration of RNA from several milliliters of plasma. When we concentrated RNA from up to 4 mL of serum, detection of cancer-related transcripts in serum from cancer patients and controls was infrequent and inconsistent.Conclusions: Extracellular RNA is most likely protected within protein or lipid vesicles, possibly apoptotic bodies, which can be disrupted by detergents. Despite optimizing many aspects of plasma RNA detection, we were unable to reproducibly detect cancer-related transcripts. Our data suggest that measurement of circulating RNA may not be a good approach to early cancer diagnosis. (C) 2004 American Association for Clinical Chemistry.