Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA:: A simple blood test to monitor disease in cancer patients
Real-time quantification in plasma of human telomerase reverse transcriptase (hTERT) mRNA:: A simple blood test to monitor disease in cancer patients
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DOI：
10.1038/labinvest.3780285
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发表时间：
2001-05-01
影响因子：
5
通讯作者：
Aliño, SF
中科院分区：
文献类型：
--
作者：
Dasí, F;Lledó, S;Aliño, SF
最近的报告（Chen 等，2000；Kopreski 等，1999）指出，在癌症患者的无细胞血清中可检测到肿瘤来源的 RNA，这为癌症的早期检测和随访开辟了新的分子方法。由于端粒酶活性是鉴定人类癌症最通用的分子标志物，因此血清中端粒酶 mRNA 表达的分析作为潜在的肿瘤标志物很有吸引力。不同的基因编码人类端粒酶复合物的各种成分。尽管已报道端粒酶逆转录酶（hTERT）表达与端粒酶活性之间存在良好相关性（de Kok 等，1999），但迄今为止尚未在血清或血浆中进行定量研究。本研究报告了一种用于测量癌症患者血浆中 hTERT 表达的定量测定方法的开发，该测定方法基于使用甘油醛-3-磷酸脱氢酶 (GAPDH) 表达标准化到 RNA 输入量的实时定量 RT-PCR (qRT-PCR)。通过 qRT-PCR 测量了 19 名结直肠癌或滤泡性淋巴瘤患者以及 10 名健康对照受试者的血浆 hTERT mRNA。结果表明，该程序在健康受试者和癌症患者之间具有高度区分性，并强烈支持了利用血浆中的这种遗传标记可以开发出有价值的癌症诊断和预后测试的想法。我们分析了 10 名患者的原发性结直肠癌切除组织。来自同一患者的邻近正常粘膜被用作正常RNA的来源。血浆和血液单核细胞取自 18 名癌症患者（9 名结直肠癌和 9 名滤泡性淋巴瘤）和 10 名无病理证据的健康受试者，装在含有柠檬酸钠凝胶和密度梯度介质（Vacutainer CPT# 362761；BD，Franklin Lakes，New Jersey）的 8 ml 采血管中。按照制造商的说明，从 50 mg 组织和 250 μl 血浆的 1 ml TRIZOL（Life Technologies，佩斯利，英国）中分离出 RNA，并添加 5 μg 无 RNase 糖原（GIBCO-BRL，Gaithersburg，Maryland）作为载体。使用 Nucleospin RNA II 试剂盒（Macherey-Nagel，伊斯顿，宾夕法尼亚州）进一步纯化 RNA，其中包括对样品进行 DNase I 处理。通过分光光度法对不含 DNA 的 RNA 进行定量。根据制造商的说明，使用 Taq-Man 逆转录试剂盒（Applied Biosystems，Foster City，California）对纯化的 RNA（200 ng）进行逆转录。随机六聚体在 20°C 退火 10 分钟后，在 42°C 进行 cDNA 合成 45 分钟，然后在 95°C 进行 5 分钟灭活步骤。对于 PCR 分析，将 5 μl RT 反应物与 45 μl 含有 250 nM 正向 (hTERT-Fw: 5'-ACCGTCTGCGTGAGGAGATC-3') 的 TaqMan Universal Master Mix 混合，反向 （hTERT-Rv：5'-CCGGTAGAAAAAAGAGCCTGTTC-3'）引物和 125 nM TaqMan 探针（hTERT-探针：5'-FAM-TGTACGTCGTCGAGCTGCTCAGGTCTTT-TAMRA-3'）。引物
T he recent report (Chen et al, 2000; Kopreski et al, 1999) that tumor-derived RNA is detectable in cell-free serum of cancer patients has opened up a new molecular approach for the early detection and follow-up of cancer. Because telomerase activity is the most general molecular marker for the identification of human cancer, the analysis of telomerase mRNA expression in serum is attractive as a potential tumor marker. Different genes encode for the various components of the human telomerase complex. Although a good correlation between telomerase reverse transcriptase (hTERT) expression and telomerase activity has been reported (de Kok et al, 1999), no quantitative studies in serum or plasma have been performed to date. The present study reports the development of a quantitative assay for the measurement of hTERT expression in the plasma of cancer patients based on real-time quantitative RT-PCR (qRT-PCR) normalized to the amount of RNA input using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression. Plasma hTERT mRNA was measured by qRT-PCR in 19 patients with colorectal cancer or follicular lymphoma, and in 10 healthy control subjects. The results show that this procedure highly discriminates between healthy subjects and cancer patients and strongly support the idea that a valuable diagnostic and prognostic test for cancer might be developed using this genetic marker in plasma.We analyzed tissues from excised primary colorectal cancer in 10 patients. The adjacent normal mucosa from the same patients was used as sources of normal RNA. Plasma and blood mononuclear cells were obtained from 18 cancer patients (9 with colorectal cancer and 9 with follicular lymphoma) and from 10 healthy subjects with no evidence of pathology, in 8-ml blood collection tubes containing sodium citrate gel and a density gradient medium (Vacutainer CPT# 362761; BD, Franklin Lakes, New Jersey). RNA was isolated from 50 mg of tissue and 250 μl of plasma in 1 ml of TRIZOL (Life Technologies, Paisley, United Kingdom) following the instructions of the manufacturer, with the addition of 5 μg of RNase-free glycogen (GIBCO-BRL, Gaithersburg, Maryland) as carrier. RNA was further purified using the Nucleospin RNA II kit (Macherey-Nagel, Easton, Pennsylvania), which includes a DNase I treatment of the sample. DNA-free RNA was quantified spectrophotometrically. Purified RNA (200 ng) was reverse transcribed with the Taq-Man Reverse Transcription reagents kit (Applied Biosystems, Foster City, California) according to the instructions of the manufacturer. After random hexamers annealed for 10 minutes at 20 C, cDNA synthesis was performed for 45 minutes at 42 C, followed by an inactivation step for 5 minutes at 95 C. For PCR analysis, 5 μl of the RT reaction were mixed with 45 μl of TaqMan Universal Master Mix containing 250 nM of the forward (hTERT-Fw: 5'-ACCGTCTGCGTGAGGAGATC-3') and reverse (hTERT-Rv: 5'-CCGGTAGAAAAAAGAGCCTGTTC-3') primers and 125 nM of the TaqMan probe (hTERT-Probe: 5'-FAM-TGTACGTCGTCGAGCTGCTCAGGTCTTT-TAMRA-3'). Primers