Purification of total RNA from human stool samples
Purification of total RNA from human stool samples
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DOI：
10.1023/a:1026699126899
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发表时间：
1998-12-01
影响因子：
3.1
通讯作者：
Raicht, RF
中科院分区：
文献类型：
--
作者：
Alexander, RJ;Raicht, RF
虽然结肠镜检查可以发现早期结肠肿瘤，但一种侵入性更小、成本效益更高的技术将是有益的。粪便可以收集脱落的结肠上皮细胞，是对粘膜取样的理想选择；脱落肿瘤细胞可能在完整肿瘤中表现出基因表达的改变。然而，首先有必要证明RNA可以从人类粪便中分离出来，并且这种RNA含有人类基因转录物。因此，我们开发了一种从新鲜排出的人粪便中分离总RNA的方法，包括在混乱剂中裂解，用苯酚和苯酚氯仿重复提取，并用RNA结合树脂吸收。在使用RNase-free DNase I处理后，我们通过定量槽印迹、northern印迹和逆转录聚合酶链反应（RT-PCR）来检测这些制剂中人类RNA的存在，我们从癌症患者的粪便中获得了每克5-30 μ g RNA，每克对照粪便中获得了约5 μ g RNA。定量槽印迹显示，该RNA中约有10%来自人类。northern blotting和RT-PCR均证实这些样品中存在人类RNA。为了明确地证明从粪便中分离RNA，我们将大鼠细胞和对照人粪便混合在37℃下孵育24小时，从该样本中分离的RNA的RT-PCR清楚地显示了大鼠特异性mRNA的存在。这些实验表明，RNA可以从人类粪便中分离出来，并且可以在这些制剂中检测人类基因编码的信息。这一过程可能为识别有结肠癌风险的患者提供一个强有力的工具。
While colonoscopy may detect early-stage colon tumors, a less invasive and more cost-effective technique would be beneficial. Stool, which picks up sloughed-off colonic epithelial cells, would be ideal for sampling the mucosa; shed tumor cells may display alterations in gene expression observed in intact tumors. It is first necessary, however, to show that RNA can be isolated from human feces and that this RNA contains human gene transcripts. We have therefore developed a method for the isolation of total RNA from freshly passed human stool, consisting of lysis in chaotropic agents, repeated extraction with phenol and phenolchloroform, and absorption with an RNA-binding resin. After treatment with RNase-free DNase I, we assayed these preparations for the presence of human RNA by quantitative slot blotting,northern blotting, and reverse transcription-polymerase chain reaction (RT-PCR), We obtained 5-30 mu g RNA per gram of stool from cancer patients, and about 5 mu g RNA per gram of control stool. Quantitative slot blotting showed that about 10% of this RNA was of human origin. Both northern blotting and RT-PCR demonstrated the presence of human RNA in these samples. To unambiguously demonstrate the isolation of RNA from stool, we incubated a mixture of rat cells and control human stool at 37 degrees C for up to 24 hr, RT-PCR of the RNA isolated from this sample clearly revealed the presence of rat-specific mRNA. These experiments indicate that RNA can be isolated from human stool and that message encoded by human genes can be assayed in these preparations. This procedure may provide a powerful tool to identify patients at risk for colon cancer.