A two-step method for the extraction of high-quality RNA from endoscopic biopsies
A two-step method for the extraction of high-quality RNA from endoscopic biopsies
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DOI：
10.1515/cclm.2003.159
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发表时间：
2003-01-01
影响因子：
6.8
通讯作者：
Malfertheiner, P
中科院分区：
文献类型：
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作者：
Wex, T;Treiber, G;Malfertheiner, P
定量RT-PCR等分子技术的使用取决于细胞RNA的质量。特别地，从内窥镜活检中提取RNA在产量方面是困难的，特别是完整性。对从胃窦、胃体和十二指肠采集的内镜活检组织进行各种RNA提取方案，并将RNA用于定量RT-PCR。随后使用两种方法，（i）苯酚/氯仿提取和（ii）基于柱的提取方法，导致每次活检的总RNA产量为4.5 g，80%的样品质量可靠。定量RT-PCR分析表明，只有在琼脂糖凝胶电泳中清晰显示18 S和28 SRNA条带的RNA样品才适合进行定量RT-PCR，这与体特异性胃蛋白酶原CmRNA和十二指肠特异性多药耐药蛋白-1（mdr-1）mRNA的表达有关。在部分降解的RNA中，胃蛋白酶原C、mdr-1或β-肌动蛋白mRNA仍可检测到，但定量测定得到的数据不一致。当其他标准方案失败时，这里描述的两步法是从内窥镜活检中提取高质量RNA的合适选择。
The use of molecular techniques such as quantitative RT-PCR depends on the quality of cellular RNA. In particular, RNA extraction from endoscopic biopsies is difficult with respect to yield, and especially integrity. Endoscopic biopsies taken from the gastric antrum, corpus and duodenum were subjected to various RNA extraction protocols, and the RNA was used for quantitative RT-PCR. The subsequent use of two methods, (i) a phenol/chloroform extraction and (ii) a columnbased extraction method, resulted in a yield of 4.5 g total RNA per biopsy with reliable quality in 80% of samples. The quantitative RT-PCR analysis revealed that only RNA samples that clearly show both 18S and 28SRNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpusspecific pepsinogen CmRNA and the duodenum-specific multidrug resistance protein-1 (mdr-1)mRNA. In partially degraded RNA, pepsinogen C, mdr-1, or beta-actin mRNAs were still detectable, but the quantitative determination gave inconsistent data. The twostep method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.