Wex Thomas, Treiber Gerhard, Lendeckel Uwe, Malfertheiner Peter
Department of Gastroenterology, Hepatology and Infectious Diseases, Otto-von-Guericke University, Magdeburg, Germany.
Clin Chem Lab Med. 2003 Aug;41(8):1033-7. doi: 10.1515/CCLM.2003.159.
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 column-based extraction method, resulted in a yield of 4.5 microg 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 28S-RNA bands in agarose gel electrophoresis were suitable for quantitative RT-PCR as shown by expression of corpus-specific pepsinogen C-mRNA and the duodenum-specific multi-drug 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 two-step method described here is a suitable option for extracting high-quality RNA from endoscopic biopsies when other standard protocols fail.
定量逆转录聚合酶链反应（qRT-PCR）等分子技术的应用取决于细胞RNA的质量。特别是，从内镜活检组织中提取RNA在产量方面存在困难，完整性方面尤为突出。取自胃窦、胃体和十二指肠的内镜活检组织采用了各种RNA提取方案，并将提取的RNA用于定量RT-PCR。随后使用两种方法，（i）酚/氯仿提取法和（ii）基于柱的提取法，在80%的样本中，每次活检可获得产量为4.5微克的总RNA，且质量可靠。定量RT-PCR分析表明，只有在琼脂糖凝胶电泳中清晰显示18S和28S RNA条带的RNA样本才适合用于定量RT-PCR，胃体特异性胃蛋白酶原C-mRNA和十二指肠特异性多药耐药蛋白1（mdr-1）-mRNA的表达情况也证明了这一点。在部分降解的RNA中，仍可检测到胃蛋白酶原C、mdr-1或β-肌动蛋白mRNA，但定量测定给出的数据不一致。当其他标准方案失败时，这里描述的两步法是从内镜活检组织中提取高质量RNA的合适选择。
