通过实时聚合酶链反应检测胃癌中的let-7a微小核糖核酸。
Detection of let-7a microRNA by real-time PCR in gastric carcinoma.
作者信息
Zhang Hong-He, Wang Xian-Jun, Li Guo-Xiong, Yang En, Yang Ning-Min
机构信息
Diagnostic Laboratory, Hangzhou First People's Hospital, Hangzhou 310006, Zhejiang Province, China.
出版信息
World J Gastroenterol. 2007 May 28;13(20):2883-8. doi: 10.3748/wjg.v13.i20.2883.
AIM
To establish an accurate and rapid stem-loop reverse transcriptional real-time PCR (RT-PCR) method to quantify human let-7a miRNA in gastric cancer.
METHODS
According to the sequence of let-7a miRNA, the stem-loop reverse transcriptional primer, the primers and quantitative MGB probes of real-time PCR were designed and synthesized. The dynamic range and the sensitivity of quantitative reverse transcriptional real-time PCR were determined. The levels of let-7a miRNA were examined in 32 gastric carcinoma samples by stem-loop RT-PCR method.
RESULTS
The dynamic range and sensitivity of the let-7a miRNA quantification scheme were evaluated, the result showed the assay could precisely detect 10 copies of mature let-7a miRNA in as few as 0.05 ng of total RNA of gastric mucosa. The results of specificity analysis showed no fluorescence signal occurred even though 50 ng of human genomic DNA was added to the reverse transcription (RT) reaction. The expression level of let-7a miRNA in gastric tumor tissues was significantly lower compared to normal tissues in 14 samples from 32 patients.
CONCLUSION
The stem-loop RT-PCR is a reliable method to detect let-7a miRNA which may play an important role in the development of gastric carcinoma.
目的
建立一种准确、快速的茎环逆转录实时荧光定量聚合酶链反应（RT-PCR）方法，用于定量检测胃癌组织中人类let-7a微小RNA（miRNA）。
方法
根据let-7a miRNA的序列，设计并合成茎环逆转录引物、实时荧光定量PCR引物及定量MGB探针。测定定量逆转录实时荧光定量PCR的线性范围和灵敏度。采用茎环RT-PCR法检测32例胃癌组织中let-7a miRNA的表达水平。
结果
对let-7a miRNA定量检测体系的线性范围和灵敏度进行评价，结果显示该方法能在0.05 ng胃黏膜总RNA中精确检测到10个拷贝的成熟let-7a miRNA。特异性分析结果显示，即使在逆转录（RT）反应中加入50 ng人类基因组DNA，也未出现荧光信号。32例患者中14例的胃癌组织中let-7a miRNA的表达水平显著低于正常组织。
结论
茎环RT-PCR是检测let-7a miRNA的可靠方法，let-7a miRNA可能在胃癌发生发展中起重要作用。
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