He Xiang-jun, Zhang Qi, Liu Yu-jing, Pan Xiu-ying
Institute of Clinical Molecular Biology & Central Laboratory, Peking University People's Hospital, Beijing 100044, China.
Beijing Da Xue Xue Bao Yi Xue Ban. 2009 Dec 18;41(6):691-8.
To investigate the non-specific and inaccurate amplification in cases of highly similar sequences among family members and the length heterogeneity of mature microRNA (miRNA), and find a condition that discriminates maximally among similar miRNA family members and detects the accurate expression level of miRNAs.
Primers with their mismatches and/or 3' end at different positions were designed. Amplification efficiencies were compared using matched and various mismatched primers by RNA-tailing and primer-extension RT-PCR at different annealing temperatures. Expression levels of several miRNAs in mouse brain were compared using miRNA specific primers with different termini.
Raising annealing temperatures 12 degrees Celsius-14 degrees Celsius above the T(m) of the primers maximally increased amplification specificity without sacrificing sensitivity. Primers designed with their termini on or near variant positions could efficiently discriminate between miRNA isoforms. Using primers that terminated before the end of the mature miRNA did not miss the detection of shorter mature miRNA and provided accurate expression levels.
Careful primer design and higher annealing temperature can increase specificity and accuracy of real time PCR miRNA detection.
研究家庭成员间高度相似序列情况下的非特异性和不准确扩增以及成熟微小RNA（miRNA）的长度异质性，寻找一种能在相似miRNA家族成员间实现最大区分并检测miRNA准确表达水平的条件。
设计在不同位置存在错配和/或3'端的引物。通过RNA加尾和引物延伸逆转录聚合酶链反应（RT-PCR），在不同退火温度下使用匹配的和各种错配引物比较扩增效率。使用具有不同末端的miRNA特异性引物比较小鼠脑中几种miRNA的表达水平。
将退火温度提高到比引物的解链温度（Tm）高12摄氏度至14摄氏度，可在不牺牲灵敏度的情况下最大程度提高扩增特异性。在变异位置或其附近设计末端的引物能够有效区分miRNA异构体。使用在成熟miRNA末端之前终止的引物不会漏检较短的成熟miRNA，并能提供准确的表达水平。
精心设计引物和提高退火温度可提高实时PCR检测miRNA的特异性和准确性。
