Facile means for quantifying microRNA expression by real-time PCR
Facile means for quantifying microRNA expression by real-time PCR
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DOI：
10.2144/000112010
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发表时间：
2005-10-01
期刊：
影响因子：
2.7
通讯作者：
Chiang, VL
中科院分区：
文献类型：
--
作者：
Shi, R;Chiang, VL
MicroRNA（miRNA）是20-24个核苷酸RNA，预计将在动物和植物中起调节作用。在这里，我们报告了一种简单敏感的实时PCR方法，用于量化植物miRNA的表达。总RNA（包括miRNA）是聚腺苷酸化的，并使用miRNA特异性正底漆和与聚（t）衔接子作为反向引物的序列互补的聚（T）适配器对cDNA进行实时PCR进行反向转录，作为实时PCR。使用SYBR（R）绿色试剂对miRNA序列进行测试，表明该方法使用低至100 pg的总RNA可以很容易地区分具有一个核苷酸序列差异的miRNA的表达。该方法还揭示了miRNA组织特异性表达模式，这些表达模式无法通过北印迹分析来解决，因此对于表征植物和动物中的miRNA表达可能广泛有用。
MicroRNAs (miRNAs) are 20-24 nucleotide RNAs that are predicted to play regulatory roles in animals and plants. Here we report a simple and sensitive real-time PCR method for quantifying the expression of plant miRNAs. Total RNA, including miRNAs, was polyadenylated and reverse-transcribed with a poly(T) adapter into cDNAs for real-time PCR using the miRNA-specific forward primer and the sequence complementary to the poly(T) adapter as the reverse primer Several Arabidopsis miRNA sequences were tested using SYBR (R) Green reagent, demonstrating that this method, using as little as 100 pg total RNA, could readily discriminate the expression of miRNAs having as few as one nucleotide sequence difference. This method also revealed miRNA tissue-specific expression patterns that cannot be resolved by Northern blot analysis and may therefore be widely useful for characterizing miRNA expression in plants as well as in animals.