Sharbati-Tehrani Soroush, Kutz-Lohroff Barbara, Scholven Jutta, Einspanier Ralf
Institute of Veterinary-Biochemistry, Freie Universität Berlin, Oertzenweg 19b, 14163 Berlin, Germany.
Biochem Biophys Res Commun. 2008 Oct 24;375(3):484-9. doi: 10.1016/j.bbrc.2008.08.048. Epub 2008 Aug 21.
While the number of human or murine microRNAs (miRNAs) increases continuously, there are limited data available from other species. We report a novel identification method of small RNAs such as miRNAs, which allows simultaneous cloning of five RNA molecules within the same insert. First, RNA molecules <40nt were polyadenylated and five concatamerising 5' DNA adaptors were ligated to the molecules in independent reactions. Reverse transcription was carried out using oligo d(T)(18) primers with concatamerising 5' overhangs. The introduced complementary termini in the different reactions enabled the subsequent coupling of five purified antisense strands to one molecule by means of an assembly PCR. After cloning, small RNAs were identified by DNA sequencing. By means of this cloning approach, we identified 10 novel and one known porcine miRNAs. Furthermore, the endogenous expression of the cloned miRNAs was quantified in various tissues using a qRT-PCR approach.
虽然人类或小鼠微小RNA（miRNA）的数量在不断增加，但来自其他物种的数据有限。我们报告了一种新的小RNA（如miRNA）鉴定方法，该方法允许在同一插入片段中同时克隆五个RNA分子。首先，对长度小于40个核苷酸的RNA分子进行聚腺苷酸化，并在独立反应中将五个串联的5' DNA接头连接到这些分子上。使用带有串联5' 突出端的寡聚d(T)(18)引物进行逆转录。不同反应中引入的互补末端使得随后能够通过组装PCR将五个纯化的反义链偶联到一个分子上。克隆后，通过DNA测序鉴定小RNA。通过这种克隆方法，我们鉴定出10种新的和1种已知的猪miRNA。此外，使用qRT-PCR方法对克隆的miRNA在各种组织中的内源性表达进行了定量。
