应用领域
ExactSTART™小RNA克隆试剂盒*从总RNA样品开始，从小RNA转录本生成克隆的文库。该试剂盒选择性地标记并转换包含5´-单磷酸酯/ 3´-羟基末端（5´pN-OH 3´）或5´-三磷酸酯/ 3´-羟基末端（5´pppN-OH 3´）的小RNA，扩增的dsDNA。试剂盒标记的RNA种类包括miRNA，其他小的非编码RNA和小的初级转录本。将扩增的dsDNA连接到pCDC-1K™克隆就绪载体（试剂盒中提供）中，然后转化为感受态大肠杆菌宿主（由用户提供）。
The ExactSTART™ Small RNA Cloning Kit* generates a cloned library derived from small RNA transcripts, starting with a total RNA sample. The kit selectively tags and converts small RNAs that contain 5´-monophosphate/ 3´-hydroxyl ends (5´ pN—OH 3´) or 5´-triphosphate/3´-hydroxyl ends (5´ pppN—OH 3´) to amplified dsDNA. Species of RNA tagged by the kit include miRNA, other small noncoding RNAs, and small primary transcripts. The amplified dsDNA is ligated into the pCDC-1K™ Cloning-Ready Vector (provided in the kit) and then transformed into a competent E. coli host (supplied by the user).
优化的RNA连接反应用于标记5'末端，cDNA合成用于标记RNA的3'末端（图1）。一个可选的步骤是使用独特的RNA 5´多磷酸酶*酶（由EPICENTER科学家发现并表征），可以对带有5´-三磷酸化末端的小初级转录本进行标记和dsDNA合成。zui初的简单RNA大小选择程序可除去大RNA，例如mRNA，18S和28S rRNA，以确保仅将小RNA转化为dsDNA，然后进行克隆。
An optimized RNA ligation reaction is used to tag the 5´ end, and cDNA synthesis is used to tag the 3´ end of the RNAs (Fig. 1). An optional step, using a unique RNA 5´ Polyphosphatase* enzyme (discovered and characterized by EPICENTRE scientists), enables tagging and dsDNA synthesis of small primary transcripts with 5´-triphosphorylated ends. An initial, simple RNA size-selection procedure removes large RNAs such as mRNA, and 18S and 28S rRNA, to ensure that only small RNAs are converted to dsDNA and subsequently cloned.
注意：由于该RNA 3'端的2'-O-甲基基团，无法使用该试剂盒克隆植物miRNA。
Note: Plant miRNAs cannot be cloned with this kit, due to the 2´-O-methyl group at the 3´ end of the RNA.
好处
Table 1. Identification of characterized human miRNAs in a HeLa cDNA library prepared using the ExactSTART™ Small RNA Cloning Kit. Ninety-six randomly selected clones from the library were analyzed by sequencing.
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