7. microRNA Research

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''High Efficiency'' microRNA Cloning Kit
microRNA Cloning Kit Wako
Wako Catalog No. 290-66501 (8 Reactions) <for Genetic Research>
microRNA Cloning Kit Wako is patent pending. (1, 10, 2007)

The microRNA Cloning Kit Wako can prepare the cDNA encoding microRNA. The cloning procedure will be completed within 1.5 days after preparation of microRNA fraction. This kit is supported by shrimp alkaline phosphatase (SAP), thermostable single strand DNA ligase(, which is selling separately: Wako Cat. #292-65101 (500 units); #298-65103 (200 units)), and original modifi ed adaptors.
The cloning efficiency using this kit is improved higher than that of the conventional methods, which used bacterial alkaline phosphatase and T4 RNA ligase.
[Features]
1. High cloning effi ciency
2. Cloning of secondary structured microRNA
3. High reproducibility of microRNA Cloning
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[Procedure of microRNA cloning]
1) Preparation of total RNA from HeLa cells (1×107 cells) by ISOGEN (Nippon Gene #315-02504, 10mL).
2) Preparation of small RNA fraction, less than 200nt, from total RNA by microRNA Isolation Kit (Bio Chain Institute Inc. catalog #KS341025).
3) Separation of microRNA fraction by denaturing PAGE.
4) Collection of the gels of 20~23nt region after electrophoresis.
5) Cloning by using microRNA Cloning Kit Wako (Wako catalog #290-66501).
6) Construction of the plasmids harboring cDNA encoding microRNA and transformation of E. coli.
7) Random selection of the 96 transformed E. coli from selection LB agar medium.
8) Determination and verifi cation of the cDNA sequences by using Sanger miRBase.


[ Figure. 1 ]
Cloning effi ciency of microRNA from HeLa cell lysate. The cloning effi ciency of microRNA was more than 70%. Others indicate that isolated cDNA sequences were not matched miRBase. Unknowns indicate that isolated cDNA sequences were not matched human genome sequence. The contents of cloned microRNA species are indicated on Table 1.

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Wako Product Update Bio-No.1 [ page. 21 ]