**Acknowledgement**

We thank Sanga Mitra and Smarajit Das of Indian Association for the Cultivation of Science for their valuable suggestions.

### **11. References**


[12] Ren Y., Gong W., Xu Q., Zheng X., Lin D., Wang Y., Li T. (2006). siRecords: an extensive database of mammalian siRNAs with efficacy ratings. *Bioinformatics,* Vol. 22, (January 2006), pp. (1027)

**Chapter 12** 

© 2012 Mizuguchi et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

© 2012 Mizuguchi et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

**Novel microRNA Cloning Using Bioinformatics** 

Yoshiaki Mizuguchi, Takuya Mishima, Eiji Uchida and Toshihiro Takizawa

MicroRNAs (miRNAs) participate in several biological processes, including development, differentiation, apoptosis, and proliferation (**1, 2**) through imperfect pairing with target messenger RNAs (mRNAs) of protein-coding genes and transcriptional or posttranscriptional regulation of their expression (**3, 4**). Approaches to miRNA detection, such as parallel sequencing technologies may replace conventional sequencing (**5**). The GS 454 technology can produce a similar number of longer (100–150-nucleotides (nt)) sequence reads in a single analysis run, with the advantage that this method can derive the complete sequence of the mature miRNA. Moreover, recent studies on miRNA profiling performed with cloning techniques suggest that sequencing methods are suitable for the detection of novel miRNAs, modifications, and precise compositions, and that cloning frequencies calculated by clone count analysis strongly correlate with the concentrations measured by Northern blotting, and are reproducible. The achievement of comprehensive profiling of miRNA in human diseases requires exhaustive qualitative and quantitative analyses. Here we show the techniques and the some of the results of the miRNA transcriptomes in the liver using sequencing. This serves as a critical step in clarifying the functional significance

**2. Techiniques of MicroRNA cloning and bioinformatics for MiRNAome** 

The method for microRNA cloning and sequencing that we moderated from the original ones are shown in Fig1. We cloned small RNA by a modification of the published miRNA cloning protocol of Lagos-Quintana et al. **(6)**. In brief, total RNA samples were extracted using ISOGEN (Nippon Gene, Tokyo, Japan), separated in a denaturing polyacrylamide gel, and the 18–24 nt fraction was recovered. Next, 5′- and 3′-adapters were ligated to the RNAs Ligation of small RNAs with DNA\_RNA chimera linkers at both termini [3' linker

Additional information is available at the end of the chapter

of specific miRNAs as they relate to liver diseases.

**2.1. MicroRNA cloning** 

http://dx.doi.org/10.5772/53945

**1. Introduction** 

