Short Report
A useful approach to total analysis of RISC-associated RNA
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* Corresponding author: Yukinobu Hayashida hayashida.yukinobu@wako-chem.co.jp
- † Equal contributors
1 Genome Research Laboratories, Wako Pure Chemical Industries, Ltd. Takada 6-1, Amagasaki, Hyogo 661-0963, Japan
2 Department of Biology, Graduate School of Science, Kobe University, 1-1 Rokkodaicho, Nadaku, Kobe 657-8501, Japan
BMC Research Notes 2009, 2:169 doi:10.1186/1756-0500-2-169
Published: 26 August 2009Abstract
Background
Identifying the endogenous RNA induced silencing complex(RISC)-associated RNAs is essential for understanding the cellular regulatory networks by miRNAs. Recently, isolation of RISC-associated mRNAs using antibody was reported, but their method needs a large amount of initial materials. We tried to improve the protocol and constructed an efficient and convenient system for analyzing miRNA and mRNA contents in RISC.
Findings
With our protocol, it is possible to clone both miRNAs and mRNAs from the endogenous RISC-associated RNAs immunoprecipitated from less than 107 cells, and we show the ability of our system to isolate the particular target mRNAs for a specific miRNA from the RISC-associated mRNAs using well-characterized miR-122 as an example. After introduction of miR-122 into HepG2 cells, we found several cDNA clones that have miR-122 target sequences. Four of these clones that were concentrated in RISC but decreased in total RNA fraction are expected to be miR-122 target candidates. Interestingly, we found substantial amounts of Alu-related sequences, including both free Alu RNA and Alu-embedded mRNA, which might be one of the general targets for miRNA, in the cDNA clones from the RISC-associated mRNAs.
Conclusion
Our method thus enables us to examine not only dynamic changes in miRNA and mRNA contents in RISC but also the relationship of miRNA and target mRNA. We believe that our method can contribute to understanding cellular regulatory networks by miRNAs.