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Profiling of microRNA expression by mRAP

Nature Protocols volume 2, pages 3136–3145 (2007)Cite this article

Abstract

MicroRNA (miRNA) amplification profiling (mRAP) is a sensitive method for the determination of miRNA expression profiles. The method relies on a long, optimized 5′ adaptor and the SMART (switching mechanism at the 5′ end of RNA templates of reverse transcriptase) reaction to yield miRNA-derived cDNAs flanked by synthesized oligomers at each end. The cDNAs are PCR-amplified with primers corresponding to the oligomers, and the products are concatamerized for nucleotide sequencing. The expression level of each miRNA can be estimated from the frequency of the occurrence of its sequence in the data set, provided that sufficient clones of the cDNAs are sequenced. This method potentially yields millions of miRNA-derived clones from as few as 1 × 104 cells, thus allowing the characterization of miRNA expression profiles with small quantities of starting material such as those available for fresh clinical specimens or organs of developing embryos. This protocol can be completed in 10 d.

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Figure 1: Example of size selection of small RNAs.
Figure 2: Schematic representation of the reactions that constitute the microRNA (miRNA) amplification profiling (mRAP) method.
Figure 3: Example of isolation of the cDNA fraction from the dimer and trimer by-products of PCR.
Figure 4: Example of size selection of concatamers.
Figure 5: Example of a nucleotide sequence of one insert obtained from Jurkat cells by the microRNA (miRNA) amplification profiling (mRAP) procedure.

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Acknowledgements

We thank laboratory members for discussion as well as Mika Otani, Kyoko Nakamura and Sayaka Aoyagi for help in preparation of the manuscript. The present work was supported in part by a grant for Third-Term Comprehensive Control Research for Cancer from the Ministry of Health, Labor and Welfare of Japan, and by a grant for Scientific Research on Priority Areas 'Applied Genomics' from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Authors and Affiliations

  1. Division of Functional Genomics, Jichi Medical University, 3311-1 Yakushiji, Shimotsukeshi, 329-0498, Tochigi, Japan

    Shuji Takada & Hiroyuki Mano

  2. CREST, Japan Science and Technology Agency, 332-0012, Saitama, Japan

    Hiroyuki Mano

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  1. Shuji Takada
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Correspondence to Hiroyuki Mano.

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Takada, S., Mano, H. Profiling of microRNA expression by mRAP. Nat Protoc 2, 3136–3145 (2007). https://doi.org/10.1038/nprot.2007.457

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