miRNA Amplification Profiling (mRAP)

Abstract

The PCR methods described in the previous chapters focus on miRNA expression detection, but are not designed for the discovery of new miRNAs. Mano and Takada from the Division of Functional Genomics, Jichi Medical University (Shimotsukeshi, Tochigi, Japan) developed a modified PCR approach, termed miRNA amplification profiling (mRAP) (Nucleic Acids Res 34:e115, 2006; Methods 43:118–122, 2007; Nat Protoc 2:3136–3145, 2007) that relies on the traditional miRNA cloning approach (Curr Biol 12:735–739, 2002) but with the substitution of the SMART (switching mechanism at the 5′-end of RNA templates of reverse transcriptase) (Science 294:862–864, 2001) reaction for the ligation of a synthetic primer to the 5′-end of small RNAs. mRAP combines cloning for new miRNA discovery, high throughput profiling, and quantification of miRNA levels into one. This approach is highly sensitive, readily allowing the isolation of >1 × 10⁴ independent miRNA-derived cDNAs from ≤1 × 10⁴ cells. The mRAP method thus makes it possible to analyze miRNA expression profiles for small quantities of tissue or cells such as fresh clinical specimens. The mRAP method can be performed in a conventional molecular biology laboratory, it readily allows the processing of multiple samples in parallel, it is able to detect a 1- nt difference among miRNAs, and is capable of identifying both new miRNAs and mutations in known miRNAs. mRAP may currently be the best choice for obtaining expression profiles for both known and unknown miRNAs or for identification of sequence alterations in miRNAs with small amounts of starting material. However, the mRAP is a complex, multistep procedure, and may not be practical for clionical use as a diagnostic tool.