Profiling of microRNA expression by mRAP.
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 x 10(4) 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.
Takada
Shuji
S
Division of Functional Genomics, Jichi Medical University, 3311-1 Yakushiji, Shimotsukeshi, Tochigi 329-0498, Japan.
Mano
Hiroyuki
H
D020869:Gene Expression Profiling / Q000379:methods*; D035683:MicroRNAs / Q000235:genetics* / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques
D035683:MicroRNAs