mRAP, a sensitive method for determination of microRNA expression profiles.
MicroRNAs (miRNAs) are noncoding RNA molecules of 21-24 nucleotides that regulate the expression of target genes in a posttranscriptional manner. Although evidence indicates that miRNAs play essential roles in embryogenesis, cell differentiation, and pathogenesis of human diseases, extensive miRNA profiling in cells or tissues has been hampered by the lack of sensitive cloning methods. Here we describe a highly efficient profiling strategy, termed miRNA amplification profiling (mRAP), that relies on the use of a long, optimized 5' adaptor, the SMART (switching mechanism at the 5' end of RNA templates of reverse transcriptase) method, the polymerase chain reaction, and cDNA concatamerization after BanI digestion. This approach is highly sensitive, readily allowing the isolation of > 1 x 10(4) independent miRNA-derived cDNAs from < or = 1 x 10(4) 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.
Mano
Hiroyuki
H
Division of Functional Genomics, Jichi Medical University, 3311-1 Yakushiji, Shimotsukeshi, Tochigi 329-0498, Japan. hmano@jichi.ac.jp
Takada
Shuji
S
D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D020869:Gene Expression Profiling / Q000379:methods* / Q000706:statistics & numerical data; D015723:Gene Library; D006801:Humans; D035683:MicroRNAs / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000706:statistics & numerical data; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D035683:MicroRNAs