title: Rapid and direct microRNA quantification by an enzymatic luminescence assay.
abstract: A quantitative bioluminescence assay for rapid and sensitive microRNA (miRNA) expression analysis was developed. The assay uses miRNA directly as a primer for binding to a circular single-stranded DNA template, followed by rolling circle amplification. The detection of inorganic pyrophosphate (PPi) molecules released during the DNA polymerization and amplification process is performed by a multi-enzyme system. PPi is converted to ATP by ATP-sulfurylase, which provides energy for luciferase to oxidize luciferin and produce light. Experimental results show that the assay has a dynamic range exceeding three orders of magnitude and the ability to discriminate miRNAs with high-homology sequences. Quantification of nine miRNAs in human heart tissues demonstrated high cross-platform consistency between this assay and the TaqMan real-time polymerase chain reaction (PCR) assay with R(2)=0.941. The assay requires fewer reagents, can be performed at an isothermal condition without thermal cycling, and is capable of detecting miRNAs in less than 1h. Compared with the real-time PCR and microarray-based detection methods, this assay provides a simpler, faster, and less expensive platform for miRNA quantification in life science research, drug discovery, and clinical diagnosis.
authors: Sci-Tec, Inc., 156 Fairbanks Road, Oak Ridge, TN 37830, USA. ye_sun@scien-tec.com
authors: Ye
authors: Y
authors: Sun
authors: Kalvin J
authors: KJ
authors: Gregory
authors: Nelson G
authors: NG
authors: Chen
authors: Val
authors: V
authors: Golovlev
mesh_terms: D001483:Base Sequence; D011756:Diphosphates / Q000378:metabolism; D004798:Enzymes / Q000378:metabolism*; D006801:Humans; D008163:Luminescent Measurements / Q000379:methods*; D035683:MicroRNAs / Q000032:analysis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D021141:Nucleic Acid Amplification Techniques; D015345:Oligonucleotide Probes / Q000737:chemistry / Q000235:genetics; D048348:Reverse Transcription; D013997:Time Factors
chemical_list: D011756:Diphosphates; D004798:Enzymes; D035683:MicroRNAs; D015345:Oligonucleotide Probes