Rapid and direct microRNA quantification by an enzymatic luminescence assay

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)...

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Veröffentlicht in:Analytical biochemistry 2012-10, Vol.429 (1), p.11-17
Hauptverfasser: Sun, Ye, Gregory, Kalvin J., Chen, Nelson G., Golovlev, Val
Format: Artikel
Sprache:eng
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Zusammenfassung: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 R2=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.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2012.06.021