Rapid and direct microRNA quantification by an enzymatic luminescence assay

Ye Sun*, Kalvin J. Gregory, G. Chen, Val Golovlev

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

26 Scopus citations

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 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 1 h. 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.

Original languageEnglish
Pages (from-to)11-17
Number of pages7
JournalAnalytical Biochemistry
Volume429
Issue number1
DOIs
StatePublished - 1 Oct 2012

Keywords

  • Bioluminescence assay
  • MicroRNA
  • Quantification
  • Rolling circle amplification

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