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Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay

Abstract

Aberrant expression of miRNAs often correlates with various human diseases. Therefore, miRNAs have been focused as disease biomarkers. Here, a novel application of a bioluminescence (BL) assay for small RNA quantification is described. The assay is based on detecting pyrophosphate (PPi) molecules released during branched rolling circle amplification (BRCA) with a second primer in the presence of target RNA molecules. The number of released PPi molecules is correlated with the target RNA copy number. This assay was capable of detecting at least 20 amol of target RNA molecules, and the dynamic range extended over at least three orders of magnitude. Appropriate use of a second primer allowed for sensitive detection of RNA molecules with a high S/N ratio in less time. Moreover, the assay could specifically detect as low as 0.1 fmol of a target small RNA within a total RNA extract with high reproducibility. These data suggest that our assay has the potential to become a simple, rapid, and highly sensitive method to detect miRNA. Furthermore, this method combined with a BL assay, which utilizes a widely used inexpensive luminometer, could be used for a wider, versatile range of applications.

Basic concept of RNA-primed BRCA combined BL assay to detect short RNAs

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Abbreviations

ATP:

Adenosine 5′-triphosphate

BL:

Bioluminescence

BRCA:

Branched RCA

miRNAs:

MicroRNAs

PEP:

Phosphoenolpyruvate

PPDK:

Pyruvate orthophosphate dikinase

PPi:

Pyrophosphate

RCA:

Rolling circle amplification

RT:

Reverse transcription

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Acknowledgments

We thank Kikkoman Corporation for supplying BL assay reagents. We are also grateful to Keiko Gomi for her comments and help. This work was supported in part by a Grant-in-Aid for Scientific Research on Priority Areas “Lifesurveyor” Grant No.17066002 from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

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Correspondence to Eiry Kobatake.

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Mashimo, Y., Mie, M., Suzuki, S. et al. Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay. Anal Bioanal Chem 401, 221–227 (2011). https://doi.org/10.1007/s00216-011-5083-3

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Keywords

  • Bioluminometric assay
  • MicroRNA detection
  • Pyruvate orthophosphate dikinase
  • Firefly luciferase
  • Branched rolling circle amplification