Quantification of microRNAs, splicing isoforms, and homologous mRNAs with the invader assay

Peggy S. Eis, Mariano Garcia-Blanco

Research output: Chapter in Book/Report/Conference proceedingChapter

2 Scopus citations

Abstract

The understanding of physiology and pathology requires accurate quantification of intracellular concentrations of important molecules such as unique RNA species. Accurate quantification of highly homologous messenger RNAs (mRNAs) (1-3), alternatively spliced mRNAs (4), and the short microRNAs (miRNAs) (5,6) has been successfully achieved using the Invader assay. This method directly detects specific RNA molecules in preparations of pure total cellular RNA (1- 100 ng) or in crude cell lysate (10<sup>3</sup>-10<sup>4</sup> cells) samples using an isothermal signal amplification process with a fluorescence resonance energy transfer (FRET)-based fluorescence readout. Features of the Invader assay include the ability to detect 1-10 RNA molecules per cell, to discriminate between RNAs that differ by a single base, and to precisely measure 1.2-fold changes in RNA expression. Further, an isothermal format and the ability to detect two different RNA molecules with a biplex format make the Invader assay suitable for high-throughput screening applications.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press
Pages279-318
Number of pages40
Volume488
ISBN (Print)9781588294197
DOIs
StatePublished - 2008
Externally publishedYes

Publication series

NameMethods in Molecular Biology
Volume488
ISSN (Print)10643745

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Keywords

  • Alternative splicing
  • Cleavase enzyme
  • FRET
  • Gene expression
  • High-throughput screening
  • HTS
  • Invader assay
  • Invasive cleavage
  • microRNA
  • miRNA
  • mRNA
  • RNA quantification/quantitation
  • Splice variant

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

Cite this

Eis, P. S., & Garcia-Blanco, M. (2008). Quantification of microRNAs, splicing isoforms, and homologous mRNAs with the invader assay. In Methods in Molecular Biology (Vol. 488, pp. 279-318). (Methods in Molecular Biology; Vol. 488). Humana Press. https://doi.org/10.1007/978-1-60327-475-3_20