Related Experiment Video
Updated: Aug 2, 2026
High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs
Published on: August 3, 2011
An integrated ion-exchange membrane-based microfluidic device for irreversible dissociation and quantification of
Kyle P McCarthy1, David B Go1,2, Satyajyoti Senapati1
1Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, Indiana 46556, USA. hchang@nd.edu.
Abstract:
Ribonucleoproteins (RNPs), particularly microRNA-induced silencing complex (miRISC), have been associated with cancer-related gene regulation. Specific RNA-protein associations in miRISC complexes or those found in let-7 lin28A complexes can downregulate tumor-suppressing genes and can be directly linked to cancer. The high protein-RNA electrostatic binding affinity is a particular challenge for the quantification of the associated microRNAs (miRNAs). We report here the first microfluidic point-of-care assay that allows direct quantification of RNP-associated RNAs, which has the potential to greatly advance RNP profiling for liquid biopsy. Key to the technology is an integrated cation-anion exchange membrane (CEM/AEM) platform for rapid and irreversible dissociation (k = 0.0025 s-1) of the RNP (Cas9-miR-21) complex and quantification of its associated miR-21 in 40 minutes. The CEM-induced depletion front is used to concentrate the RNP at the depletion front such that the high electric field (>100 V cm-1) within the concentration boundary layer induces irreversible dissociation of the low KD (∼0.5 nM) complex, with ∼100% dissociation even though the association rate (kon = 6.1 s-1) is 1000 times higher. The high field also electrophoretically drives the dissociated RNA out of the concentrated zone without reassociation. A detection limit of 1.1 nM is achieved for Cy3 labelled miR-21.
Insights
This study introduces a novel microfluidic assay for quantifying microRNAs bound to ribonucleoproteins (RNPs). The technology enables rapid, direct measurement of RNP-associated RNAs for advanced cancer diagnostics.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Ribonucleoproteins (RNPs), including microRNA-induced silencing complex (miRISC), play a role in cancer-related gene regulation.
- Dysregulation of tumor-suppressing genes by specific RNA-protein complexes is linked to cancer development.
- Quantifying microRNAs (miRNAs) bound to RNPs is challenging due to high protein-RNA binding affinity.
Purpose of the Study:
- To develop a microfluidic point-of-care assay for direct quantification of RNP-associated RNAs.
- To enable advanced RNP profiling for applications like liquid biopsy.
- To overcome the challenge of high protein-RNA binding affinity in RNP complex analysis.
Main Methods:
- Development of a microfluidic assay utilizing an integrated cation-anion exchange membrane (CEM/AEM) platform.
- Application of a high electric field (>100 V cm⁻¹) to induce rapid and irreversible dissociation of RNP complexes (e.g., Cas9-miR-21).
- Concentration of RNPs at a depletion front for efficient dissociation and subsequent electrophoretic separation of RNA.
Main Results:
- Achieved rapid and irreversible dissociation of RNP complexes (k = 0.0025 s⁻¹) within 40 minutes.
- Demonstrated ~100% dissociation of low Kᴅ (∼0.5 nM) complexes despite high association rates (k<0xE2><0x82><0x99> = 6.1 s⁻¹).
- Established a detection limit of 1.1 nM for Cy3-labeled miR-21, with dissociated RNA driven out of the concentrated zone without reassociation.
Conclusions:
- The developed microfluidic assay allows direct quantification of RNP-associated RNAs.
- This technology offers potential for advancing RNP profiling in liquid biopsies for cancer detection.
- The CEM/AEM platform effectively dissociates stable RNP complexes, facilitating accurate miRNA quantification.
More Related Videos
13:34Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip
Published on: September 29, 2012
10:28Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs
Published on: April 14, 2015
