The MirTrap System—A powerful tool for discovering and enriching miRNA targets
- Identification of even transient miRNA targets
- Efficient pull-down of miRNA targets from all cell types
- Complete, simplified workflow
What are the challenges in studying microRNA targets?
MicroRNAs are key components in eukaryotic gene expression regulation, guiding the RNA-induced silencing complex (RISC) to a target mRNA. To properly identify and study an miRNA and its target(s), researchers typically rely on immunoprecipitation of the RISC in the hope of also pulling down the bound miRNA/mRNA pair. Herein lies the problem: this interaction is extremely transient (Figure 1), and may not survive the pull-down process. Verification and study of low-abundance miRNA/target mRNAs can be a near impossible task in the face of these circumstances.
Figure 1. Transient interaction of RISC and miRNA/mRNA pair, resulting in mRNA processing.
How does the MirTrap System solve the problem?
The MirTrap System (Figure 2) utilizes a dominant negative mutation of a RISC protein subunit (called the “MirTrap protein”). When expressed in mammalian cells, this protein locks your miRNA/mRNA pair into the RISC and limits further processing. A DYKDDDDK (FLAG epitope) tag on the MirTrap protein allows for immunoprecipitation of the complex, which can then be efficiently pulled down without losing your microRNA/target mRNA pairs along the way. Targets that would have been lost using less sensitive methods are instead ready for downstream analysis—either enriching and verifying known miRNA targets with qRT-PCR, or discovering new targets via Next Generation Sequencing (NGS).
Figure 2. Experimental workflow for microRNA transfection and isolation of target mRNAs using the MirTrap System.