miR-TRAP is performed in three basic steps. Scientists 1) produce highly photoreactive probes by conjugating psoralen, a plant molecule that can be activated by light, to an miRNA of interest, 2) perform a long-wave UV photocrosslinking reaction, and 3) pull down RNA and analyze it by RT-qPCR. In other words, researchers zap cells with UV light, freezing the miRNA/mRNA duo in place. Then, after extracting the RNA from the cells, they can take a closer look at the sequence of the bound mRNA, revealing the miRNA's target gene.
Advantages of miR-TRAP
miR-TRAP is easier and more accurate than current methods of identifying miRNA targets for three main reasons. First, miR-TRAP can directly identify miRNA targets in live cells, under normal or disease conditions. Second, this technique can spot mRNA targets that are not only reduced by miRNAs, but also those whose translation into protein is repressedtargets that aren't normally picked up by other techniques, such as qPCR or microarray analysis. Third, miR-TRAP doesn't rely on antibodies, which can lead to nonspecific background signals and complicate data interpretation.
Putting miR-TRAP to the test, Rana and his team, including postdoctoral researcher Huricha Baigude, Ph.D., analyzed 13 predicted targets of two important microRNAs. The technique not only confirmed their known gene targets, but also revealed two novel targets.
"We're now applying these methods to identify miRNA targets in a number of disease models," Rana said. "And it's our hope that miR-TRAP will soon become common practice in many labs around the world."
|Contact: Heather Buschman, Ph.D.|
Sanford-Burnham Medical Research Institute