MicroRNAs are a class of noncoding regulatory RNA molecules that affect gene expression by binding to 3´-untranslated regions of messenger RNAs (mRNAs). Although recently identified as a new class of molecules, initial studies indicate that microRNAs may regulate a significant number of genes within the genome—as many as one-third—and comprise a hidden level of gene regulation that influences a wide range of biological activities and cellular processes, such as cellular proliferation, maintenance, and death; differentiation of cell lines; developmental patterning and timing; and carcinogenesis. Recent findings further suggest that various diseases and conditions are associated with specific microRNA patterns, which may lead to the development of targeted treatments.
As the number of studies involving microRNAs increases, researchers are progressively more interested in accurate, easy-to-use technology that is specifically designed for microRNA analysis; however, microRNA analysis presents several challenges because the microRNAs are short sequences ranging from 17–23 nucleotides in length that are highly similar to each other in sequence with inherently different melting temperatures. The unique combination of a bead-based array and Tm-normalized locked nucleic acids offers a solution for these challenges.
Luminex (www.luminexcorp.com) recently introduced FlexmiR™, a line of products designed specifically for microRNA analysis. FlexmiR was developed by Exiqon (www.exiqon.com) in collaboration with the Luminex Bioscience Group. The FlexmiR microRNA products combine Luminex’ xMAP multiplexing technology with Exiqon’s Locked Nucleic Acids (LNA™) to deliver highly specific results in a simplified workflow.
The favorable reaction kinetics of the liquid bead array delivered by xMAP technology gives faster, more reproducible results than with solid, planar arrays. This “liquid array” approach also offers excellent manufacturing and assay standardization due to the nature of the microspheres when compared to competing flat arrays, which are limited by solid-phase kinetics.
LNA is a conformationally restricted nucleic acid analogue in which the ribose ring is “locked” with a methylene bridge connecting the 2´-O atom with the 4´-C atom, which increases the melting temperature of the nucleic acid duplex by 2–8°C per LNA monomer when integrated into one strand. The FlexmiR microRNA products incorporate LNA technology within the oligonucleotide capture probes to greatly increase the affinity of the probes for their complementary mature microRNA targets leading to significant increases and uniformity of assay specificity.
The FlexmiR microRNA detection panels for human and mouse/rat targets give researchers the ability to measure the expression of microRNA sequences from the public miRBase database using total RNA samples without the need for RNA size fractionation or amplification. The general flow of FlexmiR products allows researchers to biotinylate the 3´ end of total RNA, followed by a hybridization step where the labeled microRNA hybridizes specifically to LNA capture probes coupled to xMAP microspheres. The detection of the biotinylated microRNA is achieved by the reaction with Streptavidin-Phycoerythrin (SA-PE) and final read of the samples in a standard 96-well plate on a Luminex analyzer.
The experiments described here examine the capability of FlexmiR microRNA products to discriminate between two closely related microRNA family members. The capture probes have been Tm-normalized to hybridize optimally under the designed conditions by varying the LNA content and the length of the capture probes.