An altered miTRAP method for miRNA affinity purification with its pros and cons.

The major mechanisms of posttranscriptional gene regulation involve microRNAs (miRs) and RNA-binding proteins (RBPs). Recent studies not only identified functionally and characterized such factors, but rather investigated their use as biomarkers and suitability as biopharmaceuticals. Indeed, some miR-based drugs are currently tested in clinical studies as potential anti-viral and as anti-cancer agents. For the chemical application, a profound knowledge of the binding affinities of miRs and RBPs to their target RNA is essential. The authors recently identified several miRs regulating the non-classical human leukocyte antigen (HLA)-G, and characterized their binding affinity to the 3' untranslated region (UTR) of HLA-G. These miRs identified by miTRAP were classified into high affinity and low affinity miRs, which were either key regulators or fine tuners of HLA-G. While the miTRAP method has been described in detail, a novel modified miTRAP technique has been established, which completely consists of commercially available components and uses a simplified cloning strategy. This technique allows the identification and characterization of miRs and RBPs for any RNA sequence of interest.