Optimizing the purification and analysis of miRNAs and mRNAs from RIP in primary human neutrophils

Introduction: MicroRNA functional role in the inflammatory responses of human neutrophils is still poorly investigated. In fact, neutrophils are extremely difficult to work with, given their short half-life, high protease and nuclease content and tiny amount of mRNA. The aim of this work is to optimize, in human neutrophils, the ribonucleoprotein immunoprecipitation (RIP) assay, which enables identification of miRNAs/mRNAs simultaneously recruited to the RISC complex in vivo. Materials and Methods: Human neutrophils (99.8% pure) were cultured in the presence or absence of LPS (100 ng/ml) for 8h. Cells, fixed or not with 1% formaldehyde, were lysed in polysome lysis buffer (PLB) or RIPA buffer, or subjected to nitrogen cavitation. The integrity of the Ago2 protein and the detection of miRNAs and mRNAs were simultaneously analyzed in the different lysates. Accordingly, RIP assays were performed and analyzed for miRNAs and mRNAs enrichment. Results: Lysing neutrophils, fixed or not, by PLB or RIPA buffer results in a significant degree of Ago2 and RNA degradation, as identified by Western blot and Agilent bioanalyzer. Conversely, N2 cavitation preserves Ago2 from degradation. RISC-associated mRNAs were detected using primer pairs flanking the miRNA seed region target, regardless the low level of RNA degradation observed in cavitates. miR-9 and NFKB1 mRNA enrichment in RIP were successfully detected. Conclusions: By coupling N2 cavitation and Ago2-IP, we optimized a novel protocol to perform RIP assay in human neutrophils. This new strategy will be useful for a large-scale characterization of miRNA functions in human neutrophils under resting and activated conditions.