Primary neuronal cultures are a useful tool for measuring pharmacological- and transgene-regulated gene expression; however, accurate measurements can be confounded by heterogeneous cell types and inconsistent transfection efficiency. Here researchers from the University of Washington describe their adaptation of a ribosomal capture strategy that was designed to be used in transgenic mice expressing tagged ribosomal subunits (RiboTag) in specific cell types, thereby allowing measurement of translating RNAs from desired cell types within complex tissues. Using this strategy they were able to isolate and analyze neuron-specific RNA despite the presence of glia by co-transfecting experimental plasmids with plasmids that selectively express RiboTag in neurons. RiboTag immunoprecipitation was capable of recovering high integrity RNA from small numbers of transfected cells that can then be interrogated by a variety of methods (e.g., RT-qPCR, PCR array, RNA-Seq) and compared with basal RNA expression of the entire culture. Additionally, the researchers demonstrate how co-transfection of RiboTag with small hairpin RNA (shRNA) constructs can validate and accurately assess the degree of gene expression knockdown, and how RiboTag can be used to measure receptor-mediated gene regulation with transiently expressed designer receptors exclusively activated by designer drugs (DREADDs). RiboTag co-transfection represents a convenient and powerful tool to isolate RNA from a specific subset of cultured cells with a variety of applications for experiments in vitro.
Isolation of translating RNA from RiboTag-containing polyribosomes in neuronal/glial co-cultured primary neuronal cultures. (A) RiboTag expression constructs used for experiments. (B) Cartoon of RiboTag protocol for isolation of RNA under active translation as modified for cell culture. Primary neuronal/glial co-cultures were transfected on the seventh day in vitro (DIV7) with RiboTag-expressing plasmids with or without additional plasmids and then harvested on DIV9. Of this homogenate, 10% was reserved as the “Input” fraction, and hemagglutinin (HA)-specific antibody was added to the remaining homogenates (except for the no primary antibody negative control sample). The remainder of the protocol was performed as previously described.