FIGURE 1.

Workflow for high-throughput RNA experiments. Depiction of the different stages common to high-throughput RNA experiments as discussed in this review. (1) The experiment design stages should consider the scope of the project and must be designed with considerations for replicates and randomization. (2) High-throughput experiments may be performed in vitro (derived from cell culture). In vivo experiments may choose between the whole brain, subcellular fractions, or single-cells. Subcellular fractions can be obtained through a variety of different filtration methods as well as recent methods focusing on cell-cultures. This stage is especially crucial for studies in local translation as subcellular fractionation allows researchers to investigate spatial changes occurring in specific neuronal compartments. (3) RNA-Seq is typically performed on a total lysate population. Herein, we review a number of methods available for molecular extraction of RNA under various conditions (i.e., bound to proteins or the ribosome) as well as in vitro assays for assessment of RNA-binding properties. (4) After quality control assessment, the RNA may be sequenced. This is followed by a number of processing steps indicated in greater detail in Figure 3. (5) Computational analysis on RNA populations may reveal patterns and connections between processes previously unknown. Such experiments may also be followed up using protein identification techniques (detailed in Table 3). (6) Finally, validation can confirm novel findings seen in (5). Many new techniques exist allowing researchers to confirm both the spatial and temporal expression of numerous proteins and RNA systems involved in the control of synaptic plasticity through local translation.