In my last post, I discussed the advantages of RNA-seq for characterizing conditional knock out mice. The question arises for this experiment and most other biological studies: how do we isolate the cell population of interest? This is a major point for consideration when planning your transcriptome experiment since RNA populations are dynamic and sensitive to environmental factors such as being ripped out of their natural context. We want to minimize perturbations to the cell that introduce stress responses that can cloud our understanding of a biological phenomenon. Cell isolation approaches range from classic to cutting-edge. Traditional approaches include simply harvesting tissue or cells by dissection. More involved cell isolation approaches can follow, including dissociation and immunopanning for specific cell type populations. This technique was used beautifully in Cahoy and colleagues’ transcriptomics paper profiling glia and neuron development. While dissociation and immunopanning can target specific cell types, the process is inherently disruptive to the living cell, but depending on your question may be the most appropriate approach. Another popular method for isolating cells of interest is flow sorting, which in essence allows the researcher to select single cells as they flow through a channel (for example, cells that have been labeled with antibody vs no antibody). This is a suitable approach for studying immune cells, other suspended cell types, and extracellular vesicles, which can transport extracellular RNA.

Cutting-edge techniques include isolating labeled RNA from your tissue of interest with a genetic and chemical approach as described by Gay and colleagues. The advantage of this method is that the researcher can genetically target the tissue of interest with expression of a cre-induced uracil-phosphoribosyltransferase. This transferase specifically incorporates the uracil-analog, 4-thiouracil, after injection allowing the scientist to observe tissue-specific RNA expression at a time of interest. Impressively, Lovatt and team can monitor the mRNA population from single cells in vitro and in vivo with a special photoactivatable tag that targets RNA. Their protocol involves injecting the photoactivatable tag into the cells of interest and then targeting the region of interest with light. Similar to the above strategy, using light and the tag allows the researcher to target your cells of interest at the right time.

I have only briefly discussed some of the options, and mentioned some cutting edge possibilities for isolating cells that are available to researchers. Determining the right method for you depends on your experimental goals and your area of expertise. Feel free to discuss your options with our Project Scientists!