Isolation of satellite glial cells for high-quality RNA purification.

Background: Satellite glial cells (SGCs) envelope the neuronal somas in the dorsal root ganglia (DRG) and are believed to provide important neuronal support. Animal models of peripheral nerve injury, diabetes or chemotherapy all demonstrate activation of SGCs, suggesting important physiological roles for SGCs in various states of peripheral neuropathy. However, the biology of these glial cells is only poorly characterized under normal as well as pathological conditions due to suboptimal isolation methods.

New Method: The method presented here allows complete dissociation and isolation of highly pure SGCs from rat DRGs by fluorescence-activated cell sorting (FACS) using SGC-specific antibodies. The method further allows purification of high-quality RNA from the fixed and permeabilized cells.

Results: The purified RNA shows very little degradation, demonstrated by RNA integrity number (RIN) analysis with an average value of 8. The purified RNA, therefore, lends itself very well to downstream applications such as qPCR and transcriptome analysis.

Comparison With Existing Methods: Primary SGC cultures have previously been established for in vitro studies. Unfortunately, SGCs quickly change morphology and gene expression in vitro, complicating biologically meaningful interpretation of the obtained results. In contrast, this method allows the investigation of SGC gene regulation in vivo by isolation of high-quality RNA.

Conclusions: This method enables investigation of SGC transcriptional response in vivo by isolation and analysis of mRNA expression, allowing a more detailed investigation of SGC biology under normal as well as pathological conditions.