Isolation of Satellite Glial Cells for High-Quality RNA purification

Publication date: Available online 2 January 2018
Source:Journal of Neuroscience Methods
Author(s): Sara Buskbjerg Jager, Lone Tjener Pallesen, Christian Bjerggaard Vaegter
BackgroundSatellite 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 MethodThe 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.ResultsThe 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 methodsPrimary 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.ConclusionsThis 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.



http://ift.tt/2CtYSAQ