Abstract
Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population.
Highlights
IntroductionPublisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations
This combined method yielded purified microglia cells ranging between 3 × 105 and
Results and 5 × 105 cells from one adult mouse brain after double-column filtering, where the viability5. This combined method yielded purified microglia cells ranging between 3 × 10 and of purified microglia ranged between 70–80% of total cells and the purity ranged between
Summary
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. The brain’s resident immune cells, account for 5–12% of total cell population in the mouse brain and play a crucial role in adult brain neurogenesis, neuroinflammation, and overall brain homeostasis [1] This multifunctional brain cell vastly depends on environmental stimuli to maintain its homeostatic phenotype [2] and immediately adjusts its functional profile based on the physiological or pathophysiological needs of the individual. TMEM119 is another cell-surface protein recently used as a specific microglial marker that can distinguish brain microglia from invading monocytes in disease conditions. We present a combined and step-wise method adapted from previously described protocols [10,11,12,13,14,15,16,20,21] to isolate intact DNA and RNA from a highly pure microglial population from one single mouse brain using CD11b as a microglia selection marker. Isolated DNA and RNA using this method could be suitable for downstream molecular biology applications, including methylated DNA and transcriptome analysis designed for understanding genetic profiles
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
