The Ambion RNAlater® Tissue Collection: RNA Stabilization Solution is an aqueous tissue storage reagent that stabilizes and protects cellular RNA in intact, unfrozen tissue samples. RNAlater®Solution eliminates the need to process tissue samples immediately or freeze them in liquid nitrogen for later processing. Thus, more time is available for sample collection without jeopardizing RNA expression profiles in precious samples. Nishimoto and colleagues from the University of California, Irvine, describe a method utilizing RNAlater® Solution for fluorescence-activated cell sorting (FACS) that minimizes downstream perturbation of gene expression profiles in sorted cells.
While FACS is a convenient and efficient method for isolating cells, cells undergoing the FACS process can experience physiologic stress and a decrease in viability. Several external factors associated with cell sorting such as flow rate, pressure, and droplet charge may cause modulations in cellular physiology including RNA transcript levels. Conditions that cells experience when streamed into collection tubes containing buffered solutions (culture medium or PBS), along with the accompanying changes in time and temperature, can further exacerbate altered RNA expression patterns.
Changes in RNA profiles can be prevented or reduced by immediate cell lysis and protein denaturation. However, these methods are complicated by the sheath fluid accompanying the sorted cells, which significantly dilutes the disrupting/denaturing reagent and results in inadequate RNA preservation. Nishimoto and colleagues successfully demonstrated that RNAlater Solution permits the isolation of high-quality RNA from cells collected by FACS . They describe a comprehensive method that validates the use of RNAlater Solution in FACS to preserve high-quality RNA for microarray gene expression profiling of isolated cellular subsets of peripheral blood mononuclear cells. They will apply this technique in their research on pathophysiological mechanisms of host-microbe interactions.
For scientists working with Drosophila cells, details about the use of RNAlater Solution during FACS have been documented by Estrada and colleagues .
- Nishimoto KP, Newkirk D, Hou S, Fruehauf J, Nelson EL (2007) Fluorescence activated cell sorting (FACS) using RNAlater to minimize RNA degradation and perturbation of mRNA expression from cells involved in initial host microbe interactions. J Microbiol Methods70: 205–208
- Estrada B, Choe SE, Gisselbrecht SS, Michaud S, Raj L, Busser BW, Halfon MS, Church GM, Michelson AM (2006) An integrated strategy for analyzing the unique developmental programs of different myoblast subtypes. PLoS Gene2:e16.