Abstract
Here we describe a carefully optimized method for the preparation of high quality RNA by flow sorting of formaldehyde fixed senescent cells immunostained for any intracellular antigen. Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. The underlying cause of senescence due to replicative exhaustion is telomere shortening. We document here a spontaneous and apparently stochastic process that continuously generates senescent cells in cultures fully immortalized with telomerase. In the course of studying this phenomenon we developed a preparative fluorescence activated flow sorting method based on immunofluorescent staining of intracellular antigens that can also deliver RNA suitable for quantitative analysis of global gene expression. The protocols were developed using normal human diploid fibroblasts (HDF) and up to 5x107 cells could be conveniently processed in a single experiment. The methodology is based on formaldehyde crosslinking of cells, followed by permeabilization, antibody staining, flow sorting, reversal of the crosslinks, and recovery of the RNA. We explored key parameters such as crosslink reversal that affect the fragmentation of RNA. The recovered RNA is of high quality for downstream molecular applications based on short range sequence analysis, such qPCR, hybridization microarrays, and next generation sequencing. The RNA was analyzed by Affymetrix Gene Chip expression profiling and compared to RNA prepared by the direct lysis of cells. The correlation between the data sets was very high, indicating that the procedure does not introduce systematic changes in the mRNA transcriptome. The methods presented in this communication should be of interest to many investigators working in diverse model systems.
Highlights
Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions
One central mechanism is the presence of unrepaired or persistent DNA double-strand breaks (DSB), which arise from telomere dysfunction or other genotoxic insults, and signal through the DNA damage response (DDR) pathway to activate the p53 tumor suppressor, leading to the upregulation of the cyclin-dependent kinase (CDK) inhibitor p21 and cell cycle arrest [7]
While these objectives have been achieved individually (FACS based on intracellular antigens, high quality RNA prepared from intact flow sorted cells), to our knowledge they have not been combined in a carefully explored, robust protocol
Summary
Replicative cellular senescence is a phenomenon of irreversible growth arrest triggered by the accumulation of a discrete number of cell divisions. Telomere shortening was the first described cause of senescence [4], but many other triggers have since been documented, including oncogene activation, a variety of genotoxic insults, and oxidative as well as other yet poorly understood stresses [5,6]. One central mechanism is the presence of unrepaired or persistent DNA double-strand breaks (DSB), which arise from telomere dysfunction or other genotoxic insults, and signal through the DNA damage response (DDR) pathway to activate the p53 tumor suppressor, leading to the upregulation of the cyclin-dependent kinase (CDK) inhibitor p21 and cell cycle arrest [7]. The DDR can signal to p16 through mechanisms such as the activation of the p38 MAPK pathway, but the regulation of p16 is not well understood, and likely involves components that are independent of genotoxic stress [10,11]. While the expression of telomerase elongates telomeres and prevents their dysfunction and activation of the p53-p21 pathway, immortalization of some www.impactaging.com
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