Chapter 28 Flow Cytometric Analysis of Double-Stranded RNA Content Distributions

Abstract

Publisher Summary This chapter develops a simple, reproducible, and specific procedure for the staining of cellular RNA. The method is based on the well-known property of propidium iodide (PI) to form highly fluorescent intercalation complexes with double-stranded nucleic acids but not with other polyanions. This property is widely used for the flow cytometric (FCM) analysis of DNA after RNA elimination. It suggests the use of PI for RNA staining following DNA digestion with DNase. The highly specific character of PI binding to nucleic acids in different cell types is the basis for its wide application to DNA analysis. It also provides a basis for quantitating double-stranded RNA (dsRNA) in DNase-treated cell populations. The chapter discusses the relationship between dsRNA content and cell proliferation for different cell types. The fluorescence intensity of cells treated with both RNase and DNase was very low, indicating that the fluorescence of DNase-treated cells shows the relative content of dsRNA. Comparison of the fluorescence intensity between DNase-treated and DNase plus RNase-treated cells showed that 93% of the fluorescence in DNase-treated cells was eliminated with RNase.