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now: Sat Feb 24 14:43:23 2018 ... mod: Thu Jul 24 15:42:37 2014
DNA vs RNA Analysis
The metachromism of AcridineOrange
can be used to simultaneously quantify both DNA and RNA in individual cells by FlowCytometry
. The cells are gently permeablized with weak detergent under cold acidic conditions to preserve nuclear structure. The acid helps to extract histones to allow the dye to more completely intercalate with DNA. Chelation of divalent cations with EDTA encourages RNA to become fully single stranded, allowing maximal differentiation between RNA and DNA by the dye.
- Mix 490 μl cold Solution I with 10 μl of a single cell suspension on ice. Incubate for one minute on ice.
- keeping the mixture cold prevents the acid from denaturing the DNA
- Add 500 μl cold Solution II and mix gently but thoroughly
- Analyze green (DNA) vs red (RNA) fluorescence by cytometry with a blue (488 nm) laser.
- 0.1% (v/v) Triton X-100
- 0.1 N HCl
- 150 mM NaCl
- 5 μg/ml AcridineOrange
- 5 mM ETDA
- 150 mM NaCl
- 100 mM Na2HPO4
- 50 mM citric acid
store solutions refrigerated indefinitely
F. Traganos, Z. Darzynkiewicz, T. Sharpless, M.R. Melamed, Journal of Histochemistry and Cytochemistry
, 25(1): 46-56 (1977). Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluormetric system.
- Cells grown without serum may require lower concentrations of Triton X-100 in Solution I to avoid lysis (0.02% - 0.04%).