Estimation of cellular DNA content in cell lysates suitable for RNA isolation.
Methods presently available for the isolation of RNA are incompatible with conditions necessary for the measurement of either DNA or cell number, resulting in infrequent quantitation of messenger RNA in relation to the quantity of cells studied. In the present studies, a microfluorometric method has been modified from previous techniques to permit the quantitation of DNA in cell lysates prepared using an acid guanidinium thiocyanate-phenol (AGTP) solution from which RNA can subsequently be isolated. The lysate is incubated in alkaline EDTA, then neutralized with KH2PO4, followed by the addition of the fluorochrome bisbenzimidazole (Hoechst 33258), and measurement of fluorescence. DNA content is comparable in measurements by the present technique and by the diphenylamine method on parallel samples. DNA content per cell for human cells measured with this technique is comparable to that previously reported using other methods. The use of AGTP solution results in stability of measurable DNA in cell lysates for periods of at least 10 weeks (permitting batching of samples and retrospective measurement) and stability of fluorescence for at least 20 h after the addition of bisbenzimidazole making the timing of fluorescence measurement less critical. The technique described should permit quantitation of messenger RNA in relation to DNA (and hence indirectly to cell number) on a routine basis.
Department of Medicine, University of Cincinnati College of Medicine, Ohio 45267-0547.
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Z
Rymaszewski
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Abplanalp
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Cohen
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Chomczynski
D001562:Benzimidazoles; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis*; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D005453:Fluorescence; D006146:Guanidines; D006801:Humans; D010636:Phenols; D012313:RNA / Q000302:isolation & purification*; D012996:Solutions; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
D001562:Benzimidazoles; D006146:Guanidines; D010636:Phenols; D012996:Solutions; D013861:Thiocyanates; C054436:guanidine thiocyanate; D012313:RNA; D004247:DNA