Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms.
Recent developments in cellular and molecular biology require the accurate quantification of DNA and RNA in large numbers of samples at a sensitivity that enables determination on small quantities. In this study, five current methods for nucleic acid quantification were compared: (i) UV absorbance spectroscopy at 260 nm, (ii) colorimetric reaction with orcinol reagent, (iii) colorimetric reaction based on diphenylamine, (iv) fluorescence detection with Hoechst 33258 reagent, and (v) fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used, as were two different types of yeast RNA and a mixture of equal quantities of DNA and RNA. We can conclude that for nucleic acid quantification, a standard curve with DNA of the microbial strain under study is the best reference. Fluorescence detection with Hoechst 33258 reagent is a sensitive and precise method for DNA quantification if the G+C content is less than 50%. In addition, this method allows quantification of very low levels of DNA (nanogram scale). Moreover, the samples can be crude cell extracts. Also, UV absorbance at 260 nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids need to be measured.
Laboratory of Industrial Microbiology and Biocatalysis, Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, B-9000 Ghent, Belgium.
Marjan
M
De Mey
Gaspard
G
Lequeux
Jo
J
Maertens
Sofie
S
De Maeseneire
Wim
W
Soetaert
Erick
E
Vandamme
D003124:Colorimetry / Q000379:methods; D004396:Coloring Agents / Q000737:chemistry / Q000378:metabolism; D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D004271:DNA, Fungal / Q000032:analysis* / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D008954:Models, Biological; D012329:RNA, Bacterial / Q000032:analysis* / Q000737:chemistry; D012331:RNA, Fungal / Q000032:analysis* / Q000737:chemistry; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D013053:Spectrophotometry / Q000379:methods*; D013056:Spectrophotometry, Ultraviolet / Q000379:methods
D004396:Coloring Agents; D004269:DNA, Bacterial; D004271:DNA, Fungal; D005456:Fluorescent Dyes; D012329:RNA, Bacterial; D012331:RNA, Fungal