Comparison of DNA and RNA quantification methods suitable for parameter estimation in metabolic modeling of microorganisms

Abstract

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. Five current methods for nucleic acid quantification have been compared: 1) UV absorbance spectroscopy at 260nm, 2) Colorimetric reaction with the orcinol reagent, 3)Colorimetric reaction based on diphenylamine, 4) Fluorescence detection with reagent Hoechst 33258 and 5) Fluorescence detection with thiazole orange reagent. Genomic DNA of three different microbial species (with widely different G+C content) was used as well as 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 reagent Hoechst 33258 ® is a sensitive and precise method for DNA quantification if the G+C content is lower than 50%. In addition this method allows quantification of very low levels of DNA (ng-scale). Moreover, the samples can be crude cell extracts. Also UV absorbance at 260nm and fluorescence detection with thiazole orange reagent are sensitive methods for nucleic acid detection, but only if purified nucleic acids have to be measured.

Dissolved oxygen alteration of the spectrophotometric analysis and quantification of nucleic acid solutions

“…Owing to the various sources of error in quantification by the above method, fluorescence-based techniques have been introduced that employ intercalating dyes such as diaminobenzoic acid, ethidium bromide, Hoechst 33258, SYBR Green I and PicoGreen that specifically bind to DNA and fluoresce [6][7][8]. Furthermore, several studies have shown their superiority over the UV absorbance method, in terms of accuracy and sensitivity [6][7][8][9][10][11][12][13].…”

Large‐scale production of endotoxin‐free plasmids for transient expression in mammalian cell culture

“…4). The more sensitive orcinol assay could not be used for RNA analysis in AE eluate and the final product due to interference from high DNA concentration (De Mey et al, 2006). Protein contamination was reduced on this step by 90% to 0.1 mg/mg.…”

Crystal structure and RNA-binding properties of an Hfq homolog from the deep-branching Aquificae: Conservation of the lateral RNA-binding mode

“…Initial Aae Hfq purification efforts were hindered by nucleic acid contaminants. Specifically, purified protein samples exhibited A 260 /A 280 absorbance ratios of $1.65, indicative of co-purifying nucleic acids (De Mey et al, 2006;; this problem is perhaps unsurprising given the known affinity of Hfq for nucleic acids, combined with the particularly high pI of Aae Hfq. By applying systematic colorimetric assays to Aae Hfq samples with high A 260 /A 280 ratios ( Supplementary Fig.…”