Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part A. Optimisation of the assay

Abstract

SUMMARY: Assay protocols for RNA and DNA in crude plankton extracts using the fluorochrome SYBR Green II are developed here. The method is based on the fluorescence in 3 aliquots: the first measures RNA after DNA digestion; the second measures DNA after RNA digestion; and the third measures residual fluorescence after digestion of both DNA and RNA. This residual fluorescence measurement is critical for accurate calculations of the nucleic acids. Optimisation of the assay conditions are described: fluorochrome concentration, buffer composition, fluorescence stability, temperature and duration of nuclease incubation. In the optimised procedure, the assays are performed in 5 mM Tris buffer (containing 0.9 mM CaCl 2 ·2H 2 O and 0.9 mM MgCl 2 ·6H 2 O, pH 8.0); DNase and RNase incubations are conducted at 37ºC for 20 min; the fluorochrome is added to all assays at a final concentration of 3.5x10 -4 and readings are done within the 10-60 min period following the SYBR Green II addition. The study evidenced the importance of the residual fluorescence after nuclease digestion, which is especially taken into account in the calculation of the nucleic acid concentrations. Finally, the variability of the fluorescent response to different RNA and DNA standards is examined; from the performed tests, calculations are based on rRNA from calf liver and DNA from calf thymus standards. The accompanying paper (Berdalet et al., 2005) describes the development of the extraction protocol, as well as the application of both protocols in measuring RNA/DNA ratios in natural plankton samples, and a comparison with ethidium bromide based methods.Key words: SYBR Green II, DNase, RNase, RNA/DNA ratios, plankton. RESUMEN: CUANTIFICACIÓN DE ARN Y ADN EN ORGANISMOS PLANCTÓNICOS MARINOS MEDIANTE SYBR GREEN II Y NUCLE-ASES. PARTE A. OPTIMIZACIÓN DEL ENSAYO. -En este trabajo se desarrollan los protocolos para la cuantificación de ARN y ADN en extractos no purificados de plancton utilizando SYBR Green II. El método se basa en la fluorescencia de 3 alícuo-tas: la primera mide el ARN tras la digestión del ADN; la segunda mide el ADN tras la digestión del ARN y la tercera mide la fluorescencia residual tras la digestión tanto del ARN como del ADN. La medida de esta fluorescencia residual es críti-ca para obtener una buena estimación de los ácidos nucleicos. Se describen las condiciones de optimización del ensayo: concentración de fluorocromo, composición del tampón, estabilidad de la fluorescencia, temperatura y duración de la incubación con nucleasas. En el procedimiento optimizado los ensayos se realizan en tampón Tris 5 mM (0.9 mM CaCl 2 ·2H 2 O y 0.9 mM MgCl 2 ·6H 2 O, pH 8); las incubaciones con nucleasas se llevan a cabo a 37°C durante 20 min; el fluorocromo se añade a todos los ensayos a una concentración final de 3.5X10 -4 y las lecturas se realizan en los 10-60 min posteriores a la adición de SYBR Green II. Este estudio evidencia la importancia de la fluorescencia residual después de la digestión con nucleasas, la cual es especialmente incluída...

Quantifying RNA and DNA in planktonic organisms with SYBR Green II and nucleases. Part B. Quantification in natural samples

“…La metodología es compatible con el ensayo fluorimétrico para ARN y ADN desarrollado por Berdalet et al (2005). La extracción de los ácidos nucleicos se realiza combinando una rotura celular mecánica (mediante un homogeneizador manual de tejidos) con una extracción química (con 0.5% de sarcosina y 0.5 mM EDTA en tampón Tris 5 mM a pH 8).…”

“…Berdalet et al, 1992), direct comparison of data obtained from different sort of organisms is not expected a priori. However, the absolute RNA/DNA value measured in any sample using a particular procedure also depends on the standards used for the calculations (Berdalet et al, 2005). On one side, the slope and the residual fluorescence of the selected standards determine the concentration of RNA and DNA estimated in the samples; on the other, the resulting RNA/DNA ratio depends on the ratio of the slope of the RNA to that of the DNA standard (m RNA /m DNA ) used.…”

“…This experimentally determined level of sensitivity is the result of the combination of: 1) the sensitivity of the fluorochrome itself; 2) the extraction with sarcosine which requires the dilution of the sample (Section F); and 3) the nuclease digestions (with the non-negligible residual fluorescence, Berdalet et al, 2005) necessary to estimate RNA and DNA in crude extracts.…”

“…Fluorometric readings were performed according to Berdalet et al (2005: Section B) using quartz cuvettes. Some data were obtained with a Shimadzu RF-570 spectrofluorometer and others with an Aminco-Bowman 2.…”

“…Analytical sensitivity, sampling and extraction problems as well as problems in experimental design were suspected causes. Berdalet et al (2005) demonstrate the sensitivity of the fluorometric methods to subtle procedural differences and explain the analytical improvements in measuring RNA and DNA with the nucleases and the SYBR Green II based fluorometric assay. The next step was to establish an effective extraction procedure compatible with the use of this protocol.…”