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

Berdalet, Elisa; Roldán, Cristina Cruces; Olivar, M. Pilar

doi:10.3989/scimar.2005.69n117

Cited by 28 publications

(28 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…To test this hypothesis we performed the "residual fluorescence" experiment using 10 Engraulis encrasicolus larvae. Each individual larva was processed essentially as outlined in Berdalet et al (2005); from each homogenate the 5 assays illustrated in Figure 5 were run. Three aliquots were exposed to incubations with DNase (Tube A), RNase (Tube B) and both DNase and RNase (Tube C); the three aliquots were subsequently stained with SG-II.…”

Section: (J) Residual Fluorescence: Implications For the Methodsmentioning

confidence: 99%

“…Natural samples were used for the experiment indicated in Section J. The details of the manipulation procedure for natural samples is given in the accompanying paper (Berdalet et al, 2005).…”

Section: (C) Natural Samplesmentioning

confidence: 99%

“…3b). From a single homogenate (Berdalet et al, 2005), 63 aliquots were distributed to run the three assays (i.e. DNase, RNase and Residual, Section K), which were read in triplicates at 7 different times from 0 to 120 min.…”

Section: (F) Tube Selectionmentioning

confidence: 99%

“…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. …”

mentioning

confidence: 99%

See 3 more Smart Citations

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

Berdalet¹,

Roldán²,

Olivar³

et al. 2005

Sci. Mar.

Self Cite

View full text Add to dashboard Cite

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...

show abstract

Section: (J) Residual Fluorescence: Implications For the Methodsmentioning

confidence: 99%

Section: (C) Natural Samplesmentioning

confidence: 99%

Section: (F) Tube Selectionmentioning

confidence: 99%

mentioning

confidence: 99%

See 2 more Smart Citations

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

Berdalet¹,

Roldán²,

Olivar³

et al. 2005

Sci. Mar.

Self Cite

View full text Add to dashboard Cite

show abstract

“…The existence of a significant relationship between RNA/DNA ratio and SL was tested using linear regression. As proposed by Berdalet et al (2005) and Caldarone et al (2006), the RNA/DNA ratios were standardized to allow for a direct comparison of the results. The slope ratio of the calibration curves of Chícharo (1997) was DNA/RNA = 4.38 (A. Chícharo pers.…”

Section: Methodsmentioning

confidence: 99%

Biochemical composition and condition in anchovy larvae Engraulis encrasicolus during growth

Dı́az¹,

Txurruka²,

Villate³

2008

Mar. Ecol. Prog. Ser.

View full text Add to dashboard Cite

Growth and condition of larval Engraulis encrasicolus larvae in the Bay of Biscay were analysed relative to their biochemical composition. Protein, carbohydrate, lipid, DNA and RNA contents of larvae were quantified during larval ontogeny to analyse changes associated with growth. Proteins were the main organic component of E. encrasicolus at every larval stage, followed by lipids and carbohydrates. The increase in relative protein content in terms of protein percentage (P%) and the decrease in lipid relative content (L%) of the total organic matter (OM) with size observed are consistent with the definition of Type 1 larvae. Protein relative content increased until the P% in larvae reached ~71%, whereas L% and carbohydrate relative content (CH%) declined, exhibiting minimum values of ~20, and ~6%, respectively. In general terms, the feeding condition of larvae, as defined by their RNA/DNA ratio, was good. Food did not seem to be a major factor limiting larval growth in the study area throughout late spring and early summer. The RNA/DNA ratio was size-related and increased during larval growth up to an asymptotic optimal value of ~3.6 in postflexion larvae in relative protein content, in terms of protein percentage (P%). The 'actual' RNA/DNA ratios found in a given larval population when compared with the 'starving' RNA/DNA ratio, and using an 'absolutely optimal' value as complementary information, could be used to determine the overall status of the population. Parental effect of low egg quality is a possible explanation of delayed ontogenetical development of larvae from July 2000.

show abstract

The effect of egg versus seston quality on hatching success, naupliar metabolism and survival of Calanus finmarchicus in mesocosms dominated by Phaeocystis and diatoms

Koski¹,

Yebra

Dutz³

et al. 2011

Mar Biol

View full text Add to dashboard Cite

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

Cited by 28 publications

References 22 publications

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

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

Biochemical composition and condition in anchovy larvae Engraulis encrasicolus during growth

The effect of egg versus seston quality on hatching success, naupliar metabolism and survival of Calanus finmarchicus in mesocosms dominated by Phaeocystis and diatoms

Contact Info

Product

Resources

About