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A high through-put protocol for quantifying nucleic acids in individual microcrustaceans using new generation RNA and DNA specific dyes.

ABSTRACT Quantifying growth rate in microcrustacea is important to plankton biologists when determining primary productivity. RNA:DNA ratios have been widely used as an indicator of instantaneous growth rate in microcrustaceans in nutritional and environmental studies. It is important when measuring nucleic acids in these small organisms that it is done efficiently and with high sensitivity, so that small differences in nucleic acid content can be accurately detected. A technique is presented for the accurate, high-throughput determination of RNA and DNA content in individual microcrustacea, based on two new generation fluorescent dyes that bind preferentially to either RNA (Quant-iT[TM] RNA) or DNA (Quant-iT[TM] DNA). Contaminant interference (either from detergents or enzymes used in the chemical cell lysis process, or cell based contaminants such as proteins) can significantly affect fluorescence readings of these dyes. Through the development of our protocol, we show that proteinase K was an ineffective cell lysis method, whereas sarcosyl efficiently lysed cells and could be used when its final assay concentration was diluted to avoid interference with dye fluorescence. Organic solvents were also utilized to remove cell based contaminants that caused an interference with the Quant-iT[TM] DNA dye fluorescence. Through the use of specific RNA and DNA dyes and by achieving low levels of technical variation, this study developed a rapid and reliable method for nucleic acid extraction and quantification in Artemia.

KEY WORDS: RNA:DNA, Artemia, microcrustacean, RNA quantification, DNA quantification, Quant-iT[TM] RNA dye, Quant-iT[TM] DNA dye, fluorometric, nucleic acid

INTRODUCTION

Somatic growth occurs through a process of cell division and replication that requires enhanced levels of protein synthesis. Because ribonucleic acid (RNA) is the link between the genome and protein synthesis, total RNA levels in cells are expected to increase when an organism has a high requirement for protein production, such as during fast growth (Farrell 2005). In contrast, the level of deoxyribonucleic acid (DNA) remains relatively constant within a cell. The relationship (or ratio) between levels of cellular RNA and DNA therefore signify increased rates of protein synthesis and can be used as a predictor of instantaneous growth rate of an organism (Grant 1996, Fukuda et al. 2001, Caldarone et al. 2003, Caldarone 2005).

The application of RNA:DNA ratios has been particularly favored by plankton biologists, who use variation in growth and nutritional condition of microcrustacea as a biological indicator of the productivity of a water body (Ferron & Leggett 1994). Methods to accurately obtain these ratios in microcrustacea have been described in the literature, whereby RNA and DNA can be quantified from cell homogenates through the use of nucleic acid receptive fluorochrome dyes (Wagner et al. 1998, Gorokhova & Kyle 2002). The problem is, however, that despite the current dyes being highly sensitive to the detection of nucleic acids, they are nonspecific and bind to RNA and DNA. Binding efficiency can be unequal and influenced by in vitro conditions under which quantification takes place. Ethidium bromide is one such nonspecific dye, which has been used to quantify RNA and DNA in individual copepods (Wagner et al. 1998, Wagner et al. 2001). This carcinogenic and mutagenic dye was later replaced by the less hazardous and more sensitive RiboGreen flurochrome (Molecular Probes) to quantify nucleic acids in individual Daphnia (Gorokhova & Kyle 2002, Vrede et al. 2002) and Artemia (Gorokhova 2005). However, ethidium bromide and Ribogreen dyes exhibit nonspecific binding and require the use of enzymatic reactions to remove one nucleic acid to quantify the other (i.e., three assays are required per sample, one for DNA, one for RNA and one for background fluorescence). Highly pure nucleases are also required to avoid degrading the nontarget nucleic acid.

Quant-iT[TM] RNA and Quant-iT[TM] DNA (Molecular Probes) are two new generation flurochrome dyes that bind preferentially to either RNA and DNA, even in the presence of equal quantities of both nucleic acids. Quant-iT[TM] dyes may allow for rapid and reliable quantification by eliminating the need for enzymatic reactions to remove either the RNA or DNA component. Before these dyes are used routinely, however, it is important to test their reliability and sensitivity for quantifying microcrustacean RNA and DNA. In particular, the manufacturers caution about possible interference of chemical reagents like sodium dodecal sulfate (SDS) on dye fluorescence/emission levels (Quant-iT[TM] dye information sheets, Molecular Probes). Therefore, many residual chemicals commonly used in RNA and DNA extractions, such as detergents and enzymes, or even cellular constituents like proteins, have the potential to interfere with nucleic acid quantification based on fluorescence of these dyes.

This study evaluated the suitability of Quant-iT[TM] DNA and Quant-iT[TM] RNA dyes for estimating Artemia cellular nucleic acid levels, and in particular, reports on the effect common chemical reagents used in nucleic acid extraction may have on dye fluorescence. Based on our findings, we report a new protocol using these dyes, which allows rapid, high-throughput and sensitive nucleic acid quantification in individual microcrustaceans.

MATERIALS AND METHODS

Because of the labile nature of RNA, all solutions were made up with 0.1% DEPC (diethylpyrocarbonate) treated water and all work stations and equipment that came into contact with the samples were cleaned with an RNase removing detergent (RNase zapp, Ambion) to eliminate RNA degrading enzymes. Reagents certified to be nuclease free were used throughout.

Before quantifying RNA and DNA in microerustaceans there are steps in the nucleic acid extraction process that need to be optimized including physical disruption of tissue (homogenization) and chemical cell lysis. Following the chemical cell lysis extraction process there is the potential for residual chemical or cellular based contaminants to artificially alter fluorescence through the interference of dye binding efficiency during quantification. It was deemed therefore necessary to investigate the potential impact of such chemical additives and cell based contaminants on the fluorescence of dyes in both pure standards and organism sample extracts, and if they did interfere, whether it was beneficial to add an additional sample clean-up step to remove the interfering compounds from crude extracts using organic solvents. Later we describe the steps taken to evaluate the effects of contaminants on overall fluorescence of RNA and DNA when bound to Quant-iT[TM] dyes.

Optimization of Tissue Homogenization

Microcrustaceans have a hard exoskeleton, which first must be completely disrupted. Complete dissociation of tissues is required because if nonhomogenous cell populations are released from different individuals then tissue specific production of RNA may bias measurements of nucleic acid content (Houlihan et al. 1988). As part of our protocol development we initially trialed several different published mechanical techniques to break open the exoskeleton, including vigorous shaking (vortexing) (Wagner et al. 1998, Gorokhova & Kyle 2002, Gorokhova 2005), crushing with a disposable pestle (Vrede et al. 2002), or sonication with high frequency sound waves (Kyle et al. 2003). Observation of Artemia under the microscope showed high variability in the degree to which the exoskeleton was dissociated among individuals, with no more than 25% of Artemia fully homogenized by these techniques (unpublished data). We therefore trialed a new mechanical digestion technique. Here, 100 [micro]l of TE buffer was added to each sample and homogenized along with five silica beads for 15 sec on ice in a microsilica bead-beater. This technique was highly effective at rapidly dissociating the Artemia exoskeleton, with 100% of individuals showing complete tissue dissociation. This technique was therefore adopted to break open the exoskeleton in this study.

Nucleic Acid Quantification

For clarity in understanding our experimental approach we present how we quantified nucleic acids through fluorescence first.

Two new nucleic acid specific assay kits were used, Quant-iT[TM] DNA and Quant-iT[TM] RNA (Invitrogen). Either 2 [[micro]L of RNA or DNA sample was added to 50 [micro]L of the respective dye (1:200 diluted Quant-iT[TM] DNA and Quant-iT[TM] RNA dye), which was subsequently mixed on a vortex (Mini shaker with plate head, 1,000 g, 10 sec). Samples were assayed in triplicate along with a set of commercial standards in a 96 well black shell/ white well microtitre plate (BIORAD). Standards were those included with the Quant-iT[TM] dye kits and consisted of commercially prepared pure Lambda ([lambda]) DNA and E. coli RNA of known concentrations. In those experiments where we were examining the effect on fluorescence of chemical reagents commonly used for cell lysis, the nucleic acid standards were spiked at concentrations that would be used in sample extracts to account for interference. We also controlled for background fluorescence as a result of the chemical reagents themselves by adding these chemicals at concentrations matching those used in the final assay of samples to triplicate wells containing Quant-iT[TM] RNA or DNA dye only. Mean fluorescence values were obtained from triplicate assays and the coefficient of determination was calculated for each sample and the standard.

Fluorescence to quantify RNA and DNA content was measured in a real-time PCR machine (MJR DNA engine with Chromo 4 detector, Applied Biosystems). The wavelengths used were from settings for already established dye sets (SYBR Green and CY5) with similar excitation/emission spectra to Quant-iT[TM] DNA (510-527 nm) and Quant-iT[TM] RNA dyes (644-673 nm). The average background fluorescence value caused by chemical reagents used was then subtracted from each sample on the plate.

Optimization of Chemical Cell Lysis and Nucleic Acid Quantification

Following tissue homogenization it is necessary to lyse the cell to release cellular contents into solution. Proteinase K (an enzyme) and N-laurosarcosine (sarcosyl) (a detergent) are two previously accepted cell lysis chemical reagents used for this purpose in microcrustaceans. Quant-iT[TM] dye product information sheets state that detergents and proteins (enzymes) may interfere with fluorescence. Therefore, as part of the optimization of our protocol we undertook a three-step process to examine the effects these chemicals had on nucleic acid fluorescence readings. First we examined the extent of interference both these chemicals had on Quant-iT[TM] RNA and DNA dye fluorescence using commercially prepared pure nucleic acid standards only. We then tested the comparative extraction efficiency of proteinase K versus sarcosyl on the quality of RNA and DNA recovered from individual Artemia, and finally we examined the impact of these chemical additives and other cell based contaminants on Quant-iT[TM] dye fluorescence by first diluting sample extracts to minimize contaminant concentrations and secondly cleaning up samples with organic solvents.

(i) Effect of Chemicals on Dye Fluorescence (Commercial Standards Only)

Proteinase K at a concentration of 1 mg/mL and sarcosyl at concentrations up to 1% are routinely used in RNA:DNA extractions. Residual concentrations of these contaminants are carried over to the final quantification assays depending on the amount of sample and dye used. To test the effect of sarcosyl and proteinase K on dye binding or fluorescence intensity, these chemicals were added to pure standards of Lambda ([lambda]) DNA and E. coli RNA to create a standard curve for each nucleic acid (i.e., from 10-160 ng/[micro]L DNA and 1-20 ng/[micro]L RNA). Initially we trialed diluting proteinase K and sarcosyl to 4-fold dilutions (proteinase K 0.008 mg/mL, 0.0037% sarcosyl) (equivalent to 2 [micro]L of a 4-fold diluted sample in a total assay volume of 52 [micro]L) to reduce the effect on dye fluorescence. Proteinase K did not interfere with dye binding or fluorescence of commercially prepared standards when added at this concentration. In contrast, 0.0037% sarcosyl did interfere with the Quant-iT[TM] RNA dye binding or fluorescence when added to standards and this additive was therefore diluted further, to the equivalent of a 10-fold sample dilution and final assay concentration of 0.0013% (see results, Fig. 1B). Nucleic acids were quantified as described earlier.

(ii) Efficiency of Chemical Lysis Techniques

To examine the quality of RNA and DNA obtained using either proteinase K or sarcosyl cell lysis procedures, 16 juvenile Artemia were placed into separate 0.2 mL tubes before being snap frozen in liquid nitrogen. Eight individuals were then used for each of the two treatments.

Artemia from both treatments were first homogenized using the bead-beater physical disruption method. After mechanical tissue disruption, samples were incubated at room temperature with a sarcosyl concentration of 0.5 % or 1 mg/mL of proteinase K in the extract (n = 8 per treatment). During the incubation period samples were shaken vigorously on a vortex. A separate time trial was conducted using the sarcosyl treated samples to test how long these samples could be vortexed. Aliquots of the same samples (n = 8) were taken after 15 and 30 min of shaking. It was found that vortexing juvenile Artemia for 15 min gave the greatest yields of both DNA and RNA, with a 32% (DNA) and 36% (RNA) higher yield than vortexing for 30 min. After the 15 min incubation period all samples were centrifuged at 3,220 g to separate any particulate matter. The supernatant containing free nucleic acids was removed and 5 [micro]L of the raw extracts were mixed with 1X Orange G loading buffer and separated on a 1.5% agarose gel. The gel was run for 15 min at 100 V in a 1X TBE buffer system using standard electrophoresis techniques (Sambrook & Russell 200l). The remaining supernatant was retained and used for experiments outlined below.

[FIGURE 1 OMITTED]

(iii) Effect of Artemia Cell Contaminants on Dye Fluorescence

Following examination of residual cell lysis chemicals on dye fluorescence based on pure nucleic acid standards in (i) above, the next experiment determined the effects chemical reagents and Artemia cell based contaminants such as protein had on Quant-iT[TM] dyes fluorescence signal. Here, the same Artemia nucleic acid samples that were extracted in section (ii) were quantified as described above to determine if chemical additives and cell contaminants affected nucleic acid fluorescence values. We found that there was an effect (see results). Therefore, we trialed two ways to reduce their effects (a) by diluting the sample and (b) by cleaning it up using organic solvents. To evaluate the effectiveness of diluting Artemia samples on minimizing interference with Quant-iT dye fluorescence, samples were diluted four and then 10-fold to produce assays with final concentrations of sarcosyl of 0.0037% and 0.0013% and 0.008 mg/mL and 0.003 mg/mL proteinase K. Using an appropriate standard curve (with respective treatment concentrations of proteinase K or sarcosyl added), the amount of ng per mL of RNA or DNA per sample was determined from fluorescence values. Recovery per individual (ng/Artemia) was calculated for each treatment by taking into account the sample dilution factor and total extraction volume. These dilution trials suggested that cellular material and/or residual chemical contaminants, particularly proteinase K, may quench Quant-iT[TM] dye fluorescence leading to an underestimation of the amount of DNA and RNA recovered from individual Artemia {see results [b(iii)]}. To reduce the contaminant effect on dye fluorescence, further dilution of the samples was trialed. However, the already small quantities of DNA fell below the range of the standard curve at greater than 10-fold sample dilution, which meant that we could not use dilution as a way to reduce the effect of contaminants on DNA fluorescence. Therefore we assessed the removal of contaminants from extracts through sample clean-up using organic solvents.

(iv) Sample Clean-Up

To investigate the potential to clean-up sample extracts and maximize the fluorescence signal obtained two organic solvent treatments were examined, (i) phenol/chloroform (25 phenol:24 chloroform:1 isoamyl alcohol) followed by chloroform (24 chloroform:1 isoamyl alcohol) clean-up and (ii) chloroform (24 chloroform: 1 isoamyl alcohol) clean-up only. Eight juvenile Artemia for each treatment were placed into separate 0.2 mL tubes before being snap frozen in liquid nitrogen. Because proteinase K yielded poor quality DNA and quenched Quant-IT[TM] DNA and RNA dye fluorescence (see results, [b{iii}]), all samples for this experiment were extracted using the bead-beater mechanical disruption and 15 min 0.5% sarcosyl extraction method described above (i). Two microliters of each sample was diluted 10-fold to serve as the DNA control and 40-fold to serve as the RNA control. RNA samples were diluted 40 fold to fit within the range of the standard curve. Eight Artemia samples from the same family were cleaned up by phenol (pH 8)/chloroform then chloroform treatment and eight samples by chloroform only, following standard techniques (Sambrook & Russell 2001). The nucleic acids recovered were quantified as described above using a standard curve with an added appropriate concentration of sarcosyl for the controls and a pure standard curve for the organic solvent treatments.

RESULTS AND DISCUSSION

Optimization of Chemical Cell Lysis and Investigation of Quant-IT Dye Interference

(i) Effect of Chemicals on Dye Fluorescence (Pure Standards Only)

Standard curves generated from pure [lambda] DNA and E. coli RNA in the absence of any chemical additives were linear over the range examined with coefficients of determination ([R.sup.2]) greater than 0.99 (DNA: Fig. 1A, RNA: Fig. 1B). It is, however, important to investigate the potential effect of residual chemicals used to lyse cells in the extraction process on dye fluorescence (Singer et al. 1997, Jones et al. 1998). When 0.008 mg/mL proteinase K (equivalent to diluting a sample 4-fold) was added to the pure DNA standards there was little change in linearity ([R.sup.2] > 0.99) and only a slight depression of Quant-iT[TM] DNA dye fluorescence across the standard range (Fig. 1A). Adding sarcosyl to a final assay concentration of 0.0037% (equivalent to diluting a sample 4-fold) retained linearity in Quant-iT[TM] DNA fluorescence readings ([R.sup.2] > 0.98) but overall fluorescence was diminished from the pure standard values (Fig. 1A).

Fluorescence of the Quant-iT[TM] RNA dye was more extensively quenched by both of these chemical additives than was the Quant-iT[TM] DNA dye. When 0.008 mg/mL proteinase K was added to RNA standards the fluorescence was reduced although the standard curve again remained linear ([R.sup.2] = 0.93) (Fig. 1B). When sarcosyl at a 4-fold dilution concentration in the final assay (0.0037%) was added to the RNA standards there was even larger variation in the slope of the line because of greater depression of dye fluorescence and the coefficient determination was reduced to just 0.62. This demonstrated that sarcosyl at this concentration substantially interferes with Quant-iT[TM] RNA dye fluorescence. Diluting sarcosyl to a lower concentration (0.0013%, equivalent to a 10-fold dilution of sample) also substantially reduced the fluorescence of commercial standards from their original values, but at this concentration the standard curve retained linearity ([R.sup.2] = 0.93) (Fig. 1B).

Although fluorescence signals were diminished for pure RNA and DNA standards, strong linearity across the entire range of the standard curve was retained when 0.008 mg/mL proteinase K or a low (0.0013%) concentration of sarcosyl was present in either the Quant-iT[TM] DNA or Quant-iT[TM] RNA dye assay. These concentrations of cell lysis chemicals may therefore be used in the final dye assays provided that unknown samples are diluted accordingly and the same concentration of proteinase K or sarcosyl is added to commercial standards used to generate the standard curves. The fluorescence signal was likely reduced because of the effect of the detergents artificially depressing the signal, or interfering with the dye binding to the RNA and DNA molecules. According to the Quant-iT[TM] dye manufacturers, the acceptable concentration for the detergent (SDS) is 0.01% in the final assay. In our study, the highest acceptable concentration of sarcosyl, a more frequently used detergent for RNA extraction, in final dye assays was 0.0013%, almost an order of magnitude less than that recommended for SDS.

(ii) Efficiency of Chemical Lysis Techniques

Sarcosyl and proteinase K were tested for their nucleic acid extraction efficiency in individual Artemia. Few studies of RNA:DNA in microcrustaceans have reported checking RNA or DNA quality following extraction procedures. Agarose gel electrophoresis of Anemia extracts in the present study showed reasonable yields of high quality RNA with distinct ribosomal RNA bands visible when using either sarcosyl or proteinase K for chemical cell lysis (Fig. 2). In contrast, the amount of DNA obtained using proteinase K was much lower and more variable with six out of eight animals releasing negligible amounts of DNA compared with that released using sarcosyl (Fig. 2). This may be because of inefficient cell lysis with proteinase K resulting in less consistent release of the larger DNA as opposed to small RNA molecules. This suggests that the ratio of DNA to RNA is likely to be less consistent between samples (and therefore less accurate) when using proteinase K to lyse Artemia cells. A more consistent ratio between RNA and DNA was evident for all eight samples using the sarcosyl extraction procedure, as even though two samples showed reduced quantities of DNA these same samples also had lower yields of RNA (black arrows, Fig. 2). These results show that the ratio of RNA and DNA extracted from Artemia is more consistent, both within and between samples, when sarcosyl rather than proteinase K is used to lyse Artemia cells.

(iii) Effects of Artemia Cell Contaminants on Dye Fluorescence

It is important to note that our trials based on pure RNA or DNA standards and chemical additives used in extraction do not account for the effects of additional cell based contaminants present when crude nucleic acid extracts of microcrustaceans are assayed. This experiment examined the effect of both extraction reagents and possible cellular contaminants on fluorescence and nucleic acid quantification. Here a modest increase in proteinase K concentration from 0.003-0.008 mg/mL was found to reduce the fluorescence intensity of Quant-iT[TM] DNA dye and decreased the apparent recovery of DNA from individual Artemia by as much as 50% (Fig. 3A). Increasing the final assay concentration of sarcosyl from 0.0013% to 0.0037%, however, only decreased the Quant-iT[TM] DNA fluorescence and apparent DNA recovery by 21% (Fig. 3A). Overall, sarcosyl treated samples at a l0-fold dilution (0.0013%) had the greatest apparent DNA recovery, higher than that detected with either of the two proteinase K concentrations (Fig. 3A). This may be partially caused depression of Quant-IT[TM] DNA dye florescence by the proteinase K itself but is most likely because of relatively poor recovery of DNA from proteinase K treated samples (see gel image, Fig. 2). Whereas sarcosyl treated samples had the highest DNA recovery it is important to note, that the CV values for even the 10-fold diluted sarcosyl treated samples were still high, averaging 56% (see control Fig. 4A). Additional dilution of samples to reduce the final assay concentration of sarcosyl and maximize the fluorescence signal obtained to provide a more accurate estimate of total DNA recovered was not possible; this was caused by the Quant-iT[TM] DNA fluorescent values falling below the linear range of the standard curves at higher sample dilutions (unpublished data).

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

The fluorescence of the Quant-iT[TM] RNA dye was also more strongly depressed by the presence of proteinase K (0.003 mg/ mL and 0.008 mg/mL) than by the lowest of the sarcosyl concentrations tested (0.0013%). Accordingly the apparent RNA recovered was 80% lower in proteinase K treated samples compared with that detected with a low sarcosyl concentration (Fig. 3B). Agarose gel electrophoresis indicated that similar levels of RNA were, however, extracted with both proteinase K and sarcosyl at the concentrations used in extraction (Fig. 2). These results combined suggest that the low apparent recovery of RNA with proteinase K is therefore likely to be caused by physical interference between proteinase K and Quant-iT[TM] RNA dye, limiting dye binding and/or fluorescence. Increasing the concentration of sarcosyl, however, also depressed the fluorescence signal and apparent RNA recovery by 21% (Fig. 3B). Of the two concentrations tested the 0.0013% sarcosyl used in the final dye assay (equivalent to 10-fold dilution of sample) gave the greatest fluorescence signal (i.e., least interference) (Fig. 3A; 3B). Given the high sensitivity of the Quant-iT[TM] RNA dye, and the high concentrations of RNA obtained from Artemia, greater dilution of samples is possible to further reduce the impact of sarcosyl on dye fluorescence and maximize the fluorescence signal to better reflect the recovery of RNA.

[FIGURE 4 OMITTED]

(iv) Sample Clean-Up

Further dilution of nucleic acid extractions to minimize the impact of sarcosyl on Quant-iT[TM] DNA fluorescence was not possible as outlined above and the high CV values (up to 56%) obtained with crude Artemia extracts required alternative solutions to the interference problems. As an alternative to dilution, organic solvents were tested for their effectiveness at removing cell based contaminants that were interfering with the dye fluorescence. Phenol dissolves contaminants such as proteins and lipids, leaving nucleic acids in the aqueous layer, whereas chloroform enhances cell lysis and inactivates DNases (Kotlowski et al. 2004, Farrell 2005). Previous microcrustacean RNA:DNA studies did not utilize organic solvents to clean-up nucleic acids extracts, instead relying on crude extracts (e.g., Gorokhova & Kyle 2002, Wagner et al. 2001, Wagner et al. 1998). Few of these studies report CV values to assess within sample variability, and it is unclear if fluorometric readings were artificially enhanced or depressed by contaminants in crude sample extracts. In the present study Artemia extracts were progressively purified with chloroform only, or by phenol: chloroform then chloroform to remove impurities that depress the fluorescence of Quant-iT[TM] dyes.

[FIGURE 5 OMITTED]

In this organic solvent trial, the apparent recovery of DNA was lowest for one in 10 diluted and unpurified samples (containing 0.0013% sarcosyl in the final assay) and a high CV (56%) was recorded (Fig. 4A). There was a 50% increase in the apparent DNA recovery when the same extract was treated with chloroform and a 77% increase with an additional phenol/ chloroform treatment on the same extract (Fig. 4A). The average coefficient of variation following both of these treatments was less than 10% for within-sample replicates (Fig. 4A). A mixture of chloroform and phenol has previously been shown to be an effective clean-up method for DNA samples in aquatic insects, molluscs, and fish (Kotlowski et al. 2004). Treatment with organic solvents was also shown in the present study to be a highly effective method at removing interfering contaminants from DNA samples.

A large apparent recovery of RNA (32,700 ng/Artemia) was detected using the Quant-iT[TM] RNA dyes without any clean-up treatments required. As a result sample dilution of up to 40-fold was possible and under these conditions average coefficients of variation were less than 10% (Fig. 4B). Both phenol/chloroform and chloroform treatments completely removed all RNA from the samples (Fig. 4B), but this step proved unnecessary for quantifying RNA because of the high sensitivity of the assay and low technical variation (CV's < 10%).

COMMENTS AND RECOMMENDATIONS

Inaccurate RNA:DNA ratios may be obtained if sources of technical variation introduced through present extraction techniques are not investigated and minimized. The development of specific fluorophores has contributed to the rapid quantification of nucleic acids and substantially reduced the amount of technical variation. Specific fluorophores eliminate the need for sample treatment with nucleases to determine specific nucleic acid quantities. It is important, however, to minimize technical variation with these sensitive dyes to obtain measures of true biological variation. This is especially true for microcrustacean studies where small quantities of nucleic acids are obtained from individual organisms. Because Quant-iT[TM] dye assays are sensitive to procedural methods, we present a list of recommendations and precautions based on the results of this study to be considered when quantifying nucleic acids in Artemia.

(1) Mechanical digestion with a bead beater is suggested to open exoskeletons and achieve homogeneity among samples.

(2) Proteinase K was insufficient for RNA and DNA extractions. It appears that it does not lyse cells sufficiently and interacts with cell based contaminants causing an interference with Quant-iT[TM] dye fluorescence.

(3) Sarcosyl was efficient in extraction but interferes with dye fluorescence at high concentrations.

(4) The interference sarcosyl has with Quant-iT[TM] RNA dye fluorescence can be overcome with dilution of sample extracts. However, the Quant-iT[TM] DNA dye is more sensitive to contamination and dilution is an ineffective way to prevent interference.

(5) Organic solvent extraction can be utilized on DNA samples to remove cell based contaminants interfering with the Quant-iT[TM] DNA dye to maximize signal fluorescence.

Through assessment of downstream effects of extraction procedures on fluorescence, the present study developed a reliable method for extracting and quantifying nucleic acids in Artemia that achieved low levels of technical variation (CV values less than 10%) and produced high quality and consistent ratios of RNA and DNA. The step-by-step protocol is summarized in Figure 5.

ACKNOWLEDGMENTS

The authors thank the staff of the Marine and Aquaculture Research Facility Unit (MARFU) for their ongoing technical assistance in this study and those members of the Aquaculture Genetics Research Group and MEEL genetics lab that offered their help in molecular analysis; especially Adrian McMahon.

LITERATURE CITED

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Kotlowski, R., A. Martin, A. Ablordey, K. Chemlal, P.-A. Fonteyne & F. Portaels. 2004. One-tube cell lysis and DNA extraction procedure for PCR-based detection of Mycobacterium ulcerans in aquatic insects, molluscs and fish. J. Med. Microbiol. 53:927-933.

Kyle, M., T. Watts, J. Schade & J. J. Elser. 2003. A microfluorometric method for quantifying RNA and DNA in terrestrial insects. J. Insect Sci. 3:1-7.

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Wagner, M., R. G. Campbell, C. A. Boudreau & E. G. Durbin. 2001. Nucleic acids and growth of Calanus finmarchicus in the laboratory under different food and temperature conditions. Mar. Ecol. Prog. Ser. 221:185-197.

ERIN L. MCGINTY, * CAROLYN SMITH-KEUNE AND DEAN R. JERRY

Aquaculture Genetics Research Group, School of Marine and Tropical Biology, James Cook University, Townsville, 4811, Queensland, Australia

* Corresponding author. E-mail: erin.mcginty@jcu.edu.au
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Title Annotation:ribonucleic acid, deoxyribonucleic acid
Author:McGinty, Erin L.; Smith-Keune, Carolyn; Jerry, Dean R.
Publication:Journal of Shellfish Research
Article Type:Report
Geographic Code:8AUST
Date:Apr 1, 2008
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