Storage stability of solutions of DNA standards

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Storage stability of solutions of DNA standards Anna Baoutina, Somanath Bhat, Lina Partis, and Kerry R. Emslie Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.9b02334 • Publication Date (Web): 29 Aug 2019 Downloaded from pubs.acs.org on August 30, 2019

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Analytical Chemistry

Storage stability of solutions of DNA standards Anna Baoutina*, Somanath Bhat, Lina Partis, and Kerry R. Emslie National Measurement Institute (NMI), Lindfield, Sydney, New South Wales, 2070, Australia ABSTRACT: High accuracy, reliability and reproducibility of genetic analyses in various applications require optimized and validated protocols and standards. Optimal procedures for storing the genetic material extracted from biological samples are equally important. In this study, we investigated the stability of dilute (4 000 cp/µL, nominal concentration, equivalent to 0.02 ng/mL) DNA solutions stored at 4°C, -20°C and -80°C in the presence or absence of nucleic acid carriers. As representative examples, we used different formulations of a linearized plasmid DNA solution considered for characterization as reference materials (RM) for specific applications. Employing droplet digital PCR, a highly accurate and precise method for quantification of nucleic acid not requiring a calibrant, we demonstrated that inclusion of a carrier nucleic acid in the formulation (at 50 ng/µL) improved the plasmid stability at -20°C and -80°C. For the case of a DNA standard used in real-time PCR assays for human erythropoietin gene, cDNA or transcript, we found that inclusion of yeast RNA in the formulation was preferred over salmon testes DNA as it had no effect on PCR amplification and provided the lowest relative expanded uncertainty for the characterized RM. RNA background may also be preferred as it is applicable to a broader range of DNA RMs. Our findings are important in production of reliable, stable DNA standards, including DNA RMs. These results can be used when selecting protocols for stable storage of DNA either extracted from biological samples or synthesized in a laboratory.

Accuracy, reliability and reproducibility of molecular genetic analyses are crucial in numerous applications. These include clinical diagnostics, prognosis and therapy, testing for genetically modified organisms in agriculture or for pathogens in the environment, pest and disease control in biosecurity, and DNA profiling in forensics. Many factors in pre-analytical and analytical processes affect testing results. To avoid errors, optimal protocols and use of rigorous controls must be employed. The use of physical nucleic acid standards is common practice in genetic analyses. For the best quality measurement results, where available, references materials (RMs) and certified reference materials (CRMs) can be used in calibration, quality control, proficiency testing, method validation and assignment of values of other materials, and, for CRMs, in establishing metrological traceability of the measurement results.1 RMs used in genetic analyses may be based on intact or fragmented genomic DNA (wild-type or mutated), amplicons, synthetic oligonucleotides or plasmid DNA (pDNA) with cloned target sequences, for example, complementary cDNA (cDNA) for a gene of interest. Characterization of RMs in accordance with ISO Guide 35 requires demonstration of their stability over short- and longterm, and this necessitates development of optimized protocols for DNA storage to ensure a shelf life which is fit-for-purpose. Optimal procedures are also important for storing DNA extracted from biological samples or cDNA synthesized in a laboratory as part of gene expression studies. Many factors have been investigated in relation to their contribution to DNA preservation. These include temperature, protocols used for DNA extraction, samples purity, storage buffers and tubes, DNA length and composition, and light exposure.2-4 With regard to temperature, strategies for DNA storage include refrigeration at 4°C, freezing DNA solutions at -20°C or -80°C, cryopreservation in liquid nitrogen or storage at room temperature on a dry solid matrix. Storage of oligonucleotides, DNA extracts, plasmids or cDNA frozen at either -20°C or -80°C, especially for prolonged periods, is common practice in molecular biology and forensic laboratories. Recently, while working on developing a DNA RM for specific genetic analyses,5 we observed that the

concentration of DNA in buffered solution stored frozen at either -20°C or -80°C decreased over time, whereas when stored at 4°C, it remained unchanged. The decrease of the concentration of three investigated materials (an amplicon, and circular and linearized plasmids) stored at -20°C was noticeably higher than at -80°C. Several studies had previously shown degradation of DNA formulations after single or multiple freezing cycles.4,6-10 The degree of DNA damage was linked to rate of sample cooling, length of storage, number of freeze-thaw cycles, and DNA base composition, length and concentration in solution. Despite these early reports, the observed decrease in DNA RM concentration over time when stored frozen was surprising, since other DNA RMs developed in our laboratory and by another group11 were stable at -20°C, -70°C or -80°C. Comparison of the formulations of these materials revealed that the concentration of RM solutions for which we observed a decrease in concentration after freezing was several orders of magnitude lower than in DNA solutions that remained stable. This concentration was chosen to allow the use of the RM in a specific application without additional dilution, which, unless performed gravimetrically using a calibrated balance of required accuracy, could affect accuracy and reproducibility of downstream measurements. As concentrated DNA solutions have been reported to have a ‘self-protective’ effect during freeze-thaw,9,12 we hypothesized that the addition of carrier background nucleic acid may protect RM DNA from degradation during freeze-thaw. An alternative hypothesis was that the observed decrease in DNA concentration during storage at -20°C or -80°C5 was an artefact resulting from either inhomogeneity of the solution after freezing and thawing or formation of secondary structures that affected template amplification in quantitative polymerase chain reaction (PCR). In this study we experimentally tested these hypotheses with the aim of improving stability at temperatures below 0°C of DNA preparations considered for characterization as RM and for use as standards, calibrants or quality controls in genetic analyses. In investigating the effect of carrier nucleic acids on target DNA stability, we tested two different nucleic acids, salmon testes DNA and yeast RNA. Both nucleic acids are common in molecular biology research, inexpensive and unrelated to

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human DNA. The latter feature makes them suitable for analyses of human genomic sequences. On the other hand, the two nucleic acids differ in size and we were interested to investigate whether this parameter affects stability of frozen DNA solution and its intended use. To test the second hypothesis, we compared different protocols for sample preparation prior to DNA quantification aimed at improving homogeneity of DNA solution or eliminating hypothetical DNA secondary structures that might prevent accessibility of target template in PCR amplification. We previously showed that, unlike with storage at -20°C or -80°C, concentration of the DNA solution stored at 4°C remained stable.5 Therefore, we considered non-specific binding of DNA to walls of the storage tube an unlikely reason for the observed decrease in DNA concentration in frozen solutions. Nonetheless, this possibility was indirectly tested when different protocols for sample preparation (with different heating and mixing of the sample) were compared. To measure DNA concentration, we employed digital PCR (dPCR), as this measurement method allows reliable and accurate quantification of nucleic acid target sequences without the need for a calibrant.5,13 This property, together with unprecedented levels of precision and accuracy for target nucleic acid quantification, makes dPCR superior to other methods used in many early studies of DNA stability; these include comparison of threshold cycle values in real-time PCR over time, spectrophotometry, fluorometry with intercalating dyes, HPLC and gel densitometry with calibrant markers. To characterize candidate DNA RMs in accordance with ISO 1703414 and ISO Guide 351 and to assign a reference value with associated uncertainty, in addition to testing their long-term stability, we assessed their homogeneity and shortterm stability. As an outcome of the study, we identified an optimal formulation of DNA RM for use as quality control or a calibrant in analysis of human erythropoietin (EPO) transgenes or transcript in gene therapy or gene doping. The obtained knowledge will facilitate production of future RM suited to genomic and transcriptomic measurements and is also valuable for improving preservation of DNA solutions during storage at temperatures below 0°C in other applications.

EXPERIMENTAL SECTION DNA Materials. DNA material used in this study was a linearized pUC-based plasmid DNA (pDNAl) (3 866 base pairs (bp)) that incorporates a synthetic construct comprising several sequences from cDNA for human EPO gene.5 Three formulations of the material (4 000 copies/µL (cp/µL), nominal concentration) were prepared using three diluents: Tris-EDTA buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0; referred to as TE0.1), TE0.1 containing salmon testes DNA (Calbiochem) or TE0.1 containing total RNA from baker’s yeast (Sigma). The formulations are referred to, respectively, as pDNAl/TE0.1, pDNAl/DNA and pDNAl/RNA. The two nucleic acids used in the diluents differ in size: 18S and 25S rRNAs, the two major components in yeast RNA, are 1 789 and 3 392 bp, respectively,15,16 while the average molecular weight of the DNA fragments in the commercial preparation of salmon testes DNA is 50-100 kDa, corresponding to approximately 75-150 bp in length (personal communication with the supplier). Concentration of salmon testes DNA or yeast RNA in pDNAl/DNA and pDNAl/RNA was 50 ng/µL as verified by UV spectrophotometry of the diluents.

The concentration of pDNAl in each of the three formulations was estimated by droplet dPCR (ddPCR) using three validated EPO transgene-specific PCR assays (Assays 1, 3 and 5)17,18 as described below. For each formulation, 150 µL was dispensed into 120 0.5-mL screw cap polypropylene vials (Axygen, SCT-050-SS-A-S) in a biosafety cabinet using a calibrated dispenser. Randomly selected vials of each formulation were allocated for homogeneity and stability testing. Homogeneity of pDNAl in each formulation was analyzed as recommended in ISO Guide 351 using two subsamples from each of seven vials and Assay 2, similarly to previously described.5 Preparation for ddPCR analysis of formulations that had been stored frozen. Six protocols were compared for homogenizing the formulations that had been stored at -20°C or -80°C prior to ddPCR quantification. The standard protocol (protocol 1) included samples’ equilibration at room temperature (RT) for 1 h followed by incubation at 37°C for 5 min in ThermoMixer (Eppendorf, Hamburg, Germany) at 800 rpm, then cooling of samples at RT for 30 min and spinning them at 100 rpm for 1 min in a bench-top centrifuge (Sigma 115P). Details of other protocols are described in Results and Discussion. For each protocol, prior to taking an aliquot for analysis, the solution was pipetted up and down 5 times. In one experiment, thawed pDNAl/TE0.1 formulation was subjected to a digest with restriction enzyme MspI (10 U) as per manufacturer’s instruction (New England Biolabs). The digest was confirmed by DNA fragment analysis using Bioanalyzer with the High Sensitivity DNA kit (Agilent Technologies). DNA quantification. DNA quantification was carried out by ddPCR using validated assays17,18, a QX100 Droplet Digital PCR system (Bio-Rad Laboratories Pty Ltd., Australia) and a calibrated C1000 thermal cycler (Bio-Rad, Pleasanton, CA, USA). MIQE guidelines for ddPCR and previously described protocol were followed.5,13,19 For no template control (NTC), the nucleic acid was substituted with TE0.1. NTC was negative in all experiments. Stability testing. Stability testing of three materials was performed using a classical study design following recommendations in ISO Guide 35.1 At each time point except zero, the material retrieved from its stored temperature was processed using protocol 1. For zero time point, the concentration value obtained from the homogeneity study was used. Long-term stability testing was performed at 4°C, -20°C and -80°C with time points at 3, 13, 26, 52 and 126 (rounded off to the nearest whole number) weeks of storage using a different assay at each time point (Assay 5, 3, 4, 2 or 4, respectively). Short-term stability testing was performed at 40°C and at room temperature (RT) with time points at 3, 7 and 14 days and using, respectively, Assay 5, 4 or 3. At each time point, pDNAl concentration was measured in three replicate units, each analyzed in triplicate. Data analysis was performed using linear trend analysis. A new vial was used at each time point and no material was subjected to repeat freeze-thaw. Real-Time PCR analysis. For standard curve analysis of each formulation, three ten-fold serial dilutions were gravimetrically prepared (XP205 5-figure balance, Mettler-Toledo) from the 4 000 cp/µL solution using the corresponding diluent (TE0.1, RNA in TE0.1 or DNA in TE0.1). The four solutions (with concentrations between 4 and 4 000 cp/µL, equivalent to 16 to 16 000 cp/20 µL reaction, nominal concentrations based on ddPCR quantification) and NTC were analyzed in duplicate on


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Analytical Chemistry Stratagene MX3005P (Agilent Technologies) using one of five validated assays as previously described.17,18 In all experiments, NTC was negative. MIQE guidelines for qPCR were followed.20 RM copy number concentration value assignment and uncertainty estimation. The average concentration obtained from homogeneity assessment was assigned as the copy number concentration of each RM formulation (Xchar). Homogeneity relative standard uncertainty, ub,rel, and relative standard uncertainty in characterization of the batch, uchar, rel, were calculated as previously described.5,21 Short-term stability relative standard uncertainty, usts, rel, was calculated using room temperature as the storage temperature. Long-term stability relative standard uncertainty, ults, rel, was calculated in accordance with ISO Guide 351 by multiplying the standard uncertainty of the slope of the linear regression and the time period of three years, and expressing it as a percentage of the assigned value. The combined uncertainty for the copy number concentration for each RM consisted of the uncertainties from characterization, uchar, between-unit heterogeneity, ub, shortterm storage, usts, and long-term storage, ults (equation 1). The expanded uncertainty of the assigned value (URM) was calculated by multiplying the combined standard uncertainty by a coverage factor k (equation 1). k was calculated using effective degrees of freedom derived from the WelchSatterthwaite equation22 for a level of confidence of 95% and rounded to two significant figures.

𝑈𝑅𝑀 = 𝑘. 𝑢2𝑐ℎ𝑎𝑟 + 𝑢2𝑏 + 𝑢2𝑠𝑡𝑠 + 𝑢2𝑙𝑡𝑠

(1)

RESULTS AND DISCUSSION Preparation of frozen formulation prior to DNA quantification by ddPCR. To test whether the observed decrease in concentration of pDNAl/TE0.1 solution after it was stored frozen5 was due to its inhomogeneity post thawing, we tested six protocols to homogenize the solution for analysis. Since the decrease in DNA concentration over prolonged time was only observed after storage at -20°C or -80°C and not at 4°C, it was unlikely to be due to sticking of DNA to the tube walls. Nonetheless, experiments with varying conditions for DNA thawing and homogenization indirectly tested this possibility. The tested protocols varied from the standard protocol 1 by conditions for DNA thawing (temperature and duration) and mixing (speed, duration and temperature). In protocol 2, the first step of samples thawing was performed at 4°C overnight. Protocols 3 and 4 varied from the standard protocol in that the mixing of samples at 800 rpm was performed at 60°C instead of 37°C (Protocol 3) or by vortexing at RT for 20 s (Protocol 4). In comparisons of protocols 2 and 3 against protocol 1, one aliquot from a vial was taken for DNA quantification by ddPCR. In other comparisons, several aliquots from the same vial were analyzed to assess within-vial homogeneity. In detail, three vials of pDNAl/TE0.1, each processed using one of three protocols (1, 3 or 4), were sampled in five consecutive 30 µL aliquots from the top of the solution; the first, the third and the fifth aliquots were quantified by ddPCR. In a separate experiment, one vial processed using protocol 1 was sampled from the top and the bottom, then mixed again on ThermoMixer at 37°C for 30 min at 800 rpm, centrifuged at 92 g for 60 s and sampled again from the top and the bottom. A similar experiment on a different vial was performed to compare

Protocol 3 with or without the second mixing step on ThermoMixer; in this comparison it was performed at 60°C for 30 min. In the second mixing step in the last two comparisons, after 15 min of mixing, the sample was flicked 5 times, spun at 92 g for 60 s and returned to ThermoMixer for additional 15 min incubation. The results from these experiments (not shown) demonstrated that, regardless of the protocol used, the concentration of pDNAl after it was stored frozen was lower than before freezing. Importantly, no difference between the vials or aliquots from the same vial processed with different protocols was observed. This suggests that the decrease in pDNAl concentration in pDNAl/TE0.1 observed after storage at -20°C or -80°C was not due to inhomogeneity of the thawed DNA solution and was unlikely to be due to absorption of DNA to tubes. Next, we tested whether decreased concentration of pDNAl in pDNAl/TE0.1 after frozen storage was caused by formation of complex secondary structures that affected its amplification in ddPCR. For that, we investigated whether pDNAl fragmentation with MspI to loosen hypothetical secondary structures in a previously-frozen DNA solution affects DNA quantification. MspI was chosen because the targets for the PCR assays within pDNAl do not contain restriction sites for this enzyme. The concentration value for pDNAl in pDNAl/TE0.1 that, following freezing, was subjected to MspI digestion was not significantly different (p=0.12) from that for pDNAl/TE0.1 that was subjected to the same digestion protocol, but in the absence of the restriction enzyme. This result indicates that the lower concentration of pDNAl in pDNAl/TE0.1 observed post freezing was not attributed to formation of secondary structures. We concluded that the decrease in concentration of pDNAl in pDNAl/TE0.1 solution (initial concentration of 4 000 cp/µL in 150 µL aliquots) stored at -20°C and, to a lesser extent, at -80°C was, indeed, the result of DNA degradation. Effect of background nucleic acid on stability of linearized pDNA. Next we tested whether addition to the pDNAl solution of carrier nucleic acid, salmon testes DNA or yeast RNA, would improve stability of pDNAl in frozen solution. All three formulations were stable at 4°C for 126 weeks (Figure 1). This was in agreement with our previous observations5 and with results reported by others.8,23 At -20°C, the difference between the average concentration values at zero and 52-week time points were 64%, 7% and 2% for pDNAl/TE0.1, pDNAl/DNA and pDNAl/RNA formulations, respectively. pDNAl/TE0.1 displayed a non-linear decrease in concentration over time with the most rapid decrease occurring between time zero and the first analysis time point of 3 weeks (Figure 2). At -80°C, according to linear trend analysis, all three formulations were stable, although for pDNAl/TE0.1, 13% decrease in the average concentration values over 126 weeks was observed, compared to 2% and 4% for pDNAl/DNA and pDNAl/RNA, respectively. The observed lower concentration value of pDNAl/DNA at 52 weeks (Figure 1) was attributed to suboptimal performance in this formulation of Assay 2 used at this time point (see below in the section that describes testing of different pDNAl formulations in qPCR). The ddPCR results for pDNAl/DNA at 52 weeks were characterized by abnormally high number of droplets with intermediate fluorescence amplitude between the maximum and minimum amplitude of negative and positive droplets. This, in turn, led to underestimation of the number of



positive droplets and to lower and more variable values for pDNAl concentration.

Figure 1. Long-term stability of three pDNAl formulations (TE0.1 buffer alone or TE0.1 buffer containing either carrier DNA or RNA) at 4°C, -20°C and -80°C. The values at each time point, except zero, are the average concentration values with their standard deviation for three vials each analyzed in triplicate (n=9). A different assay was used at each time point (refer to the Experimental section). pDNAl/TE0.1 stored at -20°C was not analyzed beyond 52 weeks as we considered this temperature unsuitable for pDNAl/TE0.1 long-term storage. The value for zero time point is the average concentration value with its standard deviation obtained from characterization of the whole batch in the homogeneity study (n=42 for pDNAl/TE0.1 and pDNAl/RNA; for pDNAl/DNA, n=36, as one vial was detected an outlier by the Grubbs test). The line is the trend over time.

Lower stability of frozen DNA solution in Tris-EDTA compared to that stored at 4°C agrees with results reported by others8,9 and has been explained by mechanical DNA shearing by ice crystals during freezing. The finding of the greater decrease in the concentration value of pDNAl/TE0.1 stored at 20°C than at -80°C is consistent with our previous observation.5 A similar result was reported in another study with a plasmid formulation in Tris-EDTA buffer.8 For relatively short oligonucleotide sequences and for plasmid DNA/lipid complexes, slower freezing was also shown to be more harmful than rapid freezing.7,6,24 In addition, different degradation rates of genomic DNA from Listeria at two temperatures (0°C and 20°C) were observed by Röder and colleagues,25 who reported higher degradation in samples frozen at 0°C. An explanation for these results could be that the rate of freezing depends on the temperature at which a DNA solution is placed and this, in turn, affects the duration of the intermediate condition between a completely frozen and liquid solution. The longer this intermediate state of simultaneous presence of ice crystals and ‘cryconcentrated’ aqueous phase is, the greater the damage to the DNA integrity by ice crystals. Our results demonstrate that addition of carrier nucleic acid to a DNA solution at the concentration used in this study (4 000 cp/µL) improves its stability at -20°C or -80°C. The biggest impact of background nucleic acid was observed for storage at -20°C, as the decrease in the concentration of pDNAl/TE0.1 at this temperature was the highest (Figure 1). Similar stabilizing effect of carrier DNA was seen with dilute solutions of DNA amplicons in water.26 A ‘self-protective’ effect of concentrated DNA solutions has also been shown during repeat freeze-thaw and other physical stresses,9,12 with offered explanations including higher freezing point depression, better vitrification around DNA fragment regions, or higher compactness of DNA fragments at a higher concentration.9

Effect of background nucleic acid on amplification of pDNAl in qPCR. One intended application of the RM formulations is use as a calibrant and positive control in realtime PCR assays that we developed for analysis of cDNA or transcript for the human EPO gene.5 Therefore, it was important to test whether either nucleic acid in the pDNAl formulation has a matrix effect in these assays. Firstly, each pDNAl formulation was subjected to standard curve analysis using one of five PCR assays for cDNA-EPO (Experimental section). No trend of one formulation performing better than another was observed, although the deviation of the amplification efficiency and linearity from the values corresponding to an ‘ideal’ assay (efficiency is 100% and R2 is 1) was slightly lower for pDNAl/RNA than for pDNAl/DNA. Specifically, efficiency and linearity values for the five assays used with pDNAl/RNA ranged between 93 and 100% (efficiency), and 0.995 and 1 (R2). For pDNAl/DNA, these parameters ranged between 90 and 110% (efficiency) and 0.976 and 0.997 (R2). Secondly, for every dilution used in the standard curve analysis, we compared Cq values between three pDNA formulations. In real-time PCR, Cq value is the cycle at which the fluorescence from the reaction crosses a specified threshold level at which the signal can be distinguished from background levels.27 The Cq values for dilutions of pDNAl/RNA were similar to those for pDNAl/TE0.1 in all five assays (within variability between duplicates; an example for two dilutions in Assay 2 is shown in Figure 2). For two or more (out of four) dilutions of pDNAl/DNA, duplicate Cq values did not overlap with those for the corresponding dilutions of pDNAl/TE0.1 in Assays 1, 3, 4 and 5; the difference between the average Cq for the two formulations was between 0.4 and 1.5 Cq values. In Assay 2 Cq values for all dilutions of pDNAl/DNA were noticeably higher than for those of pDNAl/TE0.1 (average Cq differed between 3.3


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Analytical Chemistry and 4.0 values; Figure 2 shows results for two dilutions). These results indicate that, unlike yeast RNA, salmon testes DNA inhibited Assay 2 at the concentration of 200 ng per PCR. Additional product’s details from the supplier revealed that salmon testes DNA is complexed with polyethylenimine to form polyplexes with nitrogen/phosphate (N/P) molar ratio of 1.5-1.8. Binding of DNA to polyethylenimine has been shown to inhibit PCR amongst other effects.28,29 Based on these reports, the observed higher Cq values and slightly lower efficiency and linearity of pDNAl amplification in Assay 2 in the pDNAl/DNA formulation compared to the other formulations may be explained by its impurity. Thus, our results on the effect of two nucleic acids on amplification of pDNAl in specific qPCR assays suggest that yeast RNA is a better carrier nucleic acid than salmon testes DNA for to improve pDNAl stability at -20°C or -80°C.

Figure 2. Cq values for two amounts (160 and 16 cp/20 µL reaction) of pDNAl in three formulations amplified using Assay 2. Values are the average Cq values for duplicates. Error bars correspond to the difference between the duplicate measurements. Due to low resolution of the printed image, upper error bars in some conditions are not visible. TE0.1, DNA and RNA denote pDNAl/TE0.1, pDNAl/DNA and pDNAl/RNA formulations, respectively

Characterization and value assignment of RM formulations. Characterization of the three formulations of the pDNAl as RMs required assessment of their homogeneity (refer to Experimental Section) and short-term stability. In homogeneity testing, the F-test (p-values 0.27, 0.92 and 0.07 for pDNAl/TE0.1, pDNAl/DNA and pDNAl/RNA, respectively) suggested that the three formulations were homogeneous. At room temperature all three formulations were stable for two weeks. When storage was performed at 40°C, linear trend analysis (F-test) showed that pDNAl/DNA and pDNAl/RNA formulations were stable for two weeks, whilst for pDNAl/TE0.1 a statistically significant trend was observed (p=0.02). The average concentration values for the three formulations after two week storage at 40°C were slightly higher (between 5.8 and 9.4%) than at the start of the incubation. This slight increase was attributed to evaporation of diluent, as our early study using gravimetric analysis of identical vials filled with 150 µL TE0.1 and stored at 40°C for two weeks demonstrated a 12.4% (n=7) mass decrease.5 The assigned value and associated uncertainty for copy number concentration of pDNAl in each formulation was calculated as previously described5 based on a shelf life of three years from characterization (Table 1). For the pDNAl/TE0.1formulation, we did not assign the combined uncertainty when stored at -

20°C, as we reasoned that the 64% drop in concentration over 52 weeks makes this long-term storage condition unsuitable for a reference material. The relative expanded uncertainty for pDNAl/RNA RM at all three storage temperatures was two- to three-fold lower than for pDNAl/DNA RM: 6.0% to 8.0% vs 16% to 20%, respectively. The relative expanded uncertainty for pDNAl/RNA RM was also lower when compared with pDNAl/TE0.1 RM for two storage temperatures: 6.0% vs 10% for 4°C and 8.0% vs 24% for -80°C. For all three formulations, the largest relative contribution in the combined uncertainty was from the uncertainty associated with long term stability (Table 1); between the formulations, this contribution was the lowest for pDNAl/RNA RM. In summary, using dPCR as a highly accurate and precise method for DNA quantification, we investigated different factors affecting stability at -20°C and -80°C of linearized pDNA in a dilute (4 000 cp/µL, nominal concentration, equivalent to approximately 0.02 ng/mL) solution. Our results suggest that a decrease in the amount of DNA in TE0.1 buffer following freeze-thaw was due to DNA degradation and not to inhomogeneity of the solution or topological constraints of DNA molecules that reduced accessibility of amplifiable sequences to PCR oligonucleotides. We demonstrated that including carrier nucleic acid in the formulation, either DNA or RNA, improves stability of the pDNA in solution stored frozen. This is consistent with results from previous studies 9,12 that showed concentrated DNA solutions have a ‘self-protective’ effect during freeze-thaw. We found that, unlike yeast RNA, salmon testes DNA can inhibit PCR amplification in some assays, possibly due to additives present in the commercial preparation used. Therefore, addition of yeast RNA to improve stability of the DNA RM at -20°C or -80°C is preferred over addition of this salmon testes DNA. The three pDNA formulations used in this study (pDNA in TE0.1 buffer or with addition of one of two nucleic acids as a background) were characterized as RMs following recommendations of ISO Guide 35. The stability component and, subsequently, overall relative expanded uncertainty for the formulation with yeast RNA was the lowest, suggesting that yeast RNA is a better background nucleic acid for RM formulations. RNA as a background nucleic acid could also be preferred because it is likely to be applicable to a broader range of DNA RMs. This is because, in some applications, DNA used as a background nucleic acid in RMs formulations may have homology with target DNA sequences and could interfere with their analyses due to cross-reactivity.

CONCLUSION The findings of this study are important in production of reliable, stable DNA standards, including DNA RMs. They are also valuable when selecting protocols for storing DNA extracted from biological samples or synthesized in a laboratory (for example, cDNA) to ensure their stability.

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Phone: +61 2 8467 3672

Author Contributions The manuscript was written through contributions of all authors.



All authors have given approval to the final version of the manuscript.

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ACKNOWLEDGMENT This work was supported by the World Anti-Doping Agency. We thank Leo Pinheiro and Daniel Burke from the National Measurement Institute for critically reviewing the manuscript.

Notes The authors declare no competing financial interest.

Table 1. Assigned values and their uncertainties for the copy number concentration of the three RM formulations using a shelf life of three years and long term storage at three temperatures, 4°C, -20°C and -80°C pDNAl/TE0.1 RM* 4°C RM concentration (assigned value) (cp/µL) (Xchar)

-80°C

pDNAl/DNA RM 4°C

-20°C

-80°C

pDNAl/RNA RM 4°C

-20°C

3 950

3 850

4 000

characterization relative standard uncertainty (%) (uchar, rel)

1.6

1.7

1.3

homogeneity relative standard uncertainty (%) (ub, rel)

0.7

0.8

1.0

short-term stability relative standard uncertainty (%) (usts, rel)

0.9

1.1

-80°C

0.7

long-term stability relative standard uncertainty (%) (ults, rel)

3.7

8.5

6.0

5.9

6.4

1.9

2.2

2.8

coverage factor (k)

2.4

2.8

2.6

3.2

2.8

2.2

2.2

2.4

relative expanded uncertainty for assigned value (%) (URM, rel)

10

24

16

20

19

6.0

6.0

8.0

expanded uncertainty of assigned value (cp/µL) (URM)

410

960

630

770

720

230

250

320

*

Expanded uncertainty for pDNAl/TE0.1 RM stored at -20°C was not calculated as we considered this storage temperature unsuitable for long-term storage of this RM; expanded uncertainties are rounded to two significant figures; the assigned values are rounded to match the level of significance of the expanded uncertainty.

REFERENCES (1) ISO Guide 35, Reference materials - Guidance for characterization and assessment of homogeneity and stability: Geneva, Switzerland, 2017. (2) Lee, S. B.; Crouse, C. A.; Kline, M. C. Forensic Sci. Rev. 2010, 22, 131-144. (3) Dhanasekaran, S.; Doherty, T. M.; Kenneth, J. J. Immunol. Methods 2010, 354, 34-39. (4) Rossmanith P; Röder B; Frühwirth K; Vogl C; M, W. Appl. Microbiol. Biotechnol. 2011, 89, 407-417. (5) Baoutina, A.; Bhat, S.; Zheng, M.; Partis, L.; Dobeson, M.; Alexander, I. E.; Emslie, K. R. Gene Ther. 2016, 23, 708-717. (6) Anchordoquy, T. J.; Girouard, L. G.; Carpenter, J. F.; Kroll, D. J. J. Pharm. Sci. 1998, 87, 1046-1051. (7) Davis, D. L.; O'Brien, E. P.; Bentzley, C. M. Anal. Chem. 2000, 72, 5092-5096. (8) Podivinsky, E.; Love, J. L.; Van der Colff, L.; Samuel, L. Anal. Biochem. 2009, 394, 132-134. (9) Shao, W.; Khin, S.; Kopp, W. C. Biopreserv. and Biobanking 2012, 10, 4-11. (10) Schaudien, D.; Baumgartner, W.; Herden, C. Diagn. Mol. Pathol. 2007, 16, 153-157. (11) Deprez, L.; Mazoua, S.; Corbisier, P.; Trapmann, S.; Schimmel, H.; White, H.; Cross, N.; Emons, H., The certification of the copy number concentration of solutions of plasmid DNA containing a BCR-ABL b3a2 transcript fragment: ERM-AD623a, ERM-AD623b, ERM-AD623c, ERM-AD623d, ERM-AD623e, ERM-AD623f; European Commission, Joint Research Centre, Institute for Reference Materials and Measurements Geneva, Switzerland, 2012. (12) Yoo, H. B.; Lim, H. M.; Yang, I.; Kim, S. K.; Park, S. P. J. Biophys. Chem. 2011, 2, 102-110. (13) Pinheiro, L. B.; Coleman, V. A.; Hindson, C. M.; Herrmann, J.; Hindson, B. J.; Bhat, S.; Emslie, K. R. Anal. Chem. 2012, 84, 1003-1011.

(14) ISO 17034:2016(E), General requirements for the competence of reference material producers: Geneva, Switzerland, 2016. (15) Georgiev, O. I.; Nikolaev, N.; Hadjiolov, A. A.; Skryabin, K. G.; Zakharyev, V. M.; Bayev, A. A. Nucleic Acids Res. 1981, 9, 6953-6958. (16) Rubtsov, P. M.; Musakhanov, M. M.; Zakharyev, V. M.; Krayev, A. S.; Skryabin, K. G.; Bayev, A. A. Nucleic Acids Res. 1980, 8, 5779-5794. (17) Baoutina, A.; Coldham, T.; Bains, G. S.; Emslie, K. R. Gene Ther. 2010, 17, 1022-1032. (18) Baoutina, A.; Coldham, T.; Fuller, B.; Emslie, K. R. Hum. Gene Ther: Methods. 2013, 24, 345-354. (19) Huggett, J. F.; Foy, C. A.; Benes, V.; Emslie, K.; Garson, J. A.; Haynes, R.; Hellemans, J.; Kubista, M.; Mueller, R. D.; Nolan, T.; Pfaffl, M. W.; Shipley, G. L.; Vandesompele, J.; Wittwer, C. T.; Bustin, S. A. Clinical chemistry 2013, 59, 892902. (20) Bustin, S. A.; Benes, V.; Garson, J. A.; Hellemans, J.; Huggett, J.; Kubista, M.; Mueller, R.; Nolan, T.; Pfaffl, M. W.; Shipley, G. L.; Vandesompele, J.; Wittwer, C. T. Clinical chemistry 2009, 55, 611-622. (21) Linsinger, T. P.; Pauwels, J.; van der Veen, A. M.; Schimmel, H. G.; Lamberty, A. Accredit. Qual. Assur. 2001, 6, 20-25. (22) JCGM, JCGM 100 – Evaluation of measurement data – Guide to the expression of uncertainty in measurement (GUM 1995 with minor corrections); 2008. (23) Jerome, K. R.; Huang, M. L.; Wald, A.; Selke, S.; Corey, L. J. Clin. Microbiol. 2002, 40, 2609-2611. (24) Zelphati, O.; Nguyen, C.; Ferrari, M.; Felgner, J.; Tsai, Y.; Felgner, P. L. Gene Ther. 1998, 5, 1272-1282. (25) Röder, B.; Frühwirth, K.; Vogl, C.; Wagner, M.; Rossmanith, P. J. Clin. Microbiol. 2010, 48, 4260-4262. (26) Kohler, T.; Rost, A. K.; Remke, H. BioTechniques 1997, 23, 722-726.


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Analytical Chemistry (27) ISO 16577:2016 Molecular biomarker analysis - Terms and definitions: Geneva, Switzerland, 2014. (28) Mady, M. M.; Mohammed, W. A.; El-Guendy, N. M.; Elsayed, A. Int. J. Phys. Sci. 2011, 6, 7328-7334. (29) Fotouhi, G. Study of microtip-based extraction and purification of DNA from human samples for portable devices. Dissertation for the degree Doctor of Philosophy, University of Washington, 2015.


Analytical Chemistry

For TOC only

5,000

pDNA in TE0.1 buffer at 4°C pDNA (cp/µL)

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4,000

pDNA in TE0.1 buffer with RNA at -20°C

3,000 2,000

pDNA in TE0.1 buffer at -20°C

1,000 0

20

40

60

80

Time (weeks)

100


120

Storage stability of solutions of DNA standards

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