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Blood storage at 4Cfactors involved in DNA yield and quality

ANDREA J. RICHARDSON, NIRO NARENDRAN, ROBYN H. GUYMER, HIEN VU, and PAUL N. BAIRD
MELBOURNE AND SYDNEY, AUSTRALIA

Background: Whole blood provides one of the most common sources of both high-quality DNA and high-quantity DNA for molecular biological purposes. Typically, blood storage at 4C is short term, which ranges from a few days to a few weeks. However, long-term storage usually involves blood being frozen, with a resultant loss in DNA yield. The authors examined the effects of long-term storage at 4C. Methods: Duplicate blood samples were collected from 301 participants (aged 20 98 years) enrolled as part of ongoing studies. Samples were stored at 4C for between 11 days and 922 days, and DNA was subsequently extracted using a phenol/chloroform procedure. Results: A negative correlation of the number of storage days existed at 4C with DNA yield. The main determinant on DNA yield was the age of the participant in the study, with older persons having a lower DNA yield. Conclusions: Long-term storage of blood at 4C does have a detrimental effect on DNA yield, but this effect seemed less significant than the age of a person. The impact of age of a person or storage time has a minimal impact on DNA quality. Therefore, storage of blood at 4C offers an acceptable alternative to frozen storage. (J Lab Clin Med 2006;147:290 294)
Abbreviations: DNA deoxyriboenucleic acid; dNTP deoxynucleoside triphosphate; EDTA ethylenedinitrilotetraacetic acid; MgCl2 magnesium chloride; NaCl sodium chloride; NaOAC sodium acetate; OPTN optineurin; PCR polymerase chain reaction; RNA ribonucleic acid; SAS statistical analysis system; SD standard deviation; SDS sodium dodecyl sulphate; Taq thermus aquaticus polymerase; TE buffer tris(hydroxymethyl)aminomethane ethylenedinitrilotetraacetic acid; UV ultraviolet

he collection of whole blood from consented persons offers a readily accessible resource for the extraction of DNA or RNA and their subsequent analysis in the study of disease. Although it is
From the Centre for Eye Research Australia, University of Melbourne, East Melbourne, Australia; and the Vision CRC Ltd, Sydney, Australia. Supported by the Rebecca L. Cooper Medical Research Foundation Limited and the Australian Government Cooperative Research Centre Program. Submitted for publication August 12, 2005; revision submitted December 22, 2005; accepted for publication January 30, 2006. Reprint requests: Andrea Richardson, Centre for Eye Research Australia, 32 Gisborne Street, East Melbourne, Victoria, 3002, Australia; e-mail: andreajr@unimelb.edu.au. 0022-2143/$ see front matter 2006 Mosby, Inc. All rights reserved. doi:10.1016/j.lab.2006.01.005

possible to obtain DNA from a variety of sources such as buccal swabs, Guthrie cards, and hair samples, they are often limited in their usefulness because of the smaller DNA yields compared with whole blood.1 The current belief is that long-term storage of blood at 4C has a severe and detrimental impact on the yield and the quality of DNA. As a consequence, many articles have focused on the short-term effects of blood storage, with the temperature at which blood can be stored being the main emphasis.2 6 Cushwa and Medrano2 reported that if blood was not processed immediately, then the optimum storage temperature was 4C for up to a maximum of 4 weeks, whereas blood stored at room temperature needed to be dealt with immediately and blood stored at 37C always yielded lower DNA amounts compared with blood stored at lower temperatures. However, it has also been shown that DNA in useable quantities can be obtained from blood

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Fig 1. A 2% agarose gel shows PCR-amplied products of the OPTN gene. Lane 1 Molecular weight marker (50-bp ladder, Invitrogen). Lane 2 positive control. Lanes 319 selected samples amplied using 2 ng. Days Number of days each blood had been stored at 4C before DNA extraction. A single 719-bp product of the OPTN gene in lanes 219 is shown.

that has been subjected to multiple temperatures above 4C before being processed.5 Similarly, Lahiri and Schnabel6 observed little difference in yield or quality of extracted DNA when blood was stored at temperatures of 45C, 37C, 25C, 20C, or 70C for a 24-hour period before DNA extraction compared with blood stored at 4C. In addition, good quality DNA has also been extracted from blood stored at room temperature for 1 week.7 Typically, collected blood samples are processed as soon as possible to avoid any anticipated drop in yield and to minimize potential DNA degradation. When processing cannot be undertaken immediately, for example, in multicenter studies, blood samples are usually frozen. Freezing blood at 20C, for even a short period, has, however, been reported to lead to a lower DNA yield and an increased risk of protein contamination.2,8,9 Long-term DNA storage (several years) undertaken at 70C to 80C has been shown to have little impact on DNA yield or quality.8 However, not all laboratories processing DNA have 70C to 80C freezers available for their samples. There have been no reported studies on the impact of long-term storage of patients blood samples at 4C (conventional refrigerator) on DNA quality or quantity. Therefore, the authors investigated whether this temperature could be used as an alternative long-term storage option to assess its impact on DNA.
MATERIALS AND METHODS Duplicate 7.5-mL aliquots of whole blood were collected from 301 persons recruited from clinics for the study of eye

disease. The research was carried out according to the principles of the Declaration of Helsinki. Written informed consent was obtained from all persons, and ethics approval for the project was provided by the Human Research and Ethics Committee of the Royal Victorian Eye and Ear Hospital, Melbourne, Australia. Blood samples were collected in 9-mL sterile vacuette K3E EDTA tubes (Greiner bio-one, Kremsmnster, Austria) and stored immediately. Once in the laboratory, bloods were stored for between 11 days and 922 days (2.5 years) in a standard household 4C refrigerator (Fisher & Paykel, Derrimut, Victoria, Australia) until processed. DNA extraction was performed using a standard phenolchloroform method as previously described.10 In brief, cold (4C) blood was mixed with 40 mL of buffer A (320-mM sucrose, 10-mM tris pH 7.5, 5-mM MgCl2, 1% Triton X-100), shaken and spun at 690 g for 10 minutes at 4C. The resulting pellet was rewashed and spun in 40-mL buffer A, and the pellet was lysed with the addition of 2.5-mL buffer B (75-mM NaCl, 25-mM EDTA, pH 8.0), 20% SDS, and 25-L Proteinase K (20 mG/mL) overnight at 50C. An equivalent volume of 50% phenol/50% chloroform was added, mixed, and spun at 1350 g for 15 minutes, and the process was repeated using an equivalent volume of chloroform. Precipitation of DNA was in the presence of a one-tenth volume of 3-M NaOAc (pH 5.3) and 2.5 volumes of absolute ethanol, washed in 70% ethanol, and resuspended in 200 L of TE buffer (10-mM Tris pH 7.5, 1-mM EDTA) for subsequent storage at 4C. The yield (quantity and purity) of the DNA was calculated by spectrophotometry on a Beckman DU640B spectrophotometer (Mount Waverley, Victoria, Australia) using the ratio of UV absorbance at 260 nm and 280 nm. A UV absorbance ratio of 1.8 was considered to be good quality DNA. The effect of DNAses was limited by the use of phenol/chloroform in the extraction method.

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Molecular techniques. To assess the quality of DNA for use in molecular techniques, DNA was amplied by the PCR, which consisted of an initial cycle of 94C for 15 seconds, followed by 40 cycles of 94C for 30 seconds, 54C for 30 seconds, and 72C for 30 seconds. Each PCR reaction contained varying amounts of genomic DNA, ranging from 2 ng to 25 ng, 0.2 U of Taq (Invitrogen, Mulgrave, Australia), 10 X buffer (Invitrogen), 2-mM dNTPs (Scientix, Cheltenham, Victoria), 0.75-mM MgCl2 (Invitrogen), 1 ng/L of a primer pair of choice and distilled water to a nal volume of 25 L. The primers 5=-CTGCGACGTAAAGGAGCATTG-3= and 5=-CTGTTCATTACTAGGCTATGG-3= (Geneworks, Rundell Mall, Adelaide) amplifying a 719-bp fragment of exon 11 and 12 of the OPTN gene (assession number AF283525) were used in this study. PCR products were run on 2% agarose gels and visualized in the presence of ethidium bromide. As a quality control measure, 17 DNA samples covering a range of storage times from 11 days to 884 days underwent a standard PCR reaction. DNA samples ranging from 11 days of storage, with approximately 50-day intervals thereafter, were chosen. Selection was not based on the quality of the DNA, but on the availability of a DNA sample stored for a specic length of time. A 719-bp DNA product of the OPTN gene was successfully amplied from all 17 samples using between 50 ng and 2 ng of DNA (Fig 1). Statistical analysis. Statistical analyses were performed using SAS version 8.2 (SAS Institute, Cary, North Carolina). The authors tted the mixed models for dependent and approximately normally distributed DNA yields to obtain the overall mean DNA yields, the mean yields in each age or day storage groups, and the mean yields in each day storage groups adjusted by (continuous) age. The F-tests applied to the estimates of these mixed models gave the P-values for comparing mean DNA yields across different age or day storage groups. The mixed model was also used to assess the difference among yield means across the operators. A test giving a P-value less than 0.05 was considered to be statistically signicant.

Table I. Study characteristics of age of person together with storage time and DNA yield
Age of participant Storage (days) Yield (g/mL) (years) (n 301) (n 602) (n 602)

Minimum Maximum Mean SD

20.7 98.5 69.7 15.6

11.0 922.0 253 152

234 1583 870 364

The mean and standard deviation were obtained by fitting the mixed model without covariates. Notes: A total of 301 participants took part, each providing two tubes of blood, enabling 602 individual samples to be investigated. Storage days and yield are therefore based on 602 samples. Abbreviation: n, number in study.

Table II. Mean DNA yields from blood extracted from persons of different age ranges
Age group (years) n 602 Mean DNA Yield SD (g/mL)

2050 5080 80 P value

72 376 154

1033 41 874 18 785 28 0.0001

Abbreviations: n, number in study; SD, standard deviation. P values obtained from the F-tests for equality of DNA yield means among three age groups.

Table III. Mean DNA yield storage time (days) between collection and extraction
Storage time group (days) n 602 Mean Yield SD (g/mL)

200 200300 300 P value

238 162 202

958 21 882 24 764 23 0.0001

RESULTS

A total of 301 persons aged from 20 years to 98 years of age (mean 69 years, SD 15.6 years) were enrolled into this study. Most persons were over the age of 50 years with 188 (62.5%) aged between 50 years and 80 years, 77 (25.6%) aged over 80 years, and only 36 (12%) of the participants aged between 20 years and 50 years (Table I). Blood aliquots stored for less than 3 months were red in color, and visual inspection of supernatant plasma did not suggest signicant hemolysis, although this nding was a subjective assessment. However, blood tubes stored for periods longer than 3 months did show evidence of hemolysis, with the supernatant being more brown in color on visual inspection. No evidence existed of clotting in any of the tubes, even over extended time periods. The mean number of storage days for blood samples

Note: Values are age-adjusted. Abbreviations: n, number in study; SD, standard deviation. P values obtained from F-tests for mean of DNA yield means among three different day storage groups.

in the study was 253 days (SD 152 days) (11922 days) (Table I). DNA was successfully extracted from all samples and resuspended in 600 L of TE buffer, with the yield ranging from 234 g/mL (46.8 G) to 1583 G/mL (316.6 G), with the mean DNA yield being 870 G/mL (174 G) (SD 364 G/mL) (Table I). The maximal mean DNA yield was obtained in the 20-year to 50-year age group (1037 G/mL) (Table II). However, increasing age led to a signicant reduction in DNA yield, with the 80 age group showing a mean DNA yield of 785 G/mL, a reduction of 24% (248 G/mL) compared with the younger age group (Table II). As a result of the large sample size, extracted bloods

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Table IV. DNA yield from 301 persons at two extraction time points
Yield at time point 2 (g/mL) Yield at time point 1 (g/mL) <600 600900 9001200 1200 Total number of samples

600 600900 9001200 1200

22 26 19 7

20 44 21 14

9 28 25 13

5 6 18 24

56 104 83 58

Notes: Data shown indicate the number of cases in each yield group. Bold numbers indicate the number of samples where DNA yield was in the same category at time point 1 and 2.

were grouped into three storage groups: less than 200 days, between 200 days and 300 days, and over 300 days (Table III). Each group represented approximately one third of the total number of samples. When adjusted for age, a signicant (20.3%; 194 G/mL) decrease occurred in mean DNA yield between the 200day storage time group and the 300-day storage time group (Table III). Duplicate blood aliquots were obtained from each person. DNA was extracted at two time points to allow comparison of DNA yield at these time points (Table IV). Duplicate tubes of blood, each containing the same volume of whole blood, were collected at the same time from a participant. Time point 1 represented the rst time of DNA extraction using the rst tube of blood, and time point 2 represented the second time of DNA extraction using the second tube of collected blood. The minimum time interval between time point 1 and time point 2 was 5 days, and the maximum time interval was 756 days. The mean time interval between time point 1 and time point 2 was 120 days. The categories of 600 G/mL, 600 900 G/mL, 900 1200 G/mL, and 1200 G/mL were chosen as they each represented approximately one quarter of the sample cohort. Overall, 38.2% of samples fell within the same category of DNA yield for the rst and second extraction time points. Approximately one third (33.2%) of samples resulted in a lower DNA yield at time point 2 compared with time point 1, and 28.6% of samples resulted in a higher DNA yield at time point 2 compared with time point 1.
DISCUSSION

Maximization in quantity and quality of extracted DNA is the highest priority for any laboratory undertaking molecular analysis from a clinical sample. In this study, the authors showed that the main factors impacting on DNA yield were the age of a person (25%) followed by the length of storage time at 4C (20%). However, the overall mean yield obtained in this study was approximately 1.4 times greater than previously

described in the literature11 and similar to those yields obtainable from commercially available kits. The effect of age on DNA yield is of interest, and this decline in DNA yield may represent the known decline in total leukocyte and lymphocyte count that occurs between birth and adulthood.12,13 The study by Erkeller-Yuksel et al12 found a statistically signicant (P 0.005) halving in leukocyte count from birth (cord blood) to adults in the 18-year to 70-year age group. These ndings further extend this adult age group to persons of 98 years old and may suggest that the observed decline in DNA yield that the authors detected may represent continual decline of leukocyte number with increasing age. Further studies of adult leukocyte count based on adult age would be benecial to replicate the observations reported here. The second most signicant cause of loss of DNA yield was with the length of storage time, in which a signicant decrease in yield was identied in samples stored less than 200 days compared with samples stored over 300 days. However, the decrease in mean yield mainly seemed to be caused by those persons who had a very high DNA yield at their initial extraction time point. The quality of the DNA did not seem to be affected by storage length, and DNA fragments of less than 1000 bp, which are typically used in PCR, could be readily identied from small amounts (2 ng) of DNA from any time storage period. Additional analysis of several of these fragments via dideoxy sequencing conrmed that long-term storage at 4C did not have an adverse effect on DNA quality. Larger DNA fragments were not amplied in this study, but other factors including the quality of the components of the PCR mixture, including Taq DNA polymerase and primer design, would also need to be controlled in such a study. In conclusion, the authors demonstrated that both the age of a person and the length of storage time at 4C affected the yield of DNA. Each of these effects seemed to lead to a drop in DNA yield of 20 25%. The recommendation from this study would be to prioritize samples for DNA extraction based on the age of a person, with older persons being processed rst, and the difculty or importance of that particular sample in a study, which reects

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the nding that even storage of blood samples for up to 2.5 years will yield a viable DNA sample, but the yield will drop over time. Therefore, in summary, long-term storage of blood at 4C may provide an alternative option to current storage options for laboratories to perform molecular genetic studies from clinical samples. However, the nal choice of storage method remains with the individual laboratory.
Acknowledgments: The authors wish to thank Kelly Pertile, Andrew Bell, C. S. Mao, Robyn McNeil, and Belinda Tomlin for DNA extraction, and Dr. F. M. Amirul Islam for statistical assistance.
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