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Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva
Clinica Chimica Acta 343 (2004) 191 – 194 www.elsevier.com/locate/clinchim
Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva Daniel P.K. Ng, David Koh *, Serena G.L. Choo, Vivian Ng, Qiuyun Fu Department of Community, Occupational and Family Medicine (MD3), Faculty of Medicine, National University of Singapore, 16, Medical Drive, Singapore 117597, Singapore Received 26 November 2003; received in revised form 16 January 2004; accepted 19 January 2004
Abstract Background: Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR). Methods: Saliva was collected from healthy volunteers and 2-ml aliquots subjected to different storage conditions: S1—washing of saliva using phosphatebuffered saline (PBS) and extraction of DNA on the same day of collection; S2—washing and centrifugation to yield a pellet, which was stored at 70 jC for 1 week prior to DNA extraction; S3—storage of whole saliva at 4 jC for 7 days, followed by washing and extraction of DNA; S4—storage at 4 jC for 7 days, followed by washing and pellet formation. The pellet was stored at 70 jC for 1 month before extraction of the DNA; S5—storage at 70 jC for 1 month, followed by washing and extraction of DNA. DNA yield and purity was determined by spectrophotometry at 260 and 280 nm. Twenty nanograms of genomic DNA was used for the polymerase chain reaction, and the resulting PCR band was captured by digital photography and quantified. Results: The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction, based on OD260/280 ratios, was good and comparable. PCR resulted in the presence of a single specific product of the correct size from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions (P < 0.05). Compared to S1 samples, PCR bands from conditions S2 and S3 were not as strong, while those amplified from S4 and S5 samples were the weakest. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1 – S3. Qualitatively similar results were obtained when the PCR experiment was repeated. Conclusions: Saliva can act as a useful source of genomic DNA, even when stored under less than optimal conditions. D 2004 Elsevier B.V. All rights reserved. Keywords: Saliva; Genotyping; Storage conditions; Genomic DNA; Polymerase chain reaction
1. Background Abbreviations: ANOVA, analysis of variance; DNA, deoxyribonucleic acid; GFPT1, glutamine-fructose-6-phosphate transaminase 1; OD, optical density; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; S.D., standard deviation. * Corresponding author. Tel.: +65-6874-4988; fax: +65-67791489. E-mail address: [email protected] (D. Koh). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.01.013
Large population-based studies, involving hundreds to thousands of study subjects, are crucial in the search for the genetic determinants underlying common complex diseases such as diabetes mellitus [1]. Genetic material, in the form of genomic DNA, is typically
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D.P.K. Ng et al. / Clinica Chimica Acta 343 (2004) 191–194
obtained from blood samples, a collection method that is invasive, painful and generally disliked by study subjects. For population or community studies, especially among children, less invasive methods are preferable, as this will likely result in higher study participation rates. Recent years has seen an increased interest in the use of saliva as a diagnostic fluid [2]. Xenobiotics and their metabolites, enzymes, hormones, immunoglobulins and other molecules have successfully been quantified in saliva. For the extraction of DNA from saliva, commercial kits are already available that serve this purpose. Coupled with the ease of saliva collection (by passive drooling) and the ability to isolate sufficient DNA for genetic analyses, saliva is a potentially attractive source of genetic material. Due to logistic, financial, practical and methodological reasons in large studies, it is often necessary to store saliva samples prior to the extraction of DNA. Thus, in this study, we sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR).
2. Materials and methods 2.1. Collection and storage of saliva We recruited 10 healthy adult volunteers from the staff of the Department of Community, Occupational and Family Medicine, National University of Singapore. Each person provided 12 ml of saliva that was passively accumulated over a single time period. The subjects were advised not to smoke, eat or drink for 1 h prior to saliva collection. The collected saliva was thoroughly mixed by vortexing and inversions. Five aliquots of 2 ml each were dispensed into 15-ml tubes and subjected to different storage procedures. We opted to examine 2-ml aliquots as in our previous experience, using smaller volumes (V1 ml) of saliva yielded very limited DNA amounts in some individuals, which would have precluded accurate measurements of its yield. The storage conditions studied were as follows: S1—washing of saliva using phosphate-buffered saline (PBS) and extraction of DNA on the same day of collection;
S2—washing of saliva and centrifugation to yield a pellet, which was stored at 70 jC for 1 week prior to DNA extraction; S3—storage of whole saliva at 4 jC for 7 days, followed by washing and extraction of DNA; S4—storage of whole saliva at 4 jC for 7 days, followed by washing and pellet formation. The pellet was stored at 70 jC for one month before extraction of the DNA; and S5—storage of whole saliva at 70 jC for 1 month, followed by washing and extraction of DNA. 2.2. DNA extraction Extraction of saliva DNA was performed as detailed below. Eight milliliters of PBS was added to 2 ml of saliva, vortexed and then centrifuged at 1800g for 5 min. The supernatant was carefully removed and the pellet rinsed using an additional 1 ml of PBS, before centrifugation again at 1800g for 5 min. After removal of the supernatant, the pellet was resuspended in 180 Al of PBS. For samples stored under condition S5, two additional centrifugation steps were included in the standard protocol (used for conditions S1 – S4) as freezing of whole saliva caused cell lysis making it harder to form a pellet. DNA extractions were performed using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA), in accordance with manufacturer’s instructions; RNAse treatment was also performed following these instructions. DNA was finally eluted in 50 Al of AE buffer (provided in the kit). DNA concentration was measured by spectrophotometry at 260 nm. Purity of DNA was assessed using the ratio of OD260/280 with a ratio of 1.8 – 2.0 being of good purity [3]. Quantification of all DNA samples was performed together by the same laboratory technician using a Smartspec 3000 spectrophotometer (Bio-Rad Laboratories, USA). 2.3. PCR, agarose gel electrophoresis and quantification of PCR bands Twenty nanograms of DNA was used in each 25 Al PCR reaction. PCR was performed in the presence of 1.5 mmol/l Mg2+ using 0.6 U of Taq polymerase (Promega, Madison, WI) with the following pair of primers, which spanned 581 bp of the glutamine-
D.P.K. Ng et al. / Clinica Chimica Acta 343 (2004) 191–194
193
Table 1 Yield and quality of DNA isolated from saliva stored under different conditions Conditions
S1
S2
S3
S4
S5
Yield (ng)/2 ml whole saliva OD260 /OD280 ratio PCR product band volumea,*
4430F5546 1.69F0.27 613.1F317.7
7889F9722 1.90F0.38 562.2F234.3
2979F1421 1.95F0.23 508.7F292.2
3479F1934 1.93F0.25 422.1F236.7
6790F7616 1.81F0.06 429.9F229.8
Data is present as meanFS.D. * ANOVA with repeated measures, P < 0.05. a Post-hoc analyses using Duncan’s multiple range test showed that the means of PCR product band volume for conditions S4 and S5 were significantly different from S1 – S3.
fructose-6-phosphate transaminase 1 (GFPT1) gene: forward primer—5V-CACTGTTTGCTTCAGCTATGC-3V and reverse primer—5V-AGGAACTTTAAAGCATGACAATC-3V. The PCR program consisted of 40 amplification cycles, each consisting of denaturation at 94 jC for 45 s, annealing at 58 jC for 45 s and extension at 72 jC for 60 s, with final extension at 72 jC for 10 min. This protocol has been previously used by our group to successfully amplify the PCR product from >900 individual samples in a separate genetic study [4].
Samples from the five storage conditions were amplified together using a single PCR machine. These PCR products were then fractionated on the same 1% agarose gel containing ethidium bromide. Image of the PCR bands were captured and quantified using the GelDoc EQ digital gel documentation system (BioRad Laboratories, USA). Overexposure of the gel image was avoided as this would lead to an erroneous quantification of the PCR bands. PCR bands were quantified as band volume, which is the sum of the intensities of the pixels within the band boundary
Fig. 1. Quantification of PCR products amplified from genomic DNA isolated from saliva stored under different conditions (S1 – S5). Top panel shows typical PCR results obtained from different individuals. Samples from the five storage conditions were amplified at the same time using a single PCR machine, fractionated on the same agarose gel and digitally photographed before quantifying the PCR band volumes.
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multiplied by pixel area (in mm2), using the Quantityone software (Bio-Rad Laboratories, Hercules, CA).
The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction was good and comparable across the different storage conditions, based on OD260/280 ratios (Table 1). PCR resulted in the presence of a single specific product of the correct size (581 bp) from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions ( P < 0.05, Table 1). Compared to S1 samples which were processed immediately after collection, PCR bands from conditions S2 and S3 were not as strong (Table 1, Fig. 1). Bands amplified from samples subjected to conditions S4 and S5 were the weakest in the experiment. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1 – S3. Qualitatively similar results were obtained when the PCR experiment was repeated (data not shown).
ted tubes) or personnel training. Given the non-invasive and painless nature of saliva collection, study subjects are also more likely to consent to donating saliva as opposed to blood. This advantage may translate into better consent rates for modern genetic studies, which need large numbers of study subjects. Our results demonstrated that DNA can be successfully extracted from saliva for at least up to 1 month when stored at 70 jC. These findings have important implications with regards to measurement validity in the field of salivary biomarkers research. Although we collected 12 ml of saliva from each person, this was to provide sufficient saliva for each storage condition being tested. In practice, 2 ml of saliva can provide sufficient genomic DNA to test an average of 30 –150 genetic markers, if 20– 100 ng of DNA was used for each PCR reaction. While condition S1 was expected to yield the best quality DNA, and PCR analyses supported this conclusion, DNA obtained under conditions S2 and S3 produced comparable results as well, indicating that samples can be stored under these conditions with minimal deterioration if it is not practical to process the samples straightaway. Furthermore, although the storage of the samples at 4 jC for up to 7 days prior to extraction (i.e. condition S3) may result in some bacterial growth, this did not drastically impact the extraction of PCR-quality DNA. Compared to the other conditions, S4 and S5 were less favorable with a 17– 30% decrease in PCR products. Nevertheless, DNA extracted under these conditions can still be amplified and storage under these conditions remains a less attractive, but still viable option.
4. Discussion
References
We examined the impact of different storage conditions on the extraction of PCR-quality of genomic DNA from saliva. The impetus to look into this issue stemmed from the practical consideration that, in planning the workflow of laboratory analyses in large genetic studies, it is often required to store samples prior to their analyses. Thus, knowledge of acceptable storage conditions is important. Furthermore, our study focused on DNA extraction from saliva instead of blood as the saliva collection is quick, does not require specialized consumables (syringes, anticoagulant-trea-
[1] Risch N, Merikangas K. The future of genetic studies of complex human diseases. Science 1996;273:1516 – 7. [2] Streckfus CF, Bigler LR. Saliva as a diagnostic fluid. Oral Dis 2002;8:69 – 76. [3] Maniatis T, Sambrook J, Fritsch E. Molecular cloning: a laboratory manual. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory Press; 1989. [4] Ng DPK, Walker WH, Chia K-S, Choo S, Warram JH, Krolewski AS. Scrutiny of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) locus reveals conserved haplotype block structure not associated with diabetic nephropathy. Diabetes (In press).
2.4. Data analysis Continuous group data are presented as meanF standard deviation (S.D.). Analysis of variance (ANOVA) with repeated measures was used for comparison of means across multiple groups, with the use of Duncan’s multiple range test in post-hoc analyses.
3. Results
Effect of storage conditions on the extraction of PCR-quality genomic DNA from saliva Daniel P.K. Ng, David Koh *, Serena G.L. Choo, Vivian Ng, Qiuyun Fu Department of Community, Occupational and Family Medicine (MD3), Faculty of Medicine, National University of Singapore, 16, Medical Drive, Singapore 117597, Singapore Received 26 November 2003; received in revised form 16 January 2004; accepted 19 January 2004
Abstract Background: Saliva is a potentially useful source of genomic DNA for genetic studies since it can be collected in a painless and non-invasive manner. We sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR). Methods: Saliva was collected from healthy volunteers and 2-ml aliquots subjected to different storage conditions: S1—washing of saliva using phosphatebuffered saline (PBS) and extraction of DNA on the same day of collection; S2—washing and centrifugation to yield a pellet, which was stored at 70 jC for 1 week prior to DNA extraction; S3—storage of whole saliva at 4 jC for 7 days, followed by washing and extraction of DNA; S4—storage at 4 jC for 7 days, followed by washing and pellet formation. The pellet was stored at 70 jC for 1 month before extraction of the DNA; S5—storage at 70 jC for 1 month, followed by washing and extraction of DNA. DNA yield and purity was determined by spectrophotometry at 260 and 280 nm. Twenty nanograms of genomic DNA was used for the polymerase chain reaction, and the resulting PCR band was captured by digital photography and quantified. Results: The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction, based on OD260/280 ratios, was good and comparable. PCR resulted in the presence of a single specific product of the correct size from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions (P < 0.05). Compared to S1 samples, PCR bands from conditions S2 and S3 were not as strong, while those amplified from S4 and S5 samples were the weakest. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1 – S3. Qualitatively similar results were obtained when the PCR experiment was repeated. Conclusions: Saliva can act as a useful source of genomic DNA, even when stored under less than optimal conditions. D 2004 Elsevier B.V. All rights reserved. Keywords: Saliva; Genotyping; Storage conditions; Genomic DNA; Polymerase chain reaction
1. Background Abbreviations: ANOVA, analysis of variance; DNA, deoxyribonucleic acid; GFPT1, glutamine-fructose-6-phosphate transaminase 1; OD, optical density; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; S.D., standard deviation. * Corresponding author. Tel.: +65-6874-4988; fax: +65-67791489. E-mail address: [email protected] (D. Koh). 0009-8981/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.cccn.2004.01.013
Large population-based studies, involving hundreds to thousands of study subjects, are crucial in the search for the genetic determinants underlying common complex diseases such as diabetes mellitus [1]. Genetic material, in the form of genomic DNA, is typically
192
D.P.K. Ng et al. / Clinica Chimica Acta 343 (2004) 191–194
obtained from blood samples, a collection method that is invasive, painful and generally disliked by study subjects. For population or community studies, especially among children, less invasive methods are preferable, as this will likely result in higher study participation rates. Recent years has seen an increased interest in the use of saliva as a diagnostic fluid [2]. Xenobiotics and their metabolites, enzymes, hormones, immunoglobulins and other molecules have successfully been quantified in saliva. For the extraction of DNA from saliva, commercial kits are already available that serve this purpose. Coupled with the ease of saliva collection (by passive drooling) and the ability to isolate sufficient DNA for genetic analyses, saliva is a potentially attractive source of genetic material. Due to logistic, financial, practical and methodological reasons in large studies, it is often necessary to store saliva samples prior to the extraction of DNA. Thus, in this study, we sought to determine whether different storage conditions of saliva samples impact our ability to extract genomic DNA that is of sufficient quality for use in the polymerase chain reaction (PCR).
2. Materials and methods 2.1. Collection and storage of saliva We recruited 10 healthy adult volunteers from the staff of the Department of Community, Occupational and Family Medicine, National University of Singapore. Each person provided 12 ml of saliva that was passively accumulated over a single time period. The subjects were advised not to smoke, eat or drink for 1 h prior to saliva collection. The collected saliva was thoroughly mixed by vortexing and inversions. Five aliquots of 2 ml each were dispensed into 15-ml tubes and subjected to different storage procedures. We opted to examine 2-ml aliquots as in our previous experience, using smaller volumes (V1 ml) of saliva yielded very limited DNA amounts in some individuals, which would have precluded accurate measurements of its yield. The storage conditions studied were as follows: S1—washing of saliva using phosphate-buffered saline (PBS) and extraction of DNA on the same day of collection;
S2—washing of saliva and centrifugation to yield a pellet, which was stored at 70 jC for 1 week prior to DNA extraction; S3—storage of whole saliva at 4 jC for 7 days, followed by washing and extraction of DNA; S4—storage of whole saliva at 4 jC for 7 days, followed by washing and pellet formation. The pellet was stored at 70 jC for one month before extraction of the DNA; and S5—storage of whole saliva at 70 jC for 1 month, followed by washing and extraction of DNA. 2.2. DNA extraction Extraction of saliva DNA was performed as detailed below. Eight milliliters of PBS was added to 2 ml of saliva, vortexed and then centrifuged at 1800g for 5 min. The supernatant was carefully removed and the pellet rinsed using an additional 1 ml of PBS, before centrifugation again at 1800g for 5 min. After removal of the supernatant, the pellet was resuspended in 180 Al of PBS. For samples stored under condition S5, two additional centrifugation steps were included in the standard protocol (used for conditions S1 – S4) as freezing of whole saliva caused cell lysis making it harder to form a pellet. DNA extractions were performed using the QIAamp DNA Blood Mini Kit (Qiagen, Valencia, CA), in accordance with manufacturer’s instructions; RNAse treatment was also performed following these instructions. DNA was finally eluted in 50 Al of AE buffer (provided in the kit). DNA concentration was measured by spectrophotometry at 260 nm. Purity of DNA was assessed using the ratio of OD260/280 with a ratio of 1.8 – 2.0 being of good purity [3]. Quantification of all DNA samples was performed together by the same laboratory technician using a Smartspec 3000 spectrophotometer (Bio-Rad Laboratories, USA). 2.3. PCR, agarose gel electrophoresis and quantification of PCR bands Twenty nanograms of DNA was used in each 25 Al PCR reaction. PCR was performed in the presence of 1.5 mmol/l Mg2+ using 0.6 U of Taq polymerase (Promega, Madison, WI) with the following pair of primers, which spanned 581 bp of the glutamine-
D.P.K. Ng et al. / Clinica Chimica Acta 343 (2004) 191–194
193
Table 1 Yield and quality of DNA isolated from saliva stored under different conditions Conditions
S1
S2
S3
S4
S5
Yield (ng)/2 ml whole saliva OD260 /OD280 ratio PCR product band volumea,*
4430F5546 1.69F0.27 613.1F317.7
7889F9722 1.90F0.38 562.2F234.3
2979F1421 1.95F0.23 508.7F292.2
3479F1934 1.93F0.25 422.1F236.7
6790F7616 1.81F0.06 429.9F229.8
Data is present as meanFS.D. * ANOVA with repeated measures, P < 0.05. a Post-hoc analyses using Duncan’s multiple range test showed that the means of PCR product band volume for conditions S4 and S5 were significantly different from S1 – S3.
fructose-6-phosphate transaminase 1 (GFPT1) gene: forward primer—5V-CACTGTTTGCTTCAGCTATGC-3V and reverse primer—5V-AGGAACTTTAAAGCATGACAATC-3V. The PCR program consisted of 40 amplification cycles, each consisting of denaturation at 94 jC for 45 s, annealing at 58 jC for 45 s and extension at 72 jC for 60 s, with final extension at 72 jC for 10 min. This protocol has been previously used by our group to successfully amplify the PCR product from >900 individual samples in a separate genetic study [4].
Samples from the five storage conditions were amplified together using a single PCR machine. These PCR products were then fractionated on the same 1% agarose gel containing ethidium bromide. Image of the PCR bands were captured and quantified using the GelDoc EQ digital gel documentation system (BioRad Laboratories, USA). Overexposure of the gel image was avoided as this would lead to an erroneous quantification of the PCR bands. PCR bands were quantified as band volume, which is the sum of the intensities of the pixels within the band boundary
Fig. 1. Quantification of PCR products amplified from genomic DNA isolated from saliva stored under different conditions (S1 – S5). Top panel shows typical PCR results obtained from different individuals. Samples from the five storage conditions were amplified at the same time using a single PCR machine, fractionated on the same agarose gel and digitally photographed before quantifying the PCR band volumes.
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D.P.K. Ng et al. / Clinica Chimica Acta 343 (2004) 191–194
multiplied by pixel area (in mm2), using the Quantityone software (Bio-Rad Laboratories, Hercules, CA).
The amounts of DNA extracted from 2 ml of saliva varied widely under the different storage conditions, while purity of the DNA extraction was good and comparable across the different storage conditions, based on OD260/280 ratios (Table 1). PCR resulted in the presence of a single specific product of the correct size (581 bp) from all samples regardless of saliva storage conditions. Quantification of PCR bands showed significant differences between the various storage conditions ( P < 0.05, Table 1). Compared to S1 samples which were processed immediately after collection, PCR bands from conditions S2 and S3 were not as strong (Table 1, Fig. 1). Bands amplified from samples subjected to conditions S4 and S5 were the weakest in the experiment. Post-hoc analyses showed that the means for conditions S4 and S5 were significantly different from S1 – S3. Qualitatively similar results were obtained when the PCR experiment was repeated (data not shown).
ted tubes) or personnel training. Given the non-invasive and painless nature of saliva collection, study subjects are also more likely to consent to donating saliva as opposed to blood. This advantage may translate into better consent rates for modern genetic studies, which need large numbers of study subjects. Our results demonstrated that DNA can be successfully extracted from saliva for at least up to 1 month when stored at 70 jC. These findings have important implications with regards to measurement validity in the field of salivary biomarkers research. Although we collected 12 ml of saliva from each person, this was to provide sufficient saliva for each storage condition being tested. In practice, 2 ml of saliva can provide sufficient genomic DNA to test an average of 30 –150 genetic markers, if 20– 100 ng of DNA was used for each PCR reaction. While condition S1 was expected to yield the best quality DNA, and PCR analyses supported this conclusion, DNA obtained under conditions S2 and S3 produced comparable results as well, indicating that samples can be stored under these conditions with minimal deterioration if it is not practical to process the samples straightaway. Furthermore, although the storage of the samples at 4 jC for up to 7 days prior to extraction (i.e. condition S3) may result in some bacterial growth, this did not drastically impact the extraction of PCR-quality DNA. Compared to the other conditions, S4 and S5 were less favorable with a 17– 30% decrease in PCR products. Nevertheless, DNA extracted under these conditions can still be amplified and storage under these conditions remains a less attractive, but still viable option.
4. Discussion
References
We examined the impact of different storage conditions on the extraction of PCR-quality of genomic DNA from saliva. The impetus to look into this issue stemmed from the practical consideration that, in planning the workflow of laboratory analyses in large genetic studies, it is often required to store samples prior to their analyses. Thus, knowledge of acceptable storage conditions is important. Furthermore, our study focused on DNA extraction from saliva instead of blood as the saliva collection is quick, does not require specialized consumables (syringes, anticoagulant-trea-
[1] Risch N, Merikangas K. The future of genetic studies of complex human diseases. Science 1996;273:1516 – 7. [2] Streckfus CF, Bigler LR. Saliva as a diagnostic fluid. Oral Dis 2002;8:69 – 76. [3] Maniatis T, Sambrook J, Fritsch E. Molecular cloning: a laboratory manual. Cold Spring Harbour, NY: Cold Spring Harbour Laboratory Press; 1989. [4] Ng DPK, Walker WH, Chia K-S, Choo S, Warram JH, Krolewski AS. Scrutiny of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) locus reveals conserved haplotype block structure not associated with diabetic nephropathy. Diabetes (In press).
2.4. Data analysis Continuous group data are presented as meanF standard deviation (S.D.). Analysis of variance (ANOVA) with repeated measures was used for comparison of means across multiple groups, with the use of Duncan’s multiple range test in post-hoc analyses.
3. Results