The urinary excretion of epigenetically modified DNA as a marker of pediatric ALL status and chemotherapy response.

The active DNA demethylation process may be linked to aberrant methylation and may be involved in leukemogenesis. We investigated the role of epigenetic DNA modifications in childhood acute lymphoblastic leukemia (ALL) diagnostics and therapy monitoring. We analyzed the levels of 5-methyl-2'-deoxycytidine (5-mdC) oxidation products in the cellular DNA and urine of children with ALL (at diagnosis and during chemotherapy, n = 55) using two-dimensional ultra-performance liquid chromatography with tandem mass spectrometry (2D UPLC-MS/MS). Moreover, the expression of Ten Eleven Translocation enzymes (TETs) at the mRNA and protein levels was determined. Additionally, the ascorbate level in the blood plasma was analyzed. Before treatment, the ALL patients had profoundly higher levels of the analyzed modified DNA in their urine than the controls. After chemotherapy, we observed a statistically significant decrease in active demethylation products in urine, with a final level similar to the level characteristic of healthy children. The level of 5-hmdC in the DNA of the leukocytes in blood of the patient group was significantly lower than that of the control group. Our data suggest that urinary excretion of epigenetic DNA modification may be a marker of pediatric ALL status and a reliable marker of chemotherapy response.

Deep sequencing of DNA from urine of kidney allograft recipients to estimate donor/recipient-specific DNA fractions.

Kidney transplantation is the treatment of choice for patients with end-stage kidney failure, but transplanted allograft could be affected by viral and bacterial infections and by immune rejection. The standard test for the diagnosis of acute pathologies in kidney transplants is kidney biopsy. However, noninvasive tests would be desirable. Various methods using different techniques have been developed by the transplantation community. But these methods require improvements. We present here a cost-effective method for kidney rejection diagnosis that estimates donor/recipient-specific DNA fraction in recipient urine by sequencing urinary cell DNA. We hypothesized that in the no-pathology stage, the largest tissue types present in recipient urine are donor kidney cells, and in case of rejection, a larger number of recipient immune cells would be observed. Extensive in-silico simulation was used to tune the sequencing parameters: number of variants and depth of coverage. Sequencing of DNA mixture from 2 healthy individuals showed the method is highly predictive (maximum error < 0.04). We then demonstrated the insignificant impact of familial relationship and ethnicity using an in-house and public database. Lastly, we performed deep DNA sequencing of urinary cell pellets from 32 biopsy-matched samples representing two pathology groups: acute rejection (AR, 11 samples) and acute tubular injury (ATI, 12 samples) and 9 samples with no pathology. We found a significant association between the donor/recipient-specific DNA fraction in the two pathology groups compared to no pathology (P = 0.0064 for AR and P = 0.026 for ATI). We conclude that deep DNA sequencing of urinary cells from kidney allograft recipients offers a noninvasive means of diagnosing acute pathologies in the human kidney allograft.

Analysis of recurrently protected genomic regions in cell-free DNA found in urine.

Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented. Whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we characterized fragmentation patterns in urine cfDNA using whole-genome sequencing. Size distribution of urine cfDNA fragments showed multiple strong peaks between 40 and 120 base pairs (bp) with a modal size of 81- and sharp 10-bp periodicity, suggesting transient protection from complete degradation. These properties were robust to preanalytical perturbations, such as at-home collection and delay in processing. Genome-wide sequencing coverage of urine cfDNA fragments revealed recurrently protected regions (RPRs) conserved across individuals, with partial overlap with nucleosome positioning maps inferred from plasma cfDNA. The ends of cfDNA fragments clustered upstream and downstream of RPRs, and nucleotide frequencies of fragment ends indicated enzymatic digestion of urine cfDNA. Compared to plasma, fragmentation patterns in urine cfDNA showed greater correlation with gene expression and chromatin accessibility in epithelial cells of the urinary tract. We determined that tumor-derived urine cfDNA exhibits a higher frequency of aberrant fragments that end within RPRs. By comparing the fraction of aberrant fragments and nucleotide frequencies of fragment ends, we identified urine samples from cancer patients with an area under the curve of 0.89. Our results revealed nonrandom genomic positioning of urine cfDNA fragments and suggested that analysis of fragmentation patterns across recurrently protected genomic loci may serve as a cancer diagnostic.

Mass Spectrometry-Based Analysis of DNA Modifications: Potential Applications in Basic Research and Clinic.

Stable-isotope-dilution tandem mass spectrometry is the most advanced technique used for quantitative determination of a wide spectrum of endogenously generated DNA nucleobase modifications. It is regarded as a gold standard for such analyses. Here, we consider the requirements for reliable identification and quantification of DNA adducts/modifications, whether endogenously derived or not, and discuss how their quantification can provide information on the mechanism of action and the biological relevance of individual nucleobase modifications. A clinical application of such measurements will only be possible after a full validation of the assay and once we have gained a better understanding of the exact role that these DNA modifications play in disease pathogenesis. Once these prerequisites are satisfied, DNA modification measurements may be helpful as clinical parameters for treatment monitoring, for risk group identification and for the development of prevention strategies.

MS Analysis of DNA Modifications in Urinary/Body Fluids.

Analytical techniques based on mass spectrometry allow to analyze DNA modifications in body fluids. Here we describe two chromatographic methods that can be used for the simultaneous determination of the modified DNA bases and nucleosides in the same urine sample: isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS) and high-performance liquid chromatography coupled with gas chromatography and mass spectrometry (HPLC/GC/MS).

Urine as a high-quality source of host genomic DNA from wild populations.

The ability to generate genomic data from wild animal populations has the potential to give unprecedented insight into the population history and dynamics of species in their natural habitats. However, for many species, it is impossible legally, ethically or logistically to obtain tissue samples of quality sufficient for genomic analyses. In this study we evaluate the success of multiple sources of genetic material (faeces, urine, dentin and dental calculus) and several capture methods (shotgun, whole-genome, exome) in generating genome-scale data in wild eastern chimpanzees (Pan troglodytes schweinfurthii) from Gombe National Park, Tanzania. We found that urine harbours significantly more host DNA than other sources, leading to broader and deeper coverage across the genome. Urine also exhibited a lower rate of allelic dropout. We found exome sequencing to be far more successful than both shotgun sequencing and whole-genome capture at generating usable data from low-quality samples such as faeces and dental calculus. These results highlight urine as a promising and untapped source of DNA that can be noninvasively collected from wild populations of many species.

Prevalence of and risk factors for curable sexually transmitted infections on Bubaque Island, Guinea Bissau.

OBJECTIVES: Complications from sexually transmitted infections (STIs) can result in severe morbidity and mortality. To date, no STI population studies have been conducted on the Bijagos Islands, Guinea Bissau. Our objective was to estimate the prevalence of and identify risk factors for Chlamydia trachomatis (Ct), Neisseria gonorrhoea (Ng), Mycoplasma genitalium (Mg), Trichomonas vaginalis (Tv) and Treponema pallidum (Tp) on Bubaque, the most populated island. METHODS: A cross-sectional survey was conducted on the island of Bubaque among people aged 16-49 years. Participants were asked to answer a questionnaire on STI risk factors, to provide urine samples (men and women) and vaginal swabs (women) for PCR testing for Ct, Ng, Mg and Tv, and to provide dry blood spots for Tp particle agglutination assays. Data were analysed to estimate the prevalence of STIs and logistic regression was used to identify risk factors. RESULTS: In total, 14.9% of participants were found to have a curable STI, with the highest prevalence being observed for Tv (5.9%) followed by Ct (3.8%), Ng (3.8%), Mg (1.9%) and Tp (0.8%). Significant risk factors for having any STI included being female, younger age and concurrent partnership. Having had a previous STI that was optimally treated was a protective factor. CONCLUSIONS: This study demonstrates that there is a considerable burden of STI on the Bijagos Islands, stressing the need for diagnostic testing to facilitate early detection and treatment of these pathogens to stop ongoing transmission. Moreover, these results indicate the need to conduct further research into the STI burden on the Bijagos Islands to help inform and develop a national STI control strategy.

Reduction of oxidative stress on DNA and RNA in obese patients after Roux-en-Y gastric bypass surgery-An observational cohort study of changes in urinary markers.

Increased oxidative stress in obesity and diabetes is associated with morbidity and mortality risks. Levels of oxidative damage to DNA and RNA can be estimated through measurement of 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo) in urine. Both markers have been associated with type 2 diabetes, where especially 8-oxoGuo is prognostic for mortality risk. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery that has considerable effects on bodyweight, hyperglycemia and mortality, might be working through mechanisms that reduce oxidative stress, thereby reducing levels of the urinary markers. We used liquid chromatography coupled with tandem mass spectrometry to analyze the content of 8-oxodG and 8-oxoGuo in urinary samples from 356 obese patients treated with the RYGB-procedure. Mean age (SD) was 44.2 (9.6) years, BMI was 42.1 (5.6) kg/m2. Ninety-six (27%) of the patients had type 2 diabetes. Excretion levels of each marker before and after surgery were compared as estimates of the total 24-hour excretion, using a model based on glomerular filtration rate (calculated from cystatin C, age, height and weight), plasma- and urinary creatinine. The excretion of 8-oxodG increased in the first months after RYGB. For 8-oxoGuo, a gradual decrease was seen. Two years after RYGB and a mean weight loss of 35 kg, decreased hyperglycemia and insulin resistance, excretion levels of both markers were reduced by approximately 12% (P < 0.001). For both markers, mean excretion levels were about 30% lower in the female subgroup (P < 0.0001). Also, in this subgroup, excretion of 8-oxodG was significantly lower in patients with than without diabetes. We conclude, that oxidative damage to nucleic acids, reflected in the excretion of 8-oxodG and 8-oxoGuo, had decreased significantly two years after RYGB-indicating that reduced oxidative stress could be contributing to the many long-term benefits of RYGB-surgery in obesity and type 2 diabetes.

The Sri Lankan twin registry biobank: South Asia's first twin biobank.

Introduction: Biobanks are a valuable resource for creating advancements in science through cutting-edge omics research. Twin research methods allow us to understand the degree to which genetics and environmental factors contribute to health outcomes. Methods: The Sri Lankan Twin Registry biobank (SLTR-b) was established in 2015 as part of Colombo Twin and Singleton Follow-up Study. Venous blood and urine were collected from twins and comparative sample of singletons for clinical investigations and biobanking. Results: The SLTR-b currently houses 3369 DNA and serum samples. Biobank specimens are linked to longitudinal questionnaire data, clinical investigations, anthropometric measurements, and other data. Discussion: The SLTR-b aims to address gaps in health and genetics research. It will provide opportunities for academic collaborations, local and international, and capacity building of future research leaders in twin and omics research. This paper provides a cohort profile of the SLTR-b and its linked data, and an overview of the strategies used for biobanking.

Interpreting Mixture Profiles: Comparison between Precision ID GlobalFiler™ NGS STR Panel v2 and Traditional Methods.

Forensic investigation for the identification of offenders, recognition of human remains, and verification of family relationships requires the analysis of particular types of highly informative DNA markers, which have high discriminatory power and are efficient for typing degraded samples. These markers, called STRs (Short Tandem Repeats), can be amplified by multiplex-PCR (Polymerase Chain Reaction) allowing attainment of a unique profile through which it is possible to distinguish one individual from another with a high statistical significance. The rapid and progressive evolution of analytical techniques and the advent of Next-Generation Sequencing (NGS) have completely revolutionized the DNA sequencing approach. This technology, widely used today in the diagnostic field, has the advantage of being able to process several samples in parallel, producing a huge volume of data in a short time. At this time, although default parameters of interpretation software are available, there is no general agreement on the interpretation rules of forensic data produced via NGS technology. Here we report a pilot study aimed for a comparison between NGS (Precision ID GlobalFiler™ NGS STR Panel v2, Thermo Fisher Scientific, Waltham, MA, USA) and traditional methods in their ability to identify major and minor contributors in DNA mixtures from saliva and urine samples. A quantity of six mixed samples were prepared for both saliva and urine samples from donors. A total of 12 mixtures were obtained in the ratios of 1:2; 1:4; 1:6; 1:8; 1:10; and 1:20 between minor and major contributors. Although the number of analyzed mixtures is limited, our results confirm that NGS technology offers a huge range of additional information on samples, but cannot ensure a higher sensitivity in respect to traditional methods. Finally, the Precision ID GlobalFiler™ NGS STR Panel v2 is a powerful method for kinship analyses and typing reference samples, but its use in biological evidence should be carefully considered on the basis of the characteristics of the evidence.

Detecting HbE Gene Using DNA Extracted from Urine Sediments by Chelex-plus-Heating Technique.

Detecting ßE-allele or hemoglobin E (HbE) gene by PCR generally uses DNA prepared from blood leukocytes. However, blood drawing is invasive and prone to injury and infection. The so-called Chelex-plus-heating protocol for DNA extraction from urine sediments was performed in this study. In this protocol, urine sediments were incubated at 37°C with Chelex-100 resin, followed by heating in boiling water for 20 min, and were spun for 1 min to harvest the DNA-containing supernatant. The obtained DNA was subsequently used in amplification refractory mutation system (ARMS)-PCR for detecting ßE-allele. The ARMS-PCR results obtained from urine-DNA were compared to those produced by ARMS-PCR using blood-leukocyte DNA. It was found that the Chelex-plus-heating technique successfully released DNA of good quality with sufficient quantity from urine sediments. Twenty microliters of urine having ∼1111 cells/ml was sufficient to provide good-quality DNA for PCR reaction for HbE genotyping by ARMS-PCR. It was concluded that the Chelex-plus-heating technique was suitable for preparing the DNA from urine sediments. Being simple and less costly, this technique should promote effective control of HbE for countries having a limited budget.

Development of a multivariable risk model integrating urinary cell DNA methylation and cell-free RNA data for the detection of significant prostate cancer.

BACKGROUND: Prostate cancer exhibits severe clinical heterogeneity and there is a critical need for clinically implementable tools able to precisely and noninvasively identify patients that can either be safely removed from treatment pathways or those requiring further follow up. Our objectives were to develop a multivariable risk prediction model through the integration of clinical, urine-derived cell-free messenger RNA (cf-RNA) and urine cell DNA methylation data capable of noninvasively detecting significant prostate cancer in biopsy naïve patients. METHODS: Post-digital rectal examination urine samples previously analyzed separately for both cellular methylation and cf-RNA expression within the Movember GAP1 urine biomarker cohort were selected for a fully integrated analysis (n = 207). A robust feature selection framework, based on bootstrap resampling and permutation, was utilized to find the optimal combination of clinical and urinary markers in a random forest model, deemed ExoMeth. Out-of-bag predictions from ExoMeth were used for diagnostic evaluation in men with a clinical suspicion of prostate cancer (PSA ≥ 4 ng/mL, adverse digital rectal examination, age, or lower urinary tract symptoms). RESULTS: As ExoMeth risk score (range, 0-1) increased, the likelihood of high-grade disease being detected on biopsy was significantly greater (odds ratio = 2.04 per 0.1 ExoMeth increase, 95% confidence interval [CI]: 1.78-2.35). On an initial TRUS biopsy, ExoMeth accurately predicted the presence of Gleason score ≥3 + 4, area under the receiver-operator characteristic curve (AUC) = 0.89 (95% CI: 0.84-0.93) and was additionally capable of detecting any cancer on biopsy, AUC = 0.91 (95% CI: 0.87-0.95). Application of ExoMeth provided a net benefit over current standards of care and has the potential to reduce unnecessary biopsies by 66% when a risk threshold of 0.25 is accepted. CONCLUSION: Integration of urinary biomarkers across multiple assay methods has greater diagnostic ability than either method in isolation, providing superior predictive ability of biopsy outcomes. ExoMeth represents a more holistic view of urinary biomarkers and has the potential to result in substantial changes to how patients suspected of harboring prostate cancer are diagnosed.

Selection of a Noninvasive Source of Human DNA Envisaging Genotyping Assays in Epidemiological Studies: Urine or Saliva?

Genetic epidemiology requires an appropriate approach to measure genetic variation within the population. The aim of this study was to evaluate the characteristics and genotyping results of DNA extracted from 2 human DNA sources, selected for their rapid and noninvasive sampling, and the use of simple and standardized protocols that are essential for large-scale epidemiologic studies. Saliva and urine samples were collected at the same day from 20 subjects aged 9-10 yr. Genomic DNA was extracted using commercial kits. Quantitative and qualitative evaluation was done by assessing the yield, the purity, and integrity of the extracted DNA. As a proof-of-concept, genotyping was performed targeting CC16 A38G and uteroglobin-related protein 1 (UGRP1)-112G/A. Saliva was found to provide the highest yield and concentration of total DNA extracted. Salivary DNA showed higher purity and a significantly less degraded state compared to urinary DNA. Consequently, the salivary DNA gave better genotyping results than urinary DNA. Therefore, if the choice exists, saliva is the preferred noninvasive matrix for genotyping purposes in large-scale genetic epidemiologic studies. Only in particular cases using urine could nevertheless be considered useful, although specific limitations need to be taken into account.

A Fluorescence Sensing Method with Reduced DNA Typing and Low-Cost Instrumentation for Detection of Sample Tampering Cases in Urinalysis.

This work presents a method to unequivocally detect urine sample tampering in cases where integrity of the sample needs to be verified prior to urinalysis. The technique involves the detection of distinct patterns of a triplex short tandem repeats system in DNA extracted from human urine. The analysis is realized with single-dye fluorescence detection and using a regular smartphone camera. The experimental results had demonstrated the efficacy of the analytical approach to obtaining distinct profiles of amplicons in urine from different sample providers. Reproducibility tests with fresh and stored urine have revealed a maximum variation in the profiles within an interval of 5 to 9%. Cases of urine sample tampering via mixture were simulated in the study, and the experiments have identified patterns of mixed genotypes from dual mixtures of urine samples. Moreover, sample adulteration by mixing a non-human fluid with urine in a volume ratio over 25% can be detected. The low cost of the approach is accompanied by the compatibility of the technique to use with different DNA sample preparation protocols and PCR instrumentation. Furthermore, the possibility of realizing the method in an integrated microchip system open great perspectives to conducting sample integrity tests at the site of urine sample reception and/or at resource-limited settings.

Urinary nucleic acid oxidation product levels show differential associations with pharmacological treatment in patients with type 2 diabetes.

The relationship between RNA and DNA oxidation and pharmacological treatment has not been systematically investigated in patients with type 2 diabetes (T2D). We aimed to investigate the association between pharmacological treatments and levels of urinary markers of nucleic acid oxidation in T2D patients. Vejle Diabetes Biobank cohort data was nested into nationwide registry data. Multiple logistic regression was used to associate drug usage with risk of high (above median) RNA and DNA oxidation. Data from 2664 T2D patients (64% male, age range: 25-75) were included. Questionnaire-validated lipid lowering drug use was associated with low RNA oxidation (Odds ratio, OR 0.71, 95% CI: [0.59-0.87]). Insulin and non-specific antidiabetic drugs were associated with low DNA oxidation (insulin: OR 0.60, 95% CI [0.49-0.73]). Oral antidiabetics were associated with high DNA oxidation and RNA oxidation (OR 1.30, 95% CI [1.10-1.53] and OR 1.26, 95% CI [1.07-1.29]). Our findings indicate that diabetes-related drugs are associated with RNA and DNA oxidation and further studies are required to determine causality in T2D patients.

Phenotype Profiling for Forensic Purposes: Determining Donor Sex Based on Fourier Transform Infrared Spectroscopy of Urine Traces.

Forensic science is an important field of analytical chemistry where vibrational spectroscopy, in particular Fourier transform infrared spectroscopy and Raman spectroscopy, present advantages as they have a nondestructive nature, high selectivity, and no need for sample preparation. Herein, we demonstrate a method for determination of donor sex, based on attenuated total reflection Fourier transform infrared (ATR FT-IR) spectroscopy of dry urine traces. Trace body fluid evidence is of special importance to the modern criminal investigation as a source of individualizing DNA evidence. However, individual identification of a urine donor is generally difficult because of the small amount of DNA. Therefore, the development of an innovative method to provide phenotype information about the urine donor-including sex-is highly desirable. In this study, we developed a multivariate discriminant model for the ATR FT-IR spectra of dry urine to identify the donor sex. Rigorous selection of significant wavenumbers on the spectrum using a genetic algorithm enabled superb discrimination performance for the model and conclusively indicated a chemical origin for donor sex differences, which was supported by physiological knowledge. Although further investigations need to be conducted, this proof-of-concept study demonstrates the great potential of the developed methodology for phenotype profiling based on the analysis of urine traces.

Urogenital Chlamydia trachomatis in the environment of soldiers from the Polish Special Forces.

INTRODUCTION AND OBJECTIVE: Urogenital Chlamydia trachomatis is one of the most common sexually transmitted bacterial pathogens. The aim of the study was to present the current occurrence of chlamydial infections among Polish soldiers, sexually-active men and women at reproductive age. MATERIAL AND METHODS: The research involved 253 active duty soldiers from the Polish Special Forces, 237 men and 16 women aged 26-57, stationed in Warsaw between October - November 2016. The study participants were asked to fill a socio-demographic questionnaire and then subjected to diagnostic tests. These included a urine test for the presence of Chlamydia trachomatis DNA using the Real-Time PCR assay with fluorescently labeled markers and probes, complementary to plasmid DNA of the bacteria (DNA isolated from urine samples was used as matrix). RESULTS: Chlamydia trachomatis infection was detected in two male soldiers, non-commissioned officers, at mean age 40.5 years (total: 38.0 years); reporting sexual contacts with 2-3 partners in the last 12 months (total: 141 soldiers - 1 partner, 66 - 2-3 partners, 46 - >4 partners), with no UTI symptoms. CONCLUSIONS: Among all the study participants, of whom more than 40% reported sexual contacts with 2-4 or more partners within the last 12 months, only 0.8% were found to be infected. The low prevalence of Chlamydia trachomatis infection can be associated with a regular or frequent use of STI prevention measures during casual sex, or having a single sexual partner.

In-Vivo Degradation of DNA-Based Therapeutic BC 007 in Humans.

BACKGROUND AND OBJECTIVES: Since there is no clear evidence in the literature to show how non-modified single-stranded DNA (ssDNA) drugs are metabolized in humans, we assessed the metabolism of BC 007, an ssDNA therapeutic, under development as a neutralizer of autoantibodies against G-protein-coupled receptors. In-vitro, investigating its stability in monkey plasma and serum, a successive 3'-exonuclease degradation resulting in several n-x degradation products has been previously reported. Here, we investigated the metabolism of BC 007 in humans after intravenous application to autoantibody-positive healthy subjects, in line with Phase I safety testing. METHODS: 1H-NMR was applied for n-x degradation product search and beta-aminoisobutyric acid (bAIBA) measurement in urine; ultra-performance liquid chromatography-mass spectrometry was also used for the latter. Colorimetric assays were used for quantification of uric acid in serum and urine. RESULTS: Fast degradation prohibited the detection of the intermediate n-x degradation products in urine using 1H-NMR. Instead, NMR revealed a further downstream degradation product, bAIBA, which was also detected in serum shortly after initial application. The purine degradation product, uric acid, confirmed this finding of fast metabolism. CONCLUSION: Fast and full degradation of BC 007, shown by nucleic bases degradation products, is one of the first reports about the fate of a ssDNA product in humans.

Sensitive electrogenerated chemiluminescence biosensing method for the determination of DNA hydroxymethylation based on Ru(bpy)32+-doped silica nanoparticles labeling and MoS2-poly(acrylic acid) nanosheets modified electrode.

5-Hydroxymethylcytosine (5-hmC), an oxidation product of 5-mC (5-methylcytosine), is presented in DNA of most mammalian cells and play an important role in the alteration of cancer-related genes. Herein, a sensitive electrogenerated chemiluminescence (ECL) biosensing method for the determination of 5-hmC in DNA (5-hmC DNA) was established on the basis of chemical modification and nanomaterial amplification. First, electrochemically reduced molybdenum disulfide-poly(acrylic acid) (rMoS2-PAA) nanosheets were used to modify glassy carbon electrode (GCE) to form an ECL biosensing electrode (rMoS2-PAA/GCE) which has large accessible surface area to immobilize more DNA. Then, a capture probe with amino group was hybridized with the target 5-hmC DNA and immobilized on the surface of rMoS2-PAA/GCE via amido bond. When cysteamine was introduced, the M.HhaI methyltransferase (M.HhaI) was used as specific recognition element to replace the hydroxyl group of 5-hmC by thiol and generated the amine-derivated DNA. Finally, surface chemically activated Ru(bpy)32+-doped silica (Ru@SiO2) nanoparticles, carriers of ECL reagents, were employed as signal amplification unit which covalently bonded to the amine-derivated DNA resulting in an increased ECL intensity. The increased ECL intensity was linearity to the 5-hmC DNA concentration in a range from 5.0 × 10-14 M to 1.0 × 10-11 M, with a lower detection limit of 1.2 × 10-14 M. Besides, the proposed method also displayed a good selectivity to 5-hmC in the presence of 5-C and 5-mC. Moreover, the developed biosensing method was successfully employed to monitor human urine sample.

A protocol for urine collection and storage prior to DNA methylation analysis.

BACKGROUND: Urine poses an attractive non-invasive means for obtaining liquid biopsies for oncological diagnostics. Especially molecular analysis on urinary DNA is a rapid growing field. However, optimal and practical storage conditions that result in preservation of urinary DNA, and in particular hypermethylated DNA (hmDNA), are yet to be determined. AIM: To determine the most optimal and practical conditions for urine storage that result in adequate preservation of DNA for hmDNA analysis. METHODS: DNA yield for use in methylation analysis was determined by quantitative methylation specific PCR (qMSP) targeting the ACTB and RASSF1A genes on bisulfite modified DNA. First, DNA yield (ACTB qMSP) was determined in a pilot study on urine samples of healthy volunteers using two preservatives (Ethylenediaminetetraacetic acid (EDTA) and Urine Conditioning Buffer, Zymo Research) at four different temperatures (room temperature (RT), 4°C, -20°C, -80°C) for four time periods (1, 2, 7, 28 days). Next, hmDNA levels (RASSF1A qMSP) in stored urine samples of patients suffering from bladder cancer (n = 10) or non-small cell lung cancer (NSCLC; n = 10) were measured at day 0 and 7 upon storage with and without the addition of 40mM EDTA and/or 20 µl/ml Penicillin Streptomycin (PenStrep) at RT and 4°C. RESULTS: In the pilot study, DNA for methylation analysis was only maintained at RT upon addition of preserving agents. In urine stored at 4°C for a period of 7 days or more, the addition of either preserving agent yielded a slightly better preservation of DNA. When urine was stored at -20 °C or -80 °C for up to 28 days, DNA was retained irrespective of the addition of preserving agents. In bladder cancer and NSCLC samples stored at RT loss of DNA was significantly less if EDTA was added compared to no preserving agents (p<0.001). Addition of PenStrep did not affect DNA preservation (p>0.99). Upon storage at 4°C, no difference in DNA preservation was found after the addition of preserving agents (p = 0.18). The preservation of methylated DNA (RASSF1A) was strongly correlated to that of unmethylated DNA (ACTB) in most cases, except when PCR values became inaccurate. CONCLUSIONS: Addition of EDTA offers an inexpensive preserving agent for urine storage at RT up to seven days allowing for reliable hmDNA analysis. To avoid bacterial overgrowth PenStrep can be added without negatively affecting DNA preservation.