Methods for Separate Isolation of Cell-Free DNA and Cellular DNA from Urine-Application of Methylation-Specific PCR on both DNA Fractions

Abstract

Abstract:The analysis of genomic DNA is widely-used for research, forensic and diagnostic purposes. Here we describe reliable methods for isolation of cell-free DNA and cellular DNA from urine. Both DNA fractions are suitable for PCR and Methylation-Specific PCR (MSP) amplification, leading to consistent and reproducible results. A kinetics analysis describes the decline of efficiency of MSP performed with urinary DNA which had been stored at room temperature with and without proteinase K for various time periods.Keywords: Cell-free DNA, Diagnosis, DNA methylation, Urine.For research purposes, DNA is frequently required in order to determine genetic variability in defined populations and to identify genomic and epigenomic alterations in disease. In forensic science, DNA analyses aid enquiries associated with criminal offenses and missing persons investigations, often based on trace amounts of genetic material [1]. Forensic scientists frequently utilize repetitive DNA elements for gender and species identification or inference of human ancestry [2]. An emerging application of DNA-based assays is disease diagnostics, including the diagnosis of bladder and prostate cancers, the main urological causes of morbidity and mortality worldwide [3,4]. Novel potent biomarkers may detect these malignancies earlier and with greater accuracy and may even help to discriminate between various tumor subclasses, a step further towards individualized medicine. Thus, they could contribute as well to improve the therapeutic success.Urinary DNA derives from either cell-free DNA originating from apoptotic or-, necrotic urothelial, tumor or bloodderived cells [5,6] or from intact cells shed into urine, such as exfoliated tumor cells [7], normal uroepithelial cells and leukocytes. Depending on the circumstances, tumor cell DNA could be enriched in either one or both DNA fractions. Therefore, proper differentiation between cell-free DNA released from disintegrated cells and DNA of intact cells in urine could add an additional value for diagnostic purposes. Basic prerequisites for use in diagnostics are an appropriate amount of each DNA fraction and sufficient quality for successful application in various PCR amplification procedures. In particular, altered DNA methylation is commonly found in urological malignancies [8,9] and should represent a useful source of beneficial biomarkers. DNA, and especially *Address correspondence to this author at the Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich Heine University, Düsseldorf, Germany; Tel: +49-(0)211-81-16905; Fax: +49-(0)-211-81-04543; E-mail: santourlidis@itz.uni-duesseldorf.de # contributed equally.cell-free DNA isolated from urine should therefore be suitable for Methylation Specific PCR which includes bisulfite treatment, a routine technical step, which is inevitably associated with some chemically-induced degradation of the genetic material with diminished PCR efficiency as a consequence.Voided native urine is transferred into a 50 ml sample tube and is imme...

Differences in the Quantity of DNA Found in the Urine and Saliva of Smokers Versus Nonsmokers: Implications for the Timing of Epigenetic Events

“…DNA present in urine can either be cell free, or it can originate from intact cells. Cell-free DNA is derived from apoptotic or necrotic cells, which were thought to mainly originate from the urothelial tract, while exfoliated tumor cells, uro epithelial cells and leukocytes make up the portion of DNA derived from intact cells shed into the urine [12]. However, cell-free nucleic acids have been found to pass through the glomerular membrane in the kidneys, making their way into the urine [13].…”

Unreserved application of epigenetic methods to define differences of DNA methylation between urinary cellular and cell-free DNA

“…Recently, we have shown that cell-free DNA can be routinely isolated from urine and used in a Methylation Specific PCR (MSPCR) approach [29]. Here we present further progress and demonstrate that this highly interesting DNA fraction is unrestrictedly amenable by bisulfite genomic sequencing and global, array based DNA methylation analyses.…”

“…The converted DNA served as template for amplification of methylated LINE-1 sequences in a normalized real time MSPCR approach as reported by Beermann et al [29].…”