Cell-Free DNA Integrity Analysis in Urine Samples

Summary

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

Abstract

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of cancer, few studies have focused on the potential use of urine cell-free (UCF) DNA. Starting from the hypotheses that normal apoptotic cells produce highly fragmented DNA and that cancer cells release longer DNA, the potential role of UCF DNA integrity was evaluated as an early diagnostic marker capable of distinguishing between patients with prostate or bladder cancer and healthy individuals.

A UCF DNA integrity analysis is proposed on the basis of four quantitative real-time PCRs of four sequences longer than 250 bp: c-MYC, BCAS1, HER2, and AR. Sequences that frequently have an increased DNA copy number in bladder and prostate cancers were chosen for the analysis, but the method is flexible, and these genes could be substituted with other genes of interest. The potential utility of UCF DNA as a source of biomarkers has already been demonstrated for urologic malignancies, thus paving the way for further studies on UCF DNA characterization. The UCF DNA integrity test has the advantage of being non-invasive, rapid, and easy to perform, with only a few milliliters of urine needed to carry out the analysis.

Introduction

Cell-free DNA can be detected in blood and urine due to cell death by apoptotic or necrotic mechanisms. Cell-free DNA in blood has been widely studied for diagnostic and prognostic purposes in various diseases, especially cancer¹. However, less is known about the role of urinary cell-free (UCF) DNA. UCF DNA may originate from blood passing through the glomerular filtration system or from cells that come directly into contact with this body fluid ² (e.g., urothelial cells or prostatic cells). The use of UCF DNA as a source of biomarkers has mainly been investigated for the early diagnosis of renal, bladder, and prostate cancer due to the ....

Protocol

The protocol follows the guidelines of the IRST Human Research Ethics Committee.

NOTE: In this protocol, we isolated DNA from urine samples to perform a UCF DNA integrity analysis. Lncap and MRC cell lines were used to construct standards. Techniques such as DNA extraction, DNA quantification (spectrophotometer and real-time PCR for the control gene, STOX1), and real-time PCR for specific oncogenes were performed (Figure 1).

1. Urine Collection and Processing

1. Obtain a clean-catch first-morning urine sample in a clean, dry, plastic cup. Collect at least 50 mL of the urine sample.
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Results

The total free DNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a range of between 1.51 and 138 ng/µL. Five control samples were used for reproducibility of the data: two independent real-time experiments were performed for c-MYC, HER2, BCAS1, AR, and STOX1. The coefficients of variation (CV) were then calculated for each gene (Table 2), with a good degree of reproducibility between the .......

Discussion

UCF DNA integrity analysis is a new, non-invasive method for assessing DNA integrity in urine. It was recently proposed for the early diagnosis of bladder⁹ and prostate cancers^7,8. A number of advantages and disadvantages of the UCF DNA integrity test are discussed here, together with future prospects.

The main advantage of the approach is that it offers an inexpensive, non-invasive method and a simple protocol to study urine as a potential source of biomarkers, requiring.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
QIAamp DNA Mini Kit	Qiagen	51304
iQ SYBR Green Supermix, 100 x 50 µL rxns, 2.5 mL (2 x 1.25 mL)	Biorad	1708880
IDT custom DNA oligos	IDT		HPLC purification, 100nMole DNA oligo
NanoDrop 1000 Spectrophotometer	Thermo Scientific		Other spectrophotometric methods could also be used to quantify DNA
Rotor-Gene 6000	Corbett		Another Real Time PCR instrument could also be used
microcentrifuge
one centrifuge for 50 mL tubes
incubator
-80 °C freezer
-20 °C freezer
10 μL pipette
20 μL pipette
200 μL pipette
1,000 μL pipette
pipette tips (10; 20; 200; 1,000)
1.5 mL tubes
50 mL tubes
15 mL tubes
Rotor-Disc 72 Rotor	Corbett	9018899
Strip Tubes and Caps, 0.1 mL (250)	Qiagen	981103
Collection Tubes (2 mL)	Qiagen	19201
Buffer AL (264 mL)	Qiagen	19075
Proteinase K (10 mL)	Qiagen	19133