Name: cell-free dna integrity analysis in urine samples
Uploaded: 2017-01-05T14:00:00
Description: Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

The authors thank Gr&#225;inne Tierney and Silvia Bellissimo for their editorial assistance.

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 Pr...

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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 cancers7,8. 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...

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 high percentage of UCF DNA coming directly from urinary tract cells3,4.
Little is known about UCF DNA and the best methods for isolating and characterizing it. Given the hypothesis that tumor cells release longer DNA fragments than normal cells, the evaluation of cell-free DNA integrity has been studied in an attempt to elucidate the origin of DNA in the blood circulation5. Some studies have demonstrated that cell-free DNA integrity in blood represents a good diagnostic test for many types of cancer6, and the same hypothesis has been proposed in relation to urine7-9.
This paper describes a new method for UCF DNA integrity analysis with a potential application to bladder and prostate cancer detection. In particular, the integrity of UCF DNA fragments longer than 250 bp was tested in 4 regions known to have an increased DNA copy number in solid tumors, including prostate and bladder cancer: c-MYC (8q24.21), HER2 (17q12.1), BCAS1 (20q13.2), and AR (Xq12)10-14. Specific oncogenes, rather than random sequences, were chosen to increase the probability of finding them in the cell-free fraction of cancer patients. One of the main advantages of this method is that it is flexible and that other regions can also be selected on the basis of tumor type and characteristics.

<table border="0" cellpadding="0" cellspacing="0" width="779">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:277px;">QIAamp DNA Mini Kit 
			&#160;</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">51304</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">iQ SYBR&#160;Green Supermix, 100 x 50 &#181;l rxns, 2.5 ml (2 x 1.25 ml)</td>
			<td style="width:216px;">Biorad</td>
			<td style="width:119px;">1708880</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:277px;">IDT custom DNA oligos&#160;</td>
			<td style="width:216px;">IDT&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">HPLC purification, 100nMole DNA oligo</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">NanoDrop 1000 Spectrophotometer</td>
			<td style="width:216px;">Thermo Scientific</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Other spectrophotometric methods could also be used to quantify DNA</td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:277px;">Rotor-Gene 6000</td>
			<td style="width:216px;">Corbett</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Another Real Time PCR instrument could also be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">microcentrifuge</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">one centrifuge for 50 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">incubator</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">&#160;-80&#176;C freezer</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">-20&#176;C freezer</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">10 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">20 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">200 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1000 ul pipette</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">pipette tips (10;20;200;1000)</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">1,5 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">50ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">15 ml tubes</td>
			<td style="width:216px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Rotor-Disc 72 Rotor</td>
			<td style="width:216px;">Corbett</td>
			<td>&#160;9018899</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Strip Tubes and Caps, 0.1 ml (250)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">981103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Collection Tubes (2 ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Buffer AL (264 ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19075</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:277px;">Proteinase K (10ml)</td>
			<td style="width:216px;">Qiagen</td>
			<td style="width:119px;">19133</td>
			<td></td>
		</tr>
	</tbody>
</table>

cell-free dna integrity analysis in urine samples

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.

The total free DNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a range of between 1.51 and 138 ng/&#181;L. 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...

Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

Cell-Free DNA Integrity Analysis in Urine Samples

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.

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 ...

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