Name: serum and plasma copy number detection using real-time pcr
Uploaded: 2017-12-15T14:00:00
Description: Watch this Scientific Journal Video about Serum and Plasma Copy Number Detection Using Real-time PCR at JoVE.com

We thank Chiara Molinari and Filippo Martignano for support in data analysis.

The protocol consists of the isolation of DNA from serum or plasma samples to perform real-time PCR for copy number analysis. DNA extraction, DNA quantity control (spectrophotometer) and real-time PCR for specific targets were performed. In Figure 1, a summary of the procedures and time line are reported.
The protocol follows the guidelines of IRST Human Research Ethics Committee.
1. Serum Collection and Processing
<ol>
	<li>Collect a...

<ol>
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Cancer. 112 (10), 1717-1724 (2015).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+AR+copy+number+and+outcome+to+enzalutamide+in+docetaxel-treated+metastatic+castration-resistant+prostate+cancer.">Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer.</a> Oncotarget. 7 (25), 37839-37845 (2016).</li><li>Romanel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+AR+and+abiraterone-resistant+prostate+cancer.">Plasma AR and abiraterone-resistant prostate cancer.</a> Sci. Transl. Med. 7 (312), (2015).</li><li>Conteduca, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+receptor+gene+status+in+plasma+DNA+associates+with+worse+outcome+on+enzalutamide+or+abiraterone+for+castration-resistant+prostate+cancer:+a+multi-institution+correlative+biomarker+study.">Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer: a multi-institution correlative biomarker study.</a> Ann Oncol. , (2017).</li><li>Attard, G., Antonarakis, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+cancer:+AR+aberrations+and+resistance+to+abiraterone+or+enzalutamide.">Prostate cancer: AR aberrations and resistance to abiraterone or enzalutamide.</a> Nat. Rev. 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AR CN analysis in serum and plasma sample represents a new, non-invasive approach for the stratification of castration resistant prostate cancer (CRPC) patients. It has been recently demonstrated that the AR CN is able to predict outcomes in CRPC patients treated with abiraterone and enzalutamide, before and after chemotherapy7,8,9,10.
The main adva...

Circulating cell free DNA (cfDNA) in blood has been demonstrated to be an optimal source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance1,2. Many studies have shown a good concordance between DNA alterations (mutations, copy number variations, epigenetic modifications) in tissues and those found in corresponding plasma samples1, confirming that circulating tumor DNA (ctDNA) is informative for primary and metastatic tumor tissue alterations3. The possibility of studying ctDNA thus allows for the reconstruction of genomic rearrangements and copy number variations (CNVs) at specific oncogenes4, identifying potentially metastatic clonal and subclonal cells. CtDNA has been shown to be clinically useful especially for cancer treatment monitoring as it harbors specific mutations and CNVs, related to specific targeted therapies5,6. It also overcomes the need for tissue biopsies and allows results to be obtained at different times during a specific cancer treatment in a non-invasive manner.
With regard to prostate cancer, a significant correlation between circulating cell-free androgen receptor (AR) CNVs and treatment response to abiraterone and enzalutamide has been shown, indicating AR gene copy number (CN) in cfDNA may be a promising biomarker capable of predicting treatment resistance7,8,9,10,11. CNVs of specific genes in ctDNA can be evaluated using different approaches with different sensitivity, cost, and rapidity (e.g. real-time, Digital PCR, and Next Generation Sequencing).
Here we describe a simple and fast approach, based on duplex assays in real-time PCR technology, for evaluating AR CN in cfDNA from serum and plasma samples7,8. We considered two different PCR assays designed on two different genomic regions within intron 5 of AR (Xq12) and two other genes, as internal standard reference genes known to have a normal copy number status in prostate cancer (RNaseP, located on 14q11; AGO1, located on 1p34). We selected two reference genes, rather than one, to increase the precision and sensitivity of the results. A DNA amount of 60 ng was amplified for each assay combination (combined assay for AR-assay_1+RNaseP and for AR-assay_2+AGO1). Three serum or plasma DNA samples from healthy males were pooled and used as a calibrator. We considered cutoffs of &#62;1.5 for AR gain and &#60;0.5 for deletion. One of the main advantages of this method is that it is flexible and that other genes can also be evaluated, changing the standard internal reference genes, on the basis of tumor type and characteristics.

<table>
	<tbody>
		<tr>
			<td>BD Vacutainer Serum Tubes</td>
			<td>Becton Dickinson</td>
			<td>367814</td>
			<td>whole blood tube for serum</td>
		</tr>
		<tr>
			<td>BD Vacutainer EDTA Tubes</td>
			<td>Becton Dickinson</td>
			<td>366643</td>
			<td>whole blood tube for plasma</td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td></td>
			<td>the user could use also other companies</td>
		</tr>
		<tr>
			<td>TaqMan Copy Number assay</td>
			<td>Thermo Fisher Scientific</td>
			<td>4400291</td>
			<td>Pre-designed and validated assays with FAM-dye. We used the followig assay: AR_assay1: hs04107225 adn AR_assay2: hs04511283</td>
		</tr>
		<tr>
			<td>TaqMan Copy Number assay (modified)</td>
			<td>Thermo Fisher Scientific</td>
			<td>4467084</td>
			<td>Pre-designed assay modified with VIC-dye: AGO1: Hs02320401_cn</td>
		</tr>
		<tr>
			<td>TaqMan Copy Number Reference Assay, human, RNase P</td>
			<td>Thermo Fisher Scientific</td>
			<td>4403326</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan Universal PCR Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4326708</td>
			<td>Master mix for Real Time PCR</td>
		</tr>
		<tr>
			<td>QIAamp DNA Mini Kit (50)</td>
			<td>Qiagen</td>
			<td>51304</td>
			<td>DNA extraction kit</td>
		</tr>
		<tr>
			<td>MicroAmp 96-Well Plates</td>
			<td>Thermo Fisher Scientific</td>
			<td>N8010560</td>
			<td>plates for realt time PCR</td>
		</tr>
		<tr>
			<td>NanoDrop 1000 Spectrophotometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>-</td>
			<td>the user could use also other spectrophotometric methods to quantify DNA</td>
		</tr>
		<tr>
			<td>7500 Fast Real-Time PCR System</td>
			<td>Applied Biosystem</td>
			<td>-</td>
			<td>the user could use also other real time instrument</td>
		</tr>
		<tr>
			<td>CopyCaller Software</td>
			<td>Applied Biosystem</td>
			<td>-</td>
			<td>Software for copy number analysis</td>
		</tr>
	</tbody>
</table>

serum and plasma copy number detection using real-time pcr

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, and prediction of treatment resistance. Starting from the hypothesis that androgen receptor (AR) gene copy number (CN) gain is a frequent event in metastatic castration resistance prostate cancer (mCRPC), we propose to analyze this event in cfDNA as a potential predictive biomarker.
We evaluated AR CN in cfDNA using 2 different real-time PCR assays and 2 reference genes (RNaseP and AGO1). DNA amount of 60 ng was used for each assay combination. AR CN gain was confirmed using Digital PCR as a more accurate method. CN variation analysis has already been demonstrated to be informative for the prediction of treatment resistance in the setting of mCRPC, but it could be useful also for other purposes in different patient settings. CN analysis on cfDNA has several advantages: it is non-invasive, rapid and easy to perform, and it starts from a small volume of serum or plasma material.

Total cfDNA concentration was quantifiable by spectrophotometry for all samples analyzed, showing a median of 6.12 ng/&#181;L (range: 2.00 - 23.71 ng/&#181;L) for serum samples and a median of 3.21 ng/&#181;L (range: 2.31 - 8.49 ng/&#181;L) for plasma samples. We analyzed a total of 115 samples by real-time PCR experiments.
Test sensitivity was assessed using mixed serum DNA from patients with high or low gain for AR, a...

Watch this Scientific Journal Video about Serum and Plasma Copy Number Detection Using Real-time PCR at JoVE.com

Serum and Plasma Copy Number Detection Using Real-time PCR

This manuscript describes the copy number variation analysis performed in serum or plasma DNA using real-time PCR approach. This method is suitable for the prediction of drug resistance in castration resistant prostate cancer patients, but it could be informative also for other diseases.

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

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