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 approximately 5 mL whole blood in a serum tube without anticoagulant to obtain serum. Maintain blood tubes at 4 &#176;C until processing.</li>
	<li>Centrifuge tubes at 1,000 x g for 15 min at 4 &#176;C.</li>
	<li>Carefully transfer serum into 2 mL tubes.</li>
	<li>Discard the collecting tube and immediately freeze the supernatant at -80 &#176;C until use.</li>
</ol>
2. Plasma Collection and Processing
<ol>
	<li>Collect approximately 10 mL whole blood in a plasma tube with EDTA to obtain plasma. Fill the tube completely and then invert it 8 times. Maintain blood tubes at 4 &#176;C until processing.</li>
	<li>Centrifuge the tube at 1,800 x g for 15 min at room temperature with no brake setting on the centrifuge.</li>
	<li>Carefully transfer the upper part of the plasma into 2 mL tubes. Repeat the transfer, leaving at least 1 mL of the supernatant above the white cell layer. 
	NOTE: Transferring the upper part of the supernatant reduces the risk of contamination by cells or cell debris.</li>
	<li>Discard the collecting tube and immediately freeze the supernatant at -80 &#176;C until use.</li>
</ol>
3. DNA Isolation from Serum or Plasma
NOTE: Isolation of DNA from serum or plasma should be performed using the commercial protocol modified in the following steps.
<ol>
	<li>Thaw one aliquot (containing from 0.5 to 2 mL) of serum or plasma at room temperature.</li>
	<li>Vortex and mix the sample and transfer 0.5 mL of serum or plasma into a clear 1.5 mL tube. Freeze the rest of material at -80 &#176;C.</li>
	<li>Add 50 &#181;L of proteinase k (20 mg/mL) directly to the sample.</li>
	<li>Add 0.5 mL of lysis buffer to the sample and mix well by pipetting.</li>
	<li>Close the tubes and incubate samples at 56 &#176;C for 15 min in the heat block.</li>
	<li>Add the indicated amount of 100% ethanol at wash buffer 1 and 2, as indicated by the manufacturer&#39;s instructions.</li>
	<li>Bring the samples back to room temperature and add 0.5 mL of absolute ethanol and mix well by pipetting.</li>
	<li>Add 650 &#181;L of the mixture obtained in step 3.7 to the separation column and centrifuge at 6,000 x g for 1 min.</li>
	<li>Discard the tube containing the flow through and place the column on a new clean collection tube. Repeat steps 3.8 and 3.9 one more time.</li>
	<li>Add 500 &#181;L of wash buffer 1, without wetting the rim of the column and centrifuge at 6,000 x g for 1 min.</li>
	<li>Discard the tube containing the flow through and replace it with a new clean collection tube.</li>
	<li>Add 500 &#181;L of wash buffer 2, without wetting the rim of the column and centrifuge at full speed (20,000 x g) for 3 min.</li>
	<li>Discard the tube containing the flow through and replace it with a new clean collection tube.</li>
	<li>Centrifuge at full speed (20,000 x g) for 3 min to remove any residual washing buffer.</li>
	<li>Place the column into a clean 1.5 mL tube. Add 150 &#181;L of elution buffer and wait 7 min to ensure that buffer wets the column.</li>
	<li>Centrifuge at 8,000 x g for 1 min.</li>
	<li>Pipette the elute from step 3.16 again into the same column and centrifuge at full speed (20,000 x g) for 1 min to ensure maximum recovery of DNA.</li>
</ol>
4. DNA Quantification and Dilution
<ol>
	<li>Perform quantification of DNA using a spectrophotometer. Use 2 &#181;L of sample on a bench-top spectrophotometer per the manufacturer&#39;s instructions. 
	NOTE: If the DNA quantity is not sufficient to proceed with real-time PCR (at least 120 ng), perform a new DNA isolation process.</li>
	<li>Dilute DNA from serum or plasma to 2.5 ng/&#181;L in a final volume sufficient for all real-time PCR (about 50 &#181;L).</li>
</ol>
5. Real-time PCR
<ol>
	<li>Thaw copy number assays, reference gene assay, DNA diluted samples, and calibrators pooled in ice. Equilibrate at room temperature the master mix that is stored at 4 &#176;C.</li>
	<li>Prepare a mix of 1 &#181;L of target assay, 1 &#181;L of reference gene assay, and 10 &#181;L of master mix for 3 replicates of each sample and calibrators pooled. Prepare the mix including a negative control (water) and 2 extra samples.</li>
	<li>In a 96 well plate, aliquot 12 &#181;L of the mix into each well. Do not pipette or spin tubes.</li>
	<li>Add 8 &#181;L (concentration of 2.5 ng/&#181;L) of each DNA sample and calibrators pooled into each well. Do not pipette or spin tubes. Change the tip for each well. 
	NOTE: 30 DNA samples can be processed for each Real-time experiment using the 96-well plate: 30 x 3 samples + 1 x 3 calibrators pooled + negative control = 94.</li>
	<li>Briefly spin down the plate at 1,000 x g.</li>
	<li>Run the plate using the following protocol: hold stage at 95 &#176;C for 10 min, 40 cycles at 95 &#176;C for 15 s, and 60 &#176;C for 1 min.</li>
</ol>
6. Data Analysis and Interpretation
<ol>
	<li>In the section &#34;option&#34;, below the amplification plot results, set the Ct threshold at 0.2 for the first target gene analyzed. Repeat the set for each target or reference genes. To Export the real-time PCR file in .txt extension, press &#34;Export&#34; into toolbar. Flag only &#34;results&#34; in the select data to export &#8594; choose as file type the extension .txt &#8594; press &#34;start export&#34; &#8594; close the export tool.</li>
	<li>Open the analysis software and import the file .txt (toolbar &#8594; import).</li>
	<li>Select the file name to analyze and select the analysis setting by toolbar (play symbol).</li>
	<li>Specify the calibrator sample and the known number of copies of the target (e.g., 1 copy for AR gene). Press &#34;apply.&#34;</li>
	<li>For each sample, omit one well with different delta Ct (&#916;Ct) in comparison with other wells.</li>
	<li>Reanalyze the experiment (press play symbol).</li>
	<li>Evaluate the results by the copy number bar plot or the table. 
	NOTE: The results table contains numerous parameters like confidence, Z-score, and replicates analyzed that could be useful for results interpretation.</li>
</ol>

<ol><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-free+DNA+as+a+diagnostic+marker+for+cancer%3A+current+insights%5BTitle%5D)+AND+%22Onco+Targets+Ther%22%5BJournal%5D)">Cell-free DNA as a diagnostic marker for cancer: current insights.</a> Onco Targets Ther. 9, 6549-6559 (2016).</li>
<li>Heitzer, E., Ulz, P., Geigl, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+DNA+as+a+liquid+biopsy+for+cancer%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">Circulating tumor DNA as a liquid biopsy for cancer.</a> Clin. Chem. 61 (1), 112-123 (2015).</li>
<li>Murtaza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-invasive+analysis+of+acquired+resistance+to+cancer+therapy+by+sequencing+of+plasma+DNA%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.</a> Nature. 497 (7447), 108-112 (2013).</li>
<li>Heitzer, E., Ulz, P., Geigl, J. B., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Non-invasive+detection+of+genome-wide+somatic+copy+number+alterations+by+liquid+biopsies%5BTitle%5D)+AND+%22Mol.+Oncol%22%5BJournal%5D)">Non-invasive detection of genome-wide somatic copy number alterations by liquid biopsies.</a> Mol. Oncol. 10 (3), 494-502 (2016).</li>
<li>Diaz, L. A. Jr, et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+molecular+evolution+of+acquired+resistance+to+targeted+EGFR+blockade+in+colorectal+cancers%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.</a> Nature. 486 (7404), 537-540 (2012).</li>
<li>Dawson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer%5BTitle%5D)+AND+%22N.+Engl.+J.+Med%22%5BJournal%5D)">Analysis of circulating tumor DNA to monitor metastatic breast cancer.</a> N. Engl. J. Med. 368 (13), 1199-1209 (2013).</li>
<li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+cell-free+AR+and+CYP17A1+copy+number+variations+may+associate+with+outcome+of+metastatic+castration-resistant+prostate+cancer+patients+treated+with+abiraterone%5BTitle%5D)+AND+%22Br.+J.+Cancer%22%5BJournal%5D)">Circulating cell-free AR and CYP17A1 copy number variations may associate with outcome of metastatic castration-resistant prostate cancer patients treated with abiraterone.</a> Br. J. Cancer. 112 (10), 1717-1724 (2015).</li>
<li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+AR+copy+number+and+outcome+to+enzalutamide+in+docetaxel-treated+metastatic+castration-resistant+prostate+cancer%5BTitle%5D)+AND+%22Oncotarget%22%5BJournal%5D)">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/pubmed?term=((Plasma+AR+and+abiraterone-resistant+prostate+cancer%5BTitle%5D)+AND+%22Sci.+Transl.+Med%22%5BJournal%5D)">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/pubmed?term=((Androgen+receptor+gene+status+in+plasma+DNA+associates+with+worse+outcome+on+enzalutamide+or+abiraterone+for+castration-resistant+prostate+cancer%3A+a+multi-institution+correlative+biomarker+study%5BTitle%5D)+AND+%22Ann+Oncol%22%5BJournal%5D)">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/pubmed?term=((Prostate+cancer%3A+AR+aberrations+and+resistance+to+abiraterone+or+enzalutamide%5BTitle%5D)+AND+%22Nat.+Rev.+Urol%22%5BJournal%5D)">Prostate cancer: AR aberrations and resistance to abiraterone or enzalutamide.</a> Nat. Rev. Urol. 13 (12), 697-698 (2016).</li>
<li>Lee, T. H., Montalvo, L., Chrebtow, V., Busch, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Quantitation+of+genomic+DNA+in+plasma+and+serum+samples%3A+higher+concentrations+of+genomic+DNA+found+in+serum+than+in+plasma%5BTitle%5D)+AND+%22Transfusion%22%5BJournal%5D)">Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma.</a> Transfusion. 41 (2), 276-282 (2001).</li>
<li>Chan, K. C., Yeung, S. W., Lui, W. B., Rainer, T. H., Lo, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+preanalytical+factors+on+the+molecular+size+of+cell-free+DNA+in+blood%5BTitle%5D)+AND+%22Clin.+Chem%22%5BJournal%5D)">Effects of preanalytical factors on the molecular size of cell-free DNA in blood.</a> Clin. Chem. 51 (4), 781-784 (2005).</li>
</ol>

The authors declare no competing financial interests.

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, ...

serum-and-plasma-copy-number-detection-using-real-time-pcr

Research

JoVE Journal

Cancer Research

11.4K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 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.

Video: Serum and Plasma Copy Number Detection Using Real-time PCR

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

Genetics

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Bioengineering

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

Medicine

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Biology

Detection and Quantification of Hepatitis B Virus DNA in Human Serum

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

Author Spotlight: Advancements and Challenges in Hepatitis B Virus Detection 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.

Serum and Plasma Copy Number Detection Using Real-time PCR

Summary

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Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA