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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+as+a+diagnostic+marker+for+cancer:+current+insights.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+as+a+liquid+biopsy+for+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+analysis+of+acquired+resistance+to+cancer+therapy+by+sequencing+of+plasma+DNA.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+genome-wide+somatic+copy+number+alterations+by+liquid+biopsies.">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., 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=The+molecular+evolution+of+acquired+resistance+to+targeted+EGFR+blockade+in+colorectal+cancers.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cell-free+AR+and+CYP17A1+copy+number+variations+may+associate+with+outcome+of+metastatic+castration-resistant+prostate+cancer+patients+treated+with+abiraterone.">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/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. 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+genomic+DNA+in+plasma+and+serum+samples:+higher+concentrations+of+genomic+DNA+found+in+serum+than+in+plasma.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+preanalytical+factors+on+the+molecular+size+of+cell-free+DNA+in+blood.">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

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

11.3K 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

Amplifying and Quantifying HIV-1 RNA in HIV Infected Individuals with Viral Loads Below the Limit of Detection by Standard Clinical Assays

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below 50-75 copies/ml) is necessary to gain insight to viral dynamics and virus host interactions in patients who naturally control the infection and those who are on combination antiretroviral therapy (cART). 
Here we describe how to amplify viral genomes by single genome sequencing (the SGS protocol) 13, 19 and how to accurately quantify HIV-1 RNA in patients with low viral loads (the single-copy assay (SCA) protocol) 12, 20. 
The single-copy assay is a real-time PCR assay with sensitivity depending on the volume of plasma being assayed. If a single virus genome is detected in 7 ml of plasma, then the RNA copy number is reported to be 0.3 copies/ml. The assay has an internal control testing for the efficiency of RNA extraction, and controls for possible amplification from DNA or contamination. Patient samples are measured in triplicate.
The single-genome sequencing assay (SGS), now widely used and considered to be non-labor intensive 3, 7, 12, 14, 15,is a limiting dilution assay, in which endpoint diluted cDNA product is spread over a 96-well plate. According to a Poisson distribution, when less than 1/3 of the wells give product, there is an 80% chance of the PCR product being resultant of amplification from a single cDNA molecule. SGS has the advantage over cloning of not being subjected to resampling and not being biased by PCR-introduced recombination 19. However, the amplification success of SCA and SGS depend on primer design. Both assays were developed for HIV-1 subtype B, but can be adapted for other subtypes and other regions of the genome by changing primers, probes, and standards.

Quantifying levels of HIV-1 RNA in plasma and sequencing single HIV-1 genomes from individuals with viral loads below the limit of detection (50-75 copies/ml) is difficult. Here we describe how to extract and quantify plasma viral RNA using a real time PCR assay that reliably measures HIV-1 RNA down to 0.3 copies/ml and how to amplify viral genomes by single genome sequencing, from samples with very low viral loads.

Amplifying viral genes and quantifying HIV-1 RNA in HIV-1 infected individuals with viral loads below the limit of detection by standard assays (below ...

Immunology and Infection

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a polymorphic cluster of genes on chromosome 19. Their best-characterized ligands are the human leukocyte antigen (HLA) molecules that are encoded within the major histocompatibility complex (MHC) locus on chromosome 6. There is substantial evidence that they play a significant role in immunity, reproduction, and transplantation, making it crucial to have techniques that can accurately genotype them. However, high-sequence homology, as well as allelic and copy number variation, make it difficult to design methods that can accurately and efficiently genotype all KIR genes. Traditional methods are usually limited in the resolution of data obtained, throughput, cost-effectiveness, and the time taken for setting up and running the experiments. We describe a method called quantitative KIR semi-automated typing (qKAT), which is a high-throughput multiplex real-time polymerase chain reaction method that can determine the gene copy numbers for all genes in the KIR locus. qKAT is a simple high-throughput method that can provide high-resolution KIR copy number data, which can be further used to infer the variations in the structurally polymorphic haplotypes that encompass them. This copy number and haplotype data can be beneficial for studies on large-scale disease associations, population genetics, as well as investigations on expression and functional interactions between KIR and HLA.

Quantitative killer cell immunoglobulin-like receptor (KIR) semi-automated typing (qKAT) is a simple, high-throughput, and cost-effective method to copy number type KIR genes for their application in population and disease association studies.

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

Genetics

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 ...

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

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

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Biology

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

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 ...

Real Time RT-PCR

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target genes. The method involves converting mRNA to complementary DNA with the help of an enzyme known as reverse transcriptase, followed by the PCR amplification of the cDNA. These two processes can be performed simultaneously in a single tube or separately as a two-step reaction.
The real-time quantification of the number of amplified products is carried out either using fluorescent dyes or sequence-specific probes linked to a fluorophore. The fluorescent dyes used in this case can specifically bind to double-stranded DNA and exhibit fluorescence only when bound to DNA. Fluorescence is measured at the end of each PCR cycle after the synthesis of the complementary strand. In the case of fluorophore-labeled probes, fluorescence is exhibited when the fluorophore is cleaved from the probe during the synthesis of the complementary strand. Fluorescence exhibited by these molecules is then detected by photodetectors that convert the fluorescence signals to a readable format. Real-time RT-PCR requires a specialized PCR machine equipped with a detector to enable real-time quantification, as a traditional PCR machine is not equipped for such analysis.
The output can be analyzed in a number of ways. One way is to count the number of cycles required for the fluorescence to reach detectable levels. The threshold cycle, denoted by Ct, is when the fluorescence is detected over and above the background noise. The greater the quantity of the target in the starting material, the faster the significant increase in fluorescence will appear, resulting in a lower Ct. The Ct value finds application in the downstream quantification or detection of the presence or absence of the target sequence. Additionally, comparing the Ct values of samples of unknown concentration with a series of Ct values obtained from standard concentration can determine the amount of template in an unknown reaction. This is known as absolute quantification and can also be carried out by comparing the fluorescence to a standard curve prepared using known DNA concentrations.

Real-time RT-PCR: Principle; Quantifying Gene Expression

Real-time reverse transcription-polymerase chain reaction, or Real-time RT-PCR, is an analytical tool used to determine the expression level of target ...