Name: cell-free dna integrity analysis in urine samples
Uploaded: 2017-01-05T14:00:00.000+00:00
Duration: 7 min 58 s
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 Processing
<ol>
	<li>Obtain a clean-catch first-morning urine sample in a clean, dry, plastic cup. Collect at least 50 mL of the urine sample.</li>
	<li>Maintain the urine at 4 &#176;C for a maximum of 3 h and send it to the laboratory at the same temperature.</li>
	<li>Mix each sample by inverting it twice immediately upon arrival in the laboratory and transfer into two 50-mL conical-bottomed polypropylene tubes.</li>
	<li>Centrifuge the tubes at 850 x g for 10 min at 4 &#176;C or at room temperature.</li>
	<li>Carefully transfer 10 mL of the upper part of the urine supernatant into two 5-mL tubes, leaving at least 2 mL of the supernatant above the cell pellet. 
	NOTE: Transferring the upper part of the supernatant reduces the risk of contamination by cells or cellular debris. There is no clear delineation between the upper and lower parts.</li>
	<li>Discard the pellet and immediately freeze the supernatant at -80 &#176;C until use.</li>
</ol>
2. DNA Isolation from the Urine Supernatant and Cell Lines
NOTE: Isolate the DNA from a cell line (e.g., Lncap for prostate cancer or MRC for bladder cancer) using a commercial kit and following the manufacturer&#39;s instructions. Isolation of DNA from the urine supernatant should be performed using the commercial protocol, modified as follows:
<ol>
	<li>Thaw one aliquot of the urine supernatant at room temperature.</li>
	<li>Vortex and mix the urine sample and transfer 1 mL of the urine supernatant into a clear 5-mL tube. Freeze the residual urine at -80 &#176;C.</li>
	<li>Add 100 &#181;L of proteinase k directly to the sample.</li>
	<li>Add 1 mL of AL buffer to the sample and mix well by pipetting.</li>
	<li>Close the tubes and incubate the samples at 56 &#176;C for 15 min.</li>
	<li>During incubation, prepare one column for each sample and prepare the wash buffers, AW1 and AW2, as per the manufacturer&#39;s instructions (add the indicated amount of 100% ethanol).</li>
	<li>Bring the samples back to room temperature and add 1 mL of absolute ethanol. Mix fully by pipetting.</li>
	<li>Add 650 &#181;L of the mixture obtained in step 2.7 to the column and centrifuge it 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 2.8 and 2.9 until all of the sample mixture has been used (5 times).</li>
	<li>Add 500 &#181;L of buffer AW1 without wetting the rim of the column. Centrifuge it 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 buffer AW2 without wetting the rim of the column. Centrifuge it 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>Repeat step 2.12 to remove any residual washing buffer.</li>
	<li>Place the column into a clean 1.5-mL tube. Add 50 &#181;L of elution buffer AE and wait 7 min to ensure that the buffer wets the column.</li>
	<li>Centrifuge it at 8,000 x g for 1 min.</li>
	<li>Pipette the eluent from step 2.15 into the mini-column and centrifuge it at maximum speed for 1 min to ensure the maximum recovery of DNA.</li>
</ol>
3. DNA Quantification and Dilution
<ol>
	<li>Using a spectrophotometer, perform the quantification of DNA from both the cell line and the urine supernatant samples. Use 2 &#181;L of sample on a bench-top spectrometer, as per the manufacturer&#39;s instructions.</li>
	<li>Dilute the UCF DNA samples to obtain a concentration of 1 ng/&#181;L and store the DNA at -20 &#176;C until the DNA integrity analysis. 
	NOTE: If the DNA quantity is not sufficient to proceed with real-time PCR (at least 100 ng), perform a new DNA isolation process.</li>
	<li>Dilute the DNA from cell line samples to obtain six standards with different concentrations: 0.001, 0.01, 0.1, 1, 0.5, and 2 ng/&#181;L, each with a volume of 100 &#181;L (st1, st2, st3, st4, st5, and st6). Store the cell line DNA standards at -20 &#176;C until the DNA integrity analysis.</li>
</ol>
4. DNA Integrity Test &#8212; PCR
<ol>
	<li>Thaw the primers (the concentration depends on the type of assay), green supermix, cell-line DNA standards, and UCF DNA diluted samples on ice.</li>
	<li>Prepare strip tubes in the plate for the 72-well rotor disc. Aliquot 10 &#181;L in duplicate for each standard and diluted sample and 10 &#181;L of RNase-free water for the negative control.</li>
	<li>Prepare a mix of 1 &#181;L of each primer (the concentrations are indicated in Table 1), 12.5 &#181;L of green supermix, and 6.5 &#181;L of RNase-free water for each sample. When preparing the mix, use the following number of samples: 6 standards for 2 replicates, number of samples for 2 replicates, negative control for 2 replicates, and 2 extra samples.</li>
	<li>Aliquot 15 &#181;L of the mix into each well. Do not pipette or spin the tubes.</li>
	<li>Start the protocol with the PCR conditions indicated in Table 1. 
	NOTE: For the UCF DNA value, 29 DNA samples were processed for each real-time experiment using the 72-well rotor disc: 29 x 2 samples + 6 x 2 standards + negative control x 2 = 72 (UCF DNA value). 
	NOTE: The protocol for the UCF DNA integrity analysis can also be performed using another PCR real-time instrument (when using another device, ROX dye may need to be added).</li>
</ol>
5. DNA Integrity Test &#8212; Data Analysis and Interpretation
NOTE: The UCF DNA value for each sample was obtained by a real-time instrument-detection system software using a standard curve construction for each individual PCR gene evaluation and using standard curve interpolation, as previously described7-9 (Figure 2).
<ol>
	<li>Evaluate the replicates; sample replicates with a difference &#8805;1 Ct must be discarded and re-evaluated in a second experiment.</li>
	<li>Calculate the median Ct for each sample and consider samples with a Ct value &#8804; 36.</li>
	<li>Evaluate the specificity of the PCR products using melting analysis.</li>
	<li>Evaluate the concentration of each amplicon by interpolation with the standard curve; obtain a concentration value (ng/&#181;L) for each amplicon.</li>
</ol>

<ol>
	<li>Francis, G., Stein, S. <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+Tumour+DNA+in+the+Management+of+Cancer.">Circulating Cell-Free Tumour DNA in the Management of Cancer.</a> Int.J.Mol.Sci. 16 (6), 14122-14142 (2015).</li><li>Bryzgunova, O. E., Laktionov, P. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+Nucleic+Acids+in+Urine:+Sources,+Structure,+Diagnostic+Potential.">Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential.</a> Acta Naturae. 7 (3), 48-54 (2015).</li><li>Szarvas, T., 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=Deletion+analysis+of+tumor+and+urinary+DNA+to+detect+bladder+cancer:+urine+supernatant+versus+urine+sediment.">Deletion analysis of tumor and urinary DNA to detect bladder cancer: urine supernatant versus urine sediment.</a> Oncol.Rep. 18 (2), 405-409 (2007).</li><li>Togneri, F. 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=Genomic+complexity+of+urothelial+bladder+cancer+revealed+in+urinary+cfDNA.">Genomic complexity of urothelial bladder cancer revealed in urinary cfDNA.</a> Eur.J.Hum.Genet. , (2016).</li><li>Zonta, E., Nizard, P., Taly, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+DNA+Integrity,+Applications+for+Cancer+Research.">Assessment of DNA Integrity, Applications for Cancer Research.</a> Adv.Clin.Chem. 70, 197-246 (2015).</li><li>Hao, T. B., 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+DNA+in+serum+as+a+biomarker+for+diagnosis+and+prognostic+prediction+of+colorectal+cancer.">Circulating cell-free DNA in serum as a biomarker for diagnosis and prognostic prediction of colorectal cancer.</a> Br.J.Cancer. 111 (8), 1482-1489 (2014).</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=Urine+Cell-Free+DNA+Integrity+Analysis+for+Early+Detection+of+Prostate+Cancer+Patients.">Urine Cell-Free DNA Integrity Analysis for Early Detection of Prostate Cancer Patients.</a> Dis.Markers. 2015, 574120 (2015).</li><li>Casadio, 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=Urine+cell-free+DNA+integrity+as+a+marker+for+early+prostate+cancer+diagnosis:+a+pilot+study.">Urine cell-free DNA integrity as a marker for early prostate cancer diagnosis: a pilot study.</a> Biomed.Res.Int. 2013, 270457 (2013).</li><li>Casadio, 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=Urine+cell-free+DNA+integrity+as+a+marker+for+early+bladder+cancer+diagnosis:+preliminary+data.">Urine cell-free DNA integrity as a marker for early bladder cancer diagnosis: preliminary data.</a> Urol.Oncol. 31 (8), 1744-1750 (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>Ishkanian, A. 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The authors declare no competing financial interests.

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><tbody><tr><td>QIAamp DNA Mini Kit&lt;br/&gt; &amp;nbsp;</td><td>Qiagen</td><td>51304</td><td></td></tr><tr><td>iQ SYBR&amp;nbsp;Green Supermix, 100 x 50 &amp;micro;L rxns, 2.5 mL (2 x 1.25 mL)</td><td>Biorad</td><td>1708880</td><td></td></tr><tr><td>IDT custom DNA oligos&amp;nbsp;</td><td>IDT&amp;nbsp;</td><td></td><td>HPLC purification, 100nMole DNA oligo</td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>Other spectrophotometric methods could also be used to quantify DNA</td></tr><tr><td>Rotor-Gene 6000</td><td>Corbett</td><td></td><td>Another Real Time PCR instrument could also be used</td></tr><tr><td>microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>one centrifuge for 50 mL tubes</td><td></td><td></td><td></td></tr><tr><td>incubator</td><td></td><td></td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>-20 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>10 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>20 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>1,000 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>pipette tips (10; 20; 200; 1,000)</td><td></td><td></td><td></td></tr><tr><td>1.5 mL tubes</td><td></td><td></td><td></td></tr><tr><td>50 mL tubes</td><td></td><td></td><td></td></tr><tr><td>15 mL tubes</td><td></td><td></td><td></td></tr><tr><td>Rotor-Disc 72 Rotor</td><td>Corbett</td><td>9018899</td><td></td></tr><tr><td>Strip Tubes and Caps, 0.1 mL (250)</td><td>Qiagen</td><td>981103</td><td></td></tr><tr><td>Collection Tubes (2 mL)</td><td>Qiagen</td><td>19201</td><td></td></tr><tr><td>Buffer AL (264 mL)</td><td>Qiagen</td><td>19075</td><td></td></tr><tr><td>Proteinase K (10 mL)</td><td>Qiagen</td><td>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 ...

cell-free-dna-integrity-analysis-in-urine-samples

Research

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Cancer Research

13.7K Views.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 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.

Video: Cell-Free DNA Integrity Analysis in Urine Samples

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

Genetics

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

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 ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

Biochemistry

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Immunology and Infection

CRISPR-Cas Synthetic Urine Biomarker Test for Cancer Diagnostics

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

Author Spotlight: Engineering Molecular Tools for Disease Detection and Imaging

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for ...

Bioengineering

Collection, Processing, and Storage Consideration for Urinary Biomarker Research

There are several urinary proteins that show promise as novel markers of urinary tract infections. The identification of a novel biomarker that has greater predictive accuracy compared to current diagnostic methods has the potential to greatly improve the ability to manage patients with urinary tract infections. However, sample collection, processing, and storage can all potentially impact the results of biomarker research. Understanding the effects of each of these stages on biomarker studies is necessary to inform future, high-quality research in this area, as well as to critically review other studies in this area. Here, the study reviews the literature regarding the effects of each stage of urine sample processing and reports the effects of various conditions on urinary proteins. The protocol will focus on collection techniques, time and temperature of storage, processing techniques, use of reagents, and long-term freezing on biomarker stability. It will focus on proteins but will briefly discuss other materials that may be utilized in biomarker research. In doing so, this protocol will provide a guide to future researchers to assist in the design of urinary biomarkers studies.

This protocol aims to provide considerations for urine sample collection, processing, and storage for urine tract infection biomarker studies.

There are several urinary proteins that show promise as novel markers of urinary tract infections. The identification of a novel biomarker that has ...

Medicine

A Single Cell Dissociation Approach for Molecular Analysis of Urinary Bladder in the Mouse Following Spinal Cord Injury

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and subsequent bladder wall remodeling. To facilitate assessment of the cellular composition of the bladder wall in non-injured control and spinal cord injured mice, we developed an optimized dissociation protocol that supports high cell viability and enables the detection of discrete subpopulations by flow cytometry.
Spinal cord injury is created by complete transection of the thoracic spinal cord. At the time of tissue harvest, the animal is perfused with phosphate-buffered saline under deep anesthesia and bladders are harvested into Tyrode&#8217;s buffer. Tissues are minced prior to incubation in digestion buffer that has been optimized based on the collagen content of mouse bladder as determined by interrogation of publicly available gene expression databases. Following generation of a single cell suspension, material is analyzed by flow cytometry for assessment of cell viability, cell number and specific subpopulations. We demonstrate that the method yields cell populations with greater than 90% viability, and robust representation of cells of mesenchymal and epithelial origin. This method will enable accurate downstream analysis of discrete cell types in mouse bladder and potentially other organs.

The goal of this protocol is to apply an optimized tissue dissociation protocol to a mouse model of spinal cord injury and validate the approach for single cell analysis by flow cytometry.

We describe the implementation of spinal cord injury in mice to elicit detrusor-sphincter dyssynergia, a functional bladder outlet obstruction, and ...