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

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

프로토콜은 IRST 인간의 연구 윤리위원회의 지침을 따릅니다. 주 :이 프로토콜에서, 우리는 UCF 무결성 DNA 분석을 수행 소변 샘플에서 DNA를 격리. 된 LNCaP 및 MRC 세포 라인 표준을 구성하는 데 사용 하였다. 이러한 특정 종양 유전자에 대한 DNA 추출 (제어 유전자 분광 실시간 PCR, STOX1) DNA 정량 및 실시간 PCR 등의 기술 (도 1)를 수행 하였다. 1. 소변 ?...

<ol>
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UCF DNA 무결성 분석은 소변에서 DNA의 무결성을 평가하기위한 새로운 비 침습적 방법이다. 이는 최근 블래 9 전립선 암 7,8의 조기 진단을 위해 제안되었다. 장점과 UCF DNA 무결성 검사의 단점의 숫자는 함께 앞으로의 전망과 함께, 여기에 설명되어 있습니다. 접근법의 주된 장점은 저렴하고 비 침습적 방법 및 분자 생물학 기술의 기본 지식을 필요로하는 생체의 ...

무 세포 사멸은 DNA 또는 괴사 메커니즘 혈액 세포 사망으로 인해 소변으로 검출 할 수있다. 혈액의 무 세포 DNA 널리 각종 질병의 진단 및 예후 목적으로, 특히 암 1 연구되고있다. 그러나, 이하 소변 무 세포 (UCF) DNA의 역할에 대한 알려져있다. UCF DNA는 사구체 여과 시스템을 통해, 또는이 둘의 체액 (예 urothelial 세포 또는 전립선 세포)와 접촉 직접 제공 세포로부터 혈액 통과 기인 할 수있다. 바이오 마커의 근원으로 UCF의 DNA의 사용은 주로 요로 3,4- 세포로부터 나오는 DNA UCF의 높은 백분율 신장, 방광, 전립선 암의 조기 진단을 위해 연구되어왔다. 약간은 UCF DNA와 분리하고 특성화하기위한 최선의 방법에 대한 알려져있다. 상기 평가 종양 세포는 정상 세포보다 긴 DNA 단편을 방출한다는 가설을 감안무 세포 DNA의 무결성의 혈행 5 DNA의 출처를 규명하기위한 시도로 검토되고있다. 일부 연구는 혈중 무 세포 DNA 무결성 암 (6)의 여러 유형에 대한 좋은 진단 테스트를 나타내고, 동일한 가설 소변 7-9과 관련하여 제안 된 것으로 설명했다. 이 논문은 방광과 전립선 암 검출에 잠재적 인 응용 프로그램 UCF DNA 무결성 분석을위한 새로운 방법을 설명합니다. 특히, UCF DNA의 무결성 이상 250 bp의 전립선과 방광 암을 포함한 고형 종양에서 증가 DNA의 복제 수있는 것으로 알려져 4 지역에서 시험되었다 프래그먼트 : C-MYC (8q24.21을), HER2 (17q12.1 ), BCAS1 (20q13.2)와 AR (Xq12) 10-14. 특정 암 유전자가 아닌 임의의 순서는 암 환자의 무 세포 분획을 발견 할 확률을 증가하도록 선택되었다. 의 주요 장점 중 하나이 방법은 유연하고 다른 영역은 또한 종양의 종류 및 특성에 기초하여 선택 될 수 있다는 것이다.

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

cell-free dna integrity analysis in urine samples

혈장 또는 혈청 무 세포 DNA 순환 존재 널리 많은 종류의 암에 대한 생체의 적합한 소스 것으로 도시되었지만, 몇 가지 연구는 소변 무 세포 (UCF) DNA의 잠재적 인 사용에 초점을 맞추고있다. 더 이상 DNA, 전립선이나 방광 암과 건강한 사람과 환자를 구별 할 수있는 조기 진단 마커로 평가 하였다 UCF의 DNA 무결성의 잠재적 역할은 정상 세포 사멸 세포가 매우 조각난 DNA를 생성 가설하고 암 세포에서 눌렀다 시작. C-MYC, BCAS1, HER2 및 AR하십시오 UCF DNA 무결성 분석은 250 염기쌍 이상 네 서열 네 정량적 실시간 PCR들에 기초하여 제안되었다. 종종 방광 및 전립선 암에서 증가 된 DNA의 카피 수를 서열 분석을 위해 선택되었지만, 상기 방법은가요 성이며, 이들 유전자는 INTE 다른 유전자로 치환 될 수있는휴식. 바이오 마커의 소스로 UCF DNA의 잠재적 인 유틸리티는 이미 이렇게 UCF DNA 특성에 대한 자세한 연구를위한 방법을 포장, 비뇨 기계 악성 종양에 대한 입증되었다. UCF DNA 무결성 검사는 비 침습적 신속하고 수행하기 쉬운 분석을 수행하는 데 필요한 소변 몇 밀리리터 있다는 장점을 갖는다.

모든 샘플은 1.51 내지 138 NG / μL의 범위를 보여주는 분석을 위해 총 무료 DNA 농도는 분광 광도법에 의해 정량화 하였다. 다섯 제어 샘플 데이터의 재현성에 사용 하였다 : 두 개의 독립된 실시간 실험 C-MYC, HER2, BCAS1, AR 및 STOX1 수행 하였다. 변동 (CV)의 계수는 두 개의 독립적 인 실험 간 재현성의 좋은 정도 (표 2)와 각 유?...

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.

소변 샘플에서 셀 무료 DNA 무결성 분석

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 ...

The overall goal of this method is to assess the integrity of urine cell-free DNA at specific oncogene sequences. This method can help answer key questions in the urological cancer field, such as early non-invasive diagnosis of bladder and prostate cancer. The main advantage of this technique is that it is easy to do and to analyze.The implications of this technique extend towards diagnosis of urological malignancies, because they normally release cells and free nucleic acids in urine. Though this method can provide insight into urinary samples, it can also be applied to other systems such as serum or plasma. Generally, individuals new to this method would struggle because the concentration of urine cell-free DNA is often low.We first had the idea for this method when we tried to find a simple method for non-invasive early detection of bladder cancer. To begin the procedure, obtain a 50 milliliter clean catch first morning urine sample that has been stored at four degrees Celsius for no more than three hours. Invert the sample twice and then partition the sample into two 50 milliliter conical centrifuge tubes.Centrifuge the sample at 850 times G for 10 minutes at four degrees Celsius. Then, for each tube transfer 10 milliliters of the upper part of the supernatant to two five milliliter tubes, leaving at least two milliliters of supernatant above the pellet. Discard the pellets and store the supernatant at minus 80 degrees Celsius.Repeat this process for additional urine samples as needed. To begin DNA purification, for each sample thaw an aliquot of urine supernatant. Vortex the thawed sample and invert the tube twice.Place one milliliter of the sample into a five milliliter tube, and store the remainder at minus 80 degrees Celsius. Prepare a DNA purification kit suitable for urine samples according to the manufacturer's instructions. Add 100 microliters of proteinase K and one milliliter of lysis buffer to the sample and mix well with a pipette.Incubate the sample at 56 degrees Celsius for 15 minutes. During incubation, prepare the wash buffers in a spin column. After incubation, allow the sample to cool to room temperature and then add one milliliter of absolute ethanol and mix well by pipetting.Transfer 650 microliters of the sample to the column. Centrifuge at 6000 times G for one minute. Replace the collection tube and repeat this process until all of the sample has been loaded onto the column.Then add 500 microliters of the first wash buffer and centrifuge the column at 6000 times G for one minute. Replace the collection tube, add 500 microliters of the second wash buffer, and centrifuge the column at max speed for three minutes. Place the column in a clean collection tube and centrifuge at max speed for another three minutes to remove residual wash buffer.Transfer the column to a clean 1.5 milliliter micro centrifuge tube. Load 50 microliters of elution buffer onto the column and allow the buffer to soak into the column for seven minutes. Then centrifuge the column at 6000 times G for one minute.Pipette the eluate back into the column and centrifuge the column at max speed for one minute to obtain the urine cell-free DNA. To begin preparing the standards, first isolate DNA from an appropriate cell line using a purification kit. Quantify the DNA from each purified urine sample in the cell line with a spectrophotometer per the kit manufacturer's instructions.Dilute the UCF DNA to one nanogram per microliter and store the diluted samples at minus 20 degrees Celsius until ready for the integrity test. Dilute the cell line DNA to create six, 100 microliter standards of different concentrations. Store the standards at minus 20 degrees Celsius.To begin the DNA integrity test, thaw the UCF DNA samples, the standards, the appropriate PCR primers, and green supermix on ice. Then set up the 72-well rotor disc plate with strip tubes. Place two, 10 microliter aliquots of standard and then each sample into wells.Prepare two wells of 10 microliter RNase-free water as negative controls. For each filled well, prepare a mix of one microliter of each primer, 12.5 microliters of green supermix, and 6.5 microliters of RNase-free water. Transfer 15 microliters of the mix into each filled well, and then transfer the wells into a 72-well rotor disc.Perform the PCR with a realtime instrument according to the manufacturer's specifications. Using the instrument software, check if any replicates have a cycling threshold value difference of one or more. Discard those samples from the analysis and evaluate the median cycling threshold for the remaining samples.Perform melting analysis on each sample with a median cycling threshold value of 36 or less to evaluate the PCR product specificity. Then use the standard curve to determine a concentration in nanograms per microliter for each applicon. Urine cell-free DNA was isolated and the DNA integrity was evaluated in four regions that show increased DNA copy number in solid tumors.A control sequence was also evaluated to verify that the DNA quantity in each sample was sufficient for the integrity test. The total free DNA concentration and the coefficients of variation were calculated for each gene. This method shows 73%sensitivity and 84%specificity for detecting bladder cancer, but only 58%sensitivity and 44%specificity for prostate cancer.Once mastered, this technique can be done in one working day if it is performed properly. Following this procedure, other methods like digital PCR or next generation sequencing can be performed in order to answer additional questions like copy number inducer of the genes of interest. After watching this video you should have a good understanding of how to isolate cell free DNA from urine in a very simple manner, and how to study the integrity of specific regions of interest using real time PCR approaches.

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

Research

JoVE Journal

Cancer Research

Harnessing the DNA Dye-triggered Side Population Phenotype to Detect and Purify Cancer Stem Cells from Biological Samples

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells (CSCs) and identifying suitable molecular targets relies on methods that allow their specific discrimination in heterogeneous samples such as cell lines and ex vivo tumor tissue. Flow cytometry/FACS is a powerful technology to multi-parametrically dissect biological samples at the single cell level and is to date the method of choice to recover live cells for downstream analyses. Surface markers such as CD44 and CD133 as well as detection of aldehyde dehydrogenase enzymatic activity have often been used to define and sort out CSCs from tumor samples by FACS. A complementary approach, depicted here in methodological detail, makes use of functional dye extrusion by ABC drug transporters, which identifies a distinct population of fluorescence-dim cells commonly referred to as side population (SP). SP&#160;cancer cells exhibit canonical stem cell characteristics and can be abrogated and functionally confirmed using agents that inhibit the dye-extruding drug transporter (most frequently ABCB1/P-glycoprotein/MDR1/CD243 and ABCG2/Bcrp1/CD338). Moreover, the SP&#160;assay is compatible with other flow cytometric evaluations such as staining of surface antigens, aldehyde dehydrogenase detection and dead cell discrimination (e.g., with 7-AAD or propidium iodide (PI)). Thus, we describe a valuable and broadly applicable method for CSC identification, isolation and sub-characterization mechanistically based on a functional, rather than a phenotypic parameter. Although originally performed with Hoechst 33342 as triggering dye, we here focus on the more recent Violet dye-based SP phenotype that is resolvable on any flow cytometer equipped with a violet laser source.

Methods allowing the characterization and isolation of stem cell populations from biological samples are critical for the advance of stem cell-targeted treatments in cancer and beyond. Here, we provide a detailed protocol for cancer stem cell isolation using the dye-triggered side population phenotype.

DNA 염료에 의한 측면 집단 표현형을 활용하여 생물학적 샘플에서 암 줄기 세포를 검출하고 정제

Cancer is a stem cell-driven disease and eradication of these cells has become a major therapeutic goal. Deciphering vulnerabilities of Cancer Stem Cells ...

연구

암 연구학

Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. In the clinical setting, formalin-fixed paraffin-embedded (FFPE) tissues are widely used for genetic testing. However, FFPE DNA is generally damaged and fragmented during the fixation process with formalin. Therefore, FFPE DNA is sometimes not adequate for genetic testing because of low quality and quantity of DNA. Here we present a method of touch imprint cytology (TIC) to obtain genomic DNA from cancer cells, which can be observed under a microscope. Cell morphology and cancer cell numbers can be evaluated using TIC specimens. Furthermore, the extraction of genomic DNA from TIC samples can be completed within two days. The total amount and quality of TIC DNA obtained using this method was higher than that of FFPE DNA. This rapid and simple method allows researchers to obtain high-quality DNA for genetic testing (e.g., next generation sequencing analysis, digital PCR, and quantitative real time PCR) and to shorten the turnaround time for reporting results.

Obtaining high-quality genomic DNA from tumor tissues is an essential first step for analyzing genetic alterations using next generation sequencing. In this article, we present a simple and rapid method to enrich tumor cells and obtain intact DNA from touch imprint cytology specimens.

터치 인쇄물 세포학을 사용 하 여 병 적인 임상 표본에서 높은-품질 종양 DNA를 간단 하 고 빠른 방법

It is critical to determine the mutational status in cancer before administration and treatment of specific molecular targeted drugs for cancer patients. ...

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.

대변 DNA 무결성 검출에 의한 대장암 위험 및 유병률 평가

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Evaluating Cell Death Using Cell-Free Supernatant of Probiotics in Three-Dimensional Spheroid Cultures of Colorectal Cancer Cells

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using supernatants from Lactobacillus fermentum cell culture, considered as probiotics cultures. The use of 3D cultures to test Lactobacillus cell-free supernatant (LCFS) are a better option than testing in 2D monolayers, especially as L. fermentum can produce anti-cancer effects within the gut. L. fermentum supernatant was identified to possess increased anti-proliferative effects against several colorectal cancer (CRC) cells in 3D culture conditions. Interestingly, these effects were strongly related to the culture model, demonstrating the notable ability of L. fermentum to induce cancer cell death. Stable spheroids were generated from diverse CRCs (colorectal cancer cells) using the protocol presented below. This protocol of generating 3D spheroid is time saving and cost effective. This system was developed to easily investigate the anti-cancer effects of LCFS in multiple types of CRC spheroids. As expected, CRC spheroids treated with LCFS strongly induced cell death during the experiment and expressed specific apoptosis molecular markers as analyzed by qRT-PCR, western blotting, and FACS analysis. Therefore, this method is valuable for exploring cell viability and evaluating the efficacy of anti-cancer drugs.

Here methods are presented to understand anti-cancer effects of Lactobacillus cell-free supernatant (LCFS). Colorectal cancer cell lines show cell deaths when treated with LCFS in 3D cultures. The process of generating spheroids can be optimized depending on the scaffold and the analysis methods presented are useful for evaluating the involved signaling pathways.

대장암세포의 3차원 스페로이드 배양에서 프로바이오틱스의 세포없는 슈퍼네탄을 사용하여 세포 사멸 평가

This manuscript describes a protocol to evaluate cancer cell deaths in three dimensional (3D) spheroids of multicellular types of cancer cells using ...

Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is a useful measure to provide an accurate analysis of the methylation status of gastrointestinal (GI) malignancies. Given the multiple potential translational uses of DNA methylation analysis, practicing clinicians and others new to DNA methylation studies need to be able to understand step by step how these genome-wide analyses are performed. The goal of this protocol is to provide a detailed description of how this method is used for the biomarker identification in GI malignancies. Importantly, we describe three critical steps that are needed to obtain accurate results during genome-wide analysis. Clearly and concisely written, these three methods are often lacking and not noticeable to those new to epigenetic studies. We used 48 samples of a GI malignancy (gastric cancer) to highlight practically how genome-wide DNA methylation analysis can be performed for GI malignancies.

Herein, we describe a procedure for genome-wide analysis of DNA methylation in gastrointestinal cancers. The procedure is of relevance to studies that investigate relationships between methylation patterns of genes and factors contributing to carcinogenesis in gastrointestinal cancers.

위장암에서 DNA 메틸화의 게놈 전체 분석

DNA methylation is an important epigenetic change that is biologically meaningful and a frequent focus of cancer research. Genome-wide DNA methylation is ...

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.

암 환자의 혈장 샘플에서 세포없는 DNA 의 검출

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

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate identification and localization of proteins, glycans, and small molecules. While this technique is well-established in two-dimensional (2D) cell culture, less is known about its use in three-dimensional (3D) cell models. Ovarian cancer organoids are 3D tumor models that recapitulate tumor cell clonal heterogeneity, the tumor microenvironment, and cell-cell and cell-matrix interactions. Thus, they are superior to cell lines for the evaluation of drug sensitivity and functional biomarkers. Therefore, the ability to utilize immunofluorescence on primary ovarian cancer organoids is extremely beneficial in understanding the biology of this cancer. The current study describes the technique of immunofluorescence to detect DNA damage repair proteins in high-grade serous patient-derived ovarian cancer organoids (PDOs). After exposing the PDOs to ionizing radiation, immunofluorescence is performed on intact organoids to evaluate nuclear proteins as foci. Images are collected using z-stack imaging on confocal microscopy and analyzed using automated foci counting software. The described methods allow for the analysis of temporal and special recruitment of DNA damage repair proteins and colocalization of these proteins with cell-cycle markers.

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

The present protocol describes methods for evaluating DNA damage repair proteins in patient-derived ovarian cancer organoids. Included here are comprehensive plating and staining methods, as well as detailed, objective quantification procedures.

면역형광 분석을 통한 환자 유래 난소암 오가노이드의 DNA 손상 복구 단백질 시각화

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Multiplexed Barcoding Image Analysis for Immunoprofiling and Spatial Mapping Characterization in the Single-Cell Analysis of Paraffin Tissue Samples

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is an effective methodology for tumor evaluation that improves the understanding of the tumor microenvironment. The visualization of protein expression in formalin-fixed, paraffin-embedded&#160;tissues is achieved when a specific fluorophore is annealed to an antibody-bound barcode via complementary oligonucleotides and then sample imaging is performed; indeed, this method allows for the use of customizable panels of more than 40 antibodies in a single tissue staining reaction. This method is compatible with fresh frozen tissue, formalin-fixed, paraffin-embedded tissue, cultured cells, and peripheral blood mononuclear cells, meaning that researchers can use this technology to view a variety of sample types at single-cell resolution. This method starts with a manual staining and fixing protocol, and all the antibody barcodes are applied using an antibody cocktail. The staining fluidics instrument is fully automated and performs iterative cycles of labeling, imaging, and removing spectrally distinct fluorophores until all the biomarkers have been imaged using a standard fluorescence microscope. The images are then collected and compiled across all the imaging cycles to achieve single-cell resolution for all the markers. The single-step staining and gentle fluorophore removal not only allow for highly multiplexed biomarker analysis but also preserve the sample for additional downstream analysis if desired (e.g., hematoxylin and eosin staining). Furthermore, the image analysis software enables image processing-drift compensation, background subtraction, cell segmentation, and clustering-as well as the visualization and analysis of the images and cell phenotypes for the generation of spatial network maps. In summary, this technology employs a computerized microfluidics system and fluorescence microscope to iteratively hybridize, image, and strip fluorescently labeled DNA probes that are complementary to tissue-bound, oligonucleotide-conjugated antibodies.

Multiplexed barcoding image analysis has recently improved the characterization of the tumor microenvironment, permitting comprehensive studies of cell composition, functional state, and cell-cell interactions. Herein, we describe a staining and imaging protocol using the barcoding of oligonucleotide-conjugated antibodies and cycle imaging, which allows for the use of a high-dimensional image&#160;analysis technique.

파라핀 조직 샘플의 단일 세포 분석에서 면역 프로파일링 및 공간 매핑 특성 분석을 위한 멀티플렉스 바코드 이미지 분석

Multiplexed imaging technology using antibody barcoding with oligonucleotides, which sequentially detects multiple epitopes in the same tissue section, is ...

플라스미드 DNA의 전기천공법 기반 전달을 통한 In vivo 뇌종양 모델링

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in immunotherapy, it is even more critical to have a consistent, clinically pertinent, immunocompetent mouse model to examine the tumor and immune cell populations in the brain and their response to treatment. While most preclinical models utilize orthotopic transplantation of established tumor cell lines, the modeling system presented here allows for a "personalized" representation of patient-specific tumor mutations in a gradual, yet effective development from DNA constructs inserted into dividing neural precursor cells (NPCs) in vivo. DNA constructs feature the mosaic analysis with the dual-recombinase-mediated cassette exchange (MADR) method, allowing for single-copy, somatic mutagenesis of driver mutations. Using newborn mouse pups between birth and 3 days old, NPCs are targeted by taking advantage of these dividing cells lining the lateral ventricles. Microinjection of DNA plasmids (e.g., MADR-derived, transposons, CRISPR-directed sgRNA) into the ventricles is followed by electroporation using paddles that surround the rostral region of the head. Upon electrical stimulation, the DNA is taken up into the dividing cells, with the potential of integrating into the genome. The use of this method has successfully been demonstrated in developing both pediatric and adult brain tumors, including the most common malignant brain tumor, glioblastoma. This article discusses and demonstrates the different steps of developing a brain tumor model using this technique, including the procedure of anesthetizing young mouse pups, to microinjection of the plasmid mix, followed by electroporation. With this autochthonous, immunocompetent mouse model, researchers will have the ability to expand preclinical modeling approaches, in efforts to improve and examine efficacious cancer treatment.

저자 스포트라이트: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient mutation signatures  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures(환자 돌연변이 시그니처를 나타내는 플라스미드 DNA의 전기천공법 기반 전달을 사용한 생체 내 뇌종양 모델링)

Tumor models are critical for the preclinical testing of brain tumors in terms of exploring new, more efficacious treatments. With significant interest in ...