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

protokol IRST İnsan Araştırmaları Etik Komitesi kuralları takip eder. NOT: Bu protokol, biz bir UCF DNA bütünlüğü analizi gerçekleştirmek için idrar örneklerinden DNA izole edilmiştir. LNCaP ve MRC hücre çizgileri standart oluşturmak için kullanıldı. Örneğin belirli onkogenlerin DNA ekstraksiyonu (kontrol geni için spektrofotometre ve gerçek zamanlı PCR, STOX1) DNA miktar, ve gerçek zamanlı PCR gibi teknikler (Şekil 1) uygulandı. 1. İdrar Toplama ve İşleme <ol><li> Temiz, kuru, plastik bardak temiz bir-catch ilk sabah idrar örneği almak. idrar örneğinin en az 50 mL toplayın. </li><li> 3 saatlik bir süre için 4 ° C sıcaklıkta idrar korumak ve aynı sıcaklıkta laboratuvara gönder. </li><li> iki adet 50-ml konik tabanlı polipropilen tüpler içine laboratuar ve transfer girişte iki kez hemen evirerek her bir örnek karıştırın. </li><li> tüpleri santrifüj4 ° C sıcaklıkta ya da oda sıcaklığında 10 dakika boyunca 850 x g&#39;de. </li><li> Dikkatle hücre peleti üzerinde yüzen kısımların en az 2 mL bırakarak iki adet 5 ml tüpler içine idrar yüzer üst kısmının 10 mL aktarın. Not: yüzer üst kısmı aktarma hücreler ya da hücre döküntülerinin kirlenme riskini azaltır. Üst ve alt parçalar arasında net bir tarif bulunmamaktadır. </li><li> pelet atın ve hemen kullanıma kadar -80 ° C süpernatant dondurmak. </li></ol> İdrar süpernatant ve Hücre Hatları 2. DNA İzolasyonu NOT: Bir hücre hattından DNA izole (örneğin, LNCaP mesane kanseri için prostat kanseri ya da MRC için) bir ticari kit kullanılarak ve üreticinin yönergeleri izleyerek. aşağıdaki gibi idrar süpernatanından DNA izolasyonu değiştirilmiş ticari protokol kullanılarak yapılmalıdır: <ol><li> Oda sıcaklığında idrar yüzer bir kısım çözülme. &lt;/ Li&gt; <li> Vorteks ve idrar örneği karıştırın ve berrak bir 5 ml tüp içine idrar 1 mL süpernatan aktarın. -80 ° C&#39;de rezidüel idrar dondurun. </li><li> doğrudan numune için proteinaz K, 100 uL ekleyin. </li><li> örnek AL tamponu 1 ml ilave edilir ve pipetleme iyice karıştırın. </li><li> tüpleri kapatın ve 15 dakika süreyle 56 ° C&#39;de numune inkübe edin. </li><li> İnkübasyon sırasında, her bir örnek için bir sütun hazırlamak ve üreticinin talimatlarına göre yıkama tamponları, Aw1 ve Aw2, (% 100 etanol belirtilen miktarda ekleyebilirsiniz) hazırlar. </li><li> Lütfen, oda sıcaklığına kadar örnekleri getirin ve mutlak etanol 1 ml. Pipetleme tarafından tamamen karıştırın. </li><li> sütun aşama 2.7 &#39;de elde edilen karışımın 650 uL ilave edilir ve 1 dakika süre ile 6000 x g de o santrifüj. </li><li> flow-through içeren tüp atın ve yeni, temiz toplama tüpü sütun yerleştirin. Numune karışımının tüm (5 kez) kullanılır olmuştur yineleyerek 2.8 ve 2.9 adımları tekrarlayın. </li><li> Ekle 500# 181; sütunun jant ıslatmadan tampon Aw1 L. 1 dakika boyunca 6.000 xg&#39;de bunu santrifüj. </li><li> flow-through içeren tüp atın ve yeni, temiz toplama tüpü ile değiştirin. </li><li> Sütunun çemberinin ıslatmadan tamponu Aw2 500 uL ekleyin. 3 dakika boyunca tam hızda (20.000 xg) bunu santrifüj. </li><li> flow-through içeren tüp atın ve yeni, temiz toplama tüpü ile değiştirin. </li><li> Adımı yineleyin 2.12 herhangi bir kalıntı yıkama tamponu çıkarmak için. </li><li> Temiz bir 1.5 mL tüp içine sütun yerleştirin. elüsyon tamponu AE 50 mcL ekleyin ve tampon ıslatır o sütunu sağlamak için 7 dakika bekleyin. </li><li> 1 dakika boyunca 8.000 xg&#39;de bunu santrifüj. </li><li> mini sütuna adım 2.15 den eluent Pipet ve DNA&#39;nın maksimum iyileşme sağlamak için 1 dakika boyunca maksimum hızda bunu santrifüj. </li></ol> 3. DNA sayısallaştırılması ve Seyreltme <ol><li> Bir spektrofotometre kullanılarak, bot DNA ölçümü gerçekleştirmekH hücre çizgisi ve idrar süpernatan örnekleri. üreticinin talimatlarına uygun olarak, bir tezgah üstü spektrometresi üzerindeki örnekte 2 uL kullanın. </li><li> 1 ng / ul konsantrasyon elde DNA bütünlüğü analize kadar -20 ° C &#39;de DNA depolamak için Lake Buena Vista DNA örnekleri seyreltilir. Not: DNA miktarı gerçek zamanlı PCR (en az 100 ng) ile devam etmek için yeterli değildir, yeni bir DNA izolasyon işlemi gerçekleştirmek. </li><li> Farklı konsantrasyonlarda altı standartları elde etmek için hücre çizgisi örnekleri DNA seyreltilir: 0.001, 0.01, 0.1, 1, 0.5 ve 2 ng / ml, 100 uL hacimde (ST1, ST5, ST4, ST3, ST2, her biri ve ST6). DNA bütünlüğü analiz edilinceye kadar -20 ° C&#39;de hücre çizgisi DNA standartları saklayın. </li></ol> 4. DNA Bütünlük Testi - PCR <ol><li> primerler (konsantrasyon deneyi türüne bağlıdır), yeşil Supermix, hücre hattı, DNA standartları ve buz üzerinde UCF DNA, seyreltilmiş örnekler çözülme. </li><li> plaka f şerit tüpleri hazırlayınveya 72 oyuklu rotor diski. Kısım her bir standart ve seyreltilmiş örnek negatif kontrol için RNaz içermeyen su, 10 uL, iki kopya halinde 10 uL. </li><li> Her bir primerden 1 ul bir karışımını hazırlamak, (konsantrasyonları, Tablo 1 &#39;de gösterilmiştir), yeşil Supermix 12.5 uL, ve her bir örnek için RNaz içermeyen su 6.5 uL. 2 çoğaltır, 2 çoğaltır için negatif kontrol ve 2 ekstra numuneler için numune 2 çoğaltır 6 standartları, sayı: karışımı hazırlarken, aşağıdaki örneklerin sayısını kullanın. </li><li> her bir kuyunun içine karışımın Kısım 15 uL. pipetle veya tüpleri dönmeye etmeyin. </li><li> Tablo 1 &#39;de gösterilen PCR, aşağıdaki koşullar ile bir protokol başlatın. NOT: 29 x 2 numuneleri + 6 x 2 standartlarına + negatif kontrol x 2 = 72 (UCF DNA değeri): UCF DNA değeri için, 29 DNA örnekleri 72-kuyu rotor diskini kullanarak her gerçek zamanlı deney için işlendi. NOT: UCF DNA bütünlüğü analizi için protokol de olabilir(Başka bir cihaz kullanırken, ROX boya eklenmesi gerekebilir) başka PCR gerçek zamanlı cihazı kullanılarak yapıldı. </li></ol> 5. DNA Bütünlük Testi - Veri Analizi ve Yorumlanması Not: Daha önce 7-9 (Şekil 2) tarif edildiği gibi, her bir örnek için UCF DNA, bir değer, her bir PCR gen değerlendirilmesi için standart bir eğri yapı ile ve standart eğri interpolasyon kullanarak gerçek zamanlı cihazı algılama sistemi yazılımı ile elde edilmiştir. <ol><li> çoğaltır değerlendirin; Örnek bir fark ile çoğaltır ≥1 Ct atılmalıdır ve bir ikinci deneyde yeniden değerlendirildi. </li><li> Her numune için medyan Ct hesaplayın ve 36 ≤ bir Ct değeri örnekleri göz önünde bulundurun. </li><li> erime analizi kullanılarak PCR ürünlerinin özgüllüğünü değerlendirin. </li><li> standart eğri ile enterpolasyon her amplikon konsantrasyonunu değerlendirmek; Her amplikon için konsantrasyon değeri (ng / ml) edinin.</li>
</ol>

<ol>
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The authors declare no competing financial interests.

UCF DNA bütünlüğü analizi idrar DNA bütünlüğünü değerlendirmek için yeni bir non-invaziv bir yöntemdir. Son zamanlarda mesane 9 ve prostat kanserleri 7,8 erken teşhisi için önerilmiştir. avantajları ve UCF DNA bütünlüğü testi dezavantajları bir dizi birlikte geleceği ile burada tartışılır. yaklaşımın ana avantajı ucuz, non-invazif bir yöntem ve moleküler biyoloji teknikleri sadece temel bilgi gerektiren biyomarkerların potansiyel bir...

Hücresiz bir DNA ya apoptotik ya da nekrotik mekanizma ile kan ve hücre ölümüne bağlı idrarda tespit edilebilir. Kandaki hücre-serbest DNA çok çeşitli hastalıklarda tanısal ve prognostik amaçlarla özellikle kanser 1 çalışılmıştır. Ancak, daha az idrar hücre içermeyen (UCF) DNA&#39;nın rolü hakkında bilinmektedir. UCF DNA glomerüler filtrasyon sistemi aracılığıyla ya da bu vücut sıvısı 2 (örn ürotelyal hücreler veya prostat hücreleri) ile doğrudan temas içine gelen hücrelerden kan geçen kaynaklanabilir. Biyomarkerların kaynağı olarak UCF DNA kullanımı esas olarak idrar yolu hücrelerinden 3,4 doğrudan gelen UCF DNA yüksek yüzdesi böbrek, mesane ve prostat kanserinin erken tanısı için incelenmiştir. Küçük UCF DNA ve izole ve karakterize etmek için en iyi yöntemler hakkında bilinmektedir. Değerlendirme tümör hücreleri normal hücrelerden daha uzun DNA fragmanlarını serbest hipotezi göz önüne alındığındahücresiz DNA bütünlüğünü Kan dolaşımı, 5 DNA kökenini aydınlatmak için bir girişim olarak ele alınmıştır. Bazı çalışmalar kanda hücresiz DNA bütünlüğü kanseri 6 birçok türleri iyi bir tanı testi temsil eder ve aynı hipotez idrar 7-9 ile ilişkili olarak önerilmiştir olduğunu göstermiştir. Bu çalışma, mesane ve prostat kanserinin tespiti için potansiyel bir uygulama ile UCF DNA bütünlüğü analizi için yeni bir yöntem tarif eder. Özellikle, UCF DNA bütünlüğü daha uzun 250 bp prostat ve mesane kanseri dahil olmak üzere katı tümörlerin, artan bir DNA kopya numarası olduğu bilinen 4 bölgelerde test edildi fragmanları: c-MYC (8q24.21), HER2 (17q12.1 ), BCAS1 (20q13.2) ve Ar (Xq12) 10-14. Belirli onkogenler yerine rastgele diziler, kanser hastalarının hücresiz fraksiyonu bularak olasılığını arttırmak için seçilmiştir. ana avantajlarından biri,bu yöntem, esnek olduğu ve diğer bölgelerde tümör tipi ve özellikleri temelinde seçilebilir olmasıdır.

<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

plazma ya da serum içinde hücresiz DNA devrede olmasına yaygın birçok kanser türü için biyolojik belirteçler, uygun bir kaynağı olduğu gösterilmiş olmakla birlikte, çok az çalışma idrar hücresiz (Lake Buena Vista) DNA potansiyel kullanımı üzerinde de yoğunlaşmıştır. uzun DNA, prostat veya mesane kanseri ve sağlıklı bireyler olan hastaların ayırt edebilen bir erken tanı markeri olarak değerlendirildi UCF DNA bütünlüğü potansiyel rolü, normal apoptotik hücreler son derece parçalanmış DNA üretmek hipotez ve kanser hücrelerinden serbest bırakılmasına başlatılıyor. C-myc, BCAS1, HER2 ve Ar: Bir Lake Buena Vista DNA bütünlüğü analizi 250 bp daha uzun dört dizilerin dört gerçek zamanlı kantitatif PCR analizleri bazında önerilmiştir. Sık sık, mesane ve prostat kanserleri artan bir DNA kopya numarası dizileri analiz için seçilmiştir, ancak yöntem esnektir ve bu genler, entegrasyon bölgesinin diğer genler ile ikame edilebilirdinlenme. biyomarkerların kaynağı olarak UCF DNA&#39;nın potansiyel yarar zaten böylece UCF DNA karakterizasyonu üzerine ileri çalışmalar için önünü, ürolojik maligniteler için ortaya konmuştur. UCF DNA bütünlüğü testi non-invaziv, hızlı ve kolay uygulanabilir, analiz gerçekleştirmek için gerekli idrar sadece birkaç mililitre olma avantajına sahiptir.

Tüm numuneler 1.51 ila 138 ng / uL bir dizi gösteren analiz için toplam serbest DNA konsantrasyonu spektrofotometrik olarak ölçülebilir oldu. Beş kontrol numuneleri verilerin yeniden üretilebilirlik için kullanılmıştır: iki bağımsız gerçek zamanlı deneyleri, c-myc, HER2, BCAS1, AR ve STOX1 yapıldı. Varyasyon (CV) katsayılar, daha sonra, iki bağımsız deneyin arasında yeniden iyi bir derecede (Tablo 2), ...

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.

İdrar Örneklerinin Hücre-Free DNA Bütünlüğü Analizi

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

JoVE Journal

Cancer Research

13.7K Görüntüleme.  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

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.

Biyolojik Örneklerden Kanser Kök Hücrelerini Algılamak ve Arıtmak İçin DNA Boya Tarafından Tetiklenen Yan Populasyon Fenotipi Kullanmak

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

Kanser Araştırmaları

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

Vitro DNA hasarı kuyruklu yıldız tahlil kullanarak değerlendirmek

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

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.

Yüksek kaliteli tümör DNA klinik-patolojik numunelerin Touch Künye Sitoloji kullanarak elde etmek için basit ve hızlı yöntemi

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

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer (NSCLC). Testing for PD-L1 expression with the PD-L1 immunohistochemistry (IHC) 22C3 companion diagnostic assay, which gives a tumor proportion score (TPS), has been validated on tumor tissue. We developed an optimized laboratory-developed test (LDT) that uses the 22C3 antibody (Ab) concentrate on a widely available IHC autostainer for biopsy and cytology specimens. The PD-L1 TPS was evaluated with 120 paired whole-tumor tissue sections and biopsy samples and with 70 paired biopsy and cytology samples (bronchial washes, n = 40; pleural effusions, n = 30). The 22C3 Ab concentrate-based LDT showed a high concordance rate between biopsy (~100%) and cytology (~95%) specimens when compared to PD-L1 IHC expression determined using the PD-L1 IHC 22C3 companion assay at both TPS cut points (&#8805;1%, &#8805;50%). The optimized LDT presented here, using the 22C3 Ab concentrate to determine the PD-L1 expression in both tumor tissue and in cytology specimens, will expand the ability of laboratories worldwide to assess the eligibility of patients with NSCLC for treatment with pembrolizumab monotherapy in a reliable and reproducible manner.

To expand the ability of laboratories worldwide to assess the eligibility of patients with lung cancer for treatment with pembrolizumab, in a reliable and reproducible manner, we developed an assay that uses the 22C3 antibody concentrate on a widely available immunohistochemical autostainer, for both biopsy and cytology specimens.

22C 3 kullanarak Anti-PD-L1 antikor konsantre biyopsi ve küçük hücreli dışı akciğer kanserli hastalarda sitoloji örnekleri

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

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.

Kolorektal Kanser Riski ve Yaygınlığının Dışkı DNA Bütünlüğü Saptanması ile Değerlendirilmesi

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.

Yapısal DNA Alterasyon Analizi ve Hasta Kaynaklı Ksenogreftler Kullanılarak Kanserde Hedefli Tedavilerin Test Edilmesi

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.

Kolorektal Kanser Hücrelerinin Üç Boyutlu Küresel Kültürlerinde Probiyotiklerin Hücresiz Süpernatantı Kullanılarak Hücre Ölümünün Değerlendirilmesi

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.

Gastrointestinal Kanserde DNA Metilasyonunun Genom Çapında Analizi

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.

Kanser Hastalarının Kan Plazma Örneklerinde Hücresiz DNA Tespiti

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.