Name: simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology
Uploaded: 2018-03-21T13:00:00
Description: Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

Hastane ve hastaların tüm tıbbi ve yardımcı personel katılmayı kabul etmiş için teşekkür ediyoruz. Gabrielle White Wolf, doktora, Edanz bu raporu taslağını düzenlemek için Grup (www.edanzediting.com/ac) teşekkür ediyoruz. Bu çalışmada genom araştırma projesinden Yamanashi İli (YH ve tarzı) ve bir hibe üzerinden YASUDA tıbbi Vakfı (YH) için bir Grant-in-Aid tarafından desteklenmiştir.

1. TIC hazırlık Normal cam kullanarak hızlı mikroskopik değerlendirmesi için slaytlar
<ol><li>Klinik Patolojik doku malzemelerin mevcut olacağı sonra en kısa zamanda TIC hazırlık gerçekleştirin. TIC numuneler hemen hazır değil, serum ıslatılmış kaplı doku malzemeler steril gazlı bez tutmak ve dokuları kurutma önlemek için buzdolabında saklamak.</li>
	<li>Klinik olarak cerrahi veya endoskopi tarafından elde edilen 5 mm3 doku malzeme solid tümör (örneğin, karaciğer, akci...

<ol>
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Bu çalışmada, tümör DNA TIC kullanarak klinik patolojik numunelerin elde etmek için alternatif bir yöntem mevcuttur. TIC hazırlık çok basittir ve FFPE yöntemleri, özel aletleri10zorunluluğu olmadan ile karşılaştırıldığında daha az zaman ihtiyacı var. TIC hazırlık DNA ekstraksiyon için yordamlar (şekil 1) iki gün içinde tamamlanabilir. Bu yöntem böylece genetik test gerçekleştirmek için geri dönüş süresini kısaltır. Özellikle, bu...

Yeni nesil sıralama teknolojisi araştırmacılar genetik varyasyonları, Mendel hastalığı, kalıtsal yatkınlık ve kanser 1,2,3 genom bilgileri analiz önemli gelişmeler sağlamıştır . Kanser genom Atlas (TCGA) ve uluslararası kanser genom Konsorsiyumu (ICGC) genetik değişiklikler çeşitli yaygın kanserlerin4tanımlaması yakalamışlardır. Temel kanser sürücü genler yüzlerce başarıyla tespit edilmiştir ve bu moleküllerin bazıları ilaç geliştirme1,5,6için hedef alınmaktadır.
Klinik ortamda FFPE numuneler Patolojik tanı ve moleküler kanser de dahil olmak üzere çeşitli hastalıklar için test için yaygın olarak kullanılır. Ancak, formalin ile fiksasyon sürecinde DNA-protein veya DNA-DNA cross-linking oluşur ve DNA fragmantasyonu indüklenen. Böylece, FFPE DNA örnekleri her zaman düşük kalite ve miktar DNA7,8,9nedeniyle genetik analiz için uygun değildir. Ayrıca, o FFPE numuneler hazırlamak için birkaç gün sürer ve teknik beceri doğru bölümleri hazırlamak gereklidir. Bu nedenle, bu yüksek kaliteli bozulmamış DNA elde etmek için basit ve hızlı bir yöntem geliştirmek için arzu edilir.
Sitoloji Patolojik tanı için alternatif bir yöntemdir. Daha basit, daha az pahalı ve daha hızlı yaklaşım FFPE hazırlık10ile karşılaştırıldığında saytolojik örnek hazırlıktır. TIC tekniği sentinel lenf düğümleri ve meme kanserli hastalarda bir yıl11,12için intraoperatif hızlı tanı için marjinal dokulardan yapıldı. Ancak, yüksek kaliteli genomik DNA-ebilmek var olmak hulâsa TIC örnekler incelenmiştir ve sonraki genetik analiz için kullanılan birkaç raporları vardır. Saytolojik örnekler yaygın özelikle (Pap) veya Giemsa boyama ile lekeli ve biz daha önce miktarı ve kalitesi TIC örnekler (özellikle Giemsa lekeli örnekleri) çıkarılan DNA'ın FFPE elde edilen örnekler daha üstün olduğunu bildirdi doku13. PAP boyama ile karşılaştırıldığında, Giemsa boyama boyama daha az prosedürleri gerektiren bir avantaja sahiptir. Sonra örnekleri sabit ve lekeli, Pap boyama, onlar orta (örneğin, Malinol) tümör hücreleri, normal hücreleri ve inflamatuar hücrelerin mikroskop altında gibi örnek içeriği ayırt için montaj ile monte edilmesi gerekir. Pap numune montaj adım hazırlanan örnek kurutulur çünkü hücrelerin mikroskop altında gözlemlemek neredeyse imkansız. İçinde karşılaştırma, Giemsa boyama kurutulmuş durumda görülebilir, bu nedenle, montaj adım hızlı hücresel değerlendirme için gerekli değildir. Kuru numune gerektirdiğinden mikrodiseksiyon için Giemsa boyama daha uygundur.
Bu raporda, biz Giemsa boyama ile TIC numunelerin hazırlanması için basit ve hızlı bir yöntem tanıtmak ve TIC FFPE numuneler ile karşılaştırıldığında DNA için daha iyi bir kaynak olduğunu göstermektedir.

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			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306311</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
			<td style="width:143px;">A63881</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ethanol(99.5)</td>
			<td style="width:191px;">Nacalai Tesque</td>
			<td style="width:143px;">08948-25</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Sodium hydroxide (10M)</td>
			<td style="width:191px;">Sigma</td>
			<td style="width:143px;">72068</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">DTU-Neo</td>
			<td style="width:191px;">TAITEC</td>
			<td style="width:143px;">0063286-000</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">E-36&#160;</td>
			<td style="width:191px;">TAITEC</td>
			<td>0027765-000</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">ECLIPSE Ci-L</td>
			<td style="width:191px;">Nikon</td>
			<td style="width:143px;">704354</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-2XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014393</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-10XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014388</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-20XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014392</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-100XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014384</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-200XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014391</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Pipet-Lite LTS Pipette L-1000XLS+</td>
			<td>METTLER TOLEDO</td>
			<td>17014382</td>
			<td style="width:180px;"></td>
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		<tr height="41">
			<td height="41" style="height:41px;">petit-change</td>
			<td>WAKEN</td>
			<td>MODEL8864</td>
			<td style="width:180px;">Mini centrifuge</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">petit-incubator</td>
			<td>WAKEN</td>
			<td>WKN-2290</td>
			<td style="width:180px;">Air incubator</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">SensiCare Powder-free Nitrile Exam Gloves</td>
			<td style="width:191px;">MEDLINE</td>
			<td>SEM486802</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Sterile gauze</td>
			<td style="width:191px;">Osaki</td>
			<td style="width:143px;">11138</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Refrigerator MediCool</td>
			<td style="width:191px;">SANYO</td>
			<td style="width:143px;">MPR-312DCN-PJ</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.130</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER TRIMMING BLAD</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">No.260</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FEATHER&#160; S</td>
			<td style="width:191px;">FEATHER</td>
			<td style="width:143px;">FA-10</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Vortex Genius 3</td>
			<td style="width:191px;">IKA</td>
			<td>41-0458&#160;</td>
			<td style="width:180px;">Vortex mixer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Pincette</td>
			<td style="width:191px;">NATSUME</td>
			<td style="width:143px;">A-5</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">1.5 mL microtube</td>
			<td style="width:191px;">BIOBIK</td>
			<td style="width:143px;">RC-0150</td>
			<td style="width:180px;"></td>
		</tr>
	</tbody>
</table>

simple and rapid method to obtain high-quality tumor dna from clinical-pathological specimens using touch imprint cytology

Yönetim ve belirli moleküler hedefli uyuşturucu tedavisi kanser hastaları için önce kanser mutational durumunu belirlemek için önemlidir. Klinik ortamda parafin gömülü (FFPE) doku formalin sabit genetik test etmek için yaygın olarak kullanılır. Ancak, FFPE DNA genellikle zarar görmüş ve formalin ile fiksasyon işlemi sırasında parçalanmış. Bu nedenle, FFPE DNA bazen düşük kalite ve DNA miktarı nedeniyle genetik test için yeterli değildir. Burada mikroskop altında gözlenen dokunmatik Künye Sitoloji (TIC) genomik DNA kanser hücrelerinden elde etmek için bir yöntem mevcut. Hücre morfolojisi ve kanser hücre sayıları TIC numuneler kullanılarak değerlendirilebilir. Ayrıca, genomik DNA ekstraksiyon TIC örneklerinden iki gün içinde tamamlanabilir. Toplam miktar ve kalite TIC bu yöntemi kullanarak elde edilen DNA FFPE DNA daha yüksek. Araştırmacılar yüksek kaliteli DNA (örneğin, sonraki nesil sıralama analizi, dijital PCR ve nicel gerçek zamanlı PCR) genetik test için elde etmek için ve sonuçları raporlama için dönüş süresini kısaltmak için bu hızlı ve basit bir yöntem sağlar.

Şekil 1 TIC numuneler DNA ekstraksiyon için hazırlanıyor üzerinden tüm süreci gösterir. Özellikle, yordamı genomik DNA TIC örneklerinden elde etmek için sadece iki gün alır. Biz herhangi bir etkisi tümör depolama slayt işlemeden önce değerlendirildi. Tümör hücreleri doku örnekleri hemen slayt dokundu vardı ve dokular serum fizyolojik nemli steril-gazlı bez için 1 h (Şekil 2) tutuldu cam slayt bağlı ol...

Watch this Scientific Journal Video about Simple and Rapid Method to Obtain High-quality Tumor DNA from Clinical-pathological Specimens Using Touch Imprint Cytology at JoVE.com

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

Yüksek kaliteli genomik DNA tümör dokulardan alma genetik değişiklikler sonraki nesil sıralama kullanarak analiz etmek için önemli bir ilk adım olduğunu. Bu makalede, biz tümör hücreleri zenginleştirmek ve bozulmamış DNA touch Künye Sitoloji numune almak için basit ve hızlı bir yöntem mevcut.

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. ...

The overall goal of this protocol is to obtain high-quality tumor DNA from clinical samples. This method can help answer key questions in basic and clinical science such as cancer research, diagnosis, and treatment decisions. The main advantage of this technique is that we can obtain tumor DNA more simply and quickly than with previous methods.To begin the protocol, touch the tumor surface of a previously prepared and resected tissue onto a normal glass slide several times with gloved hands. Visually confirm that the touched area is covering more than 80%of the glass slide. Lightly press the normal glass slide against a Polyethylene Naphthalate or PEN membrane slide and rub it gently two to three times with gloved hands.Visually confirm that the cells are transferred from the normal glass slide to the PEN membrane slide. Air dry both the glass and PEN membrane slides for five minutes at room temperature. After drying, stain the normal glass slide for direct cytological examination.Dip the glass slides into fixative solution for five seconds and then stain with Giemsa staining solution for 15 seconds. Assess and screen the tumor contents and cellularity on the normal glass slide entirely with a microscope for quick assessment. Evaluate tumor cells based on several criteria.If the sample shows tumor cellularity over 60%by quick microscopic assessment from the previous section, cut the tumor-touched film of the PEN membrane slides for DNA extraction with a knife and gloved hands. Transfer the cut film to a sterile microcentrifuge tube with a pin set and gloved hands. Store the film-containing microcentrifuge tube at four degrees Celsius until DNA extraction.Open the software and plan the run condition and set the run parameter within the software. Click the plan tab and select template, then select the appropriate run method. Select the application and technique type and click next.Select the instrument, sample preparation kit, library kit type, template kit, sequencing kit, base calibration mode, chip type, control sequence, and barcode set and click next. Select plugins and click next. Select project and click next.Then select default reference and BED files of the targeted region. Type the sample name, select the barcode, and click plan run. Next, perform template preparation and chip loading in an automated instrument according to the manufacturer's instructions.Thaw the reagent cartridge at room temperature for 45 minutes before use. Dilute the undiluted library with nuclease-free water according to the library concentration calculated in step 6.8 and make 20 picomolar libraries. Prepare a pooled library for sequencing and store it on ice.Add 25 microliters of the pooled library with a 100 microliter pipette to the bottom of the sample tube. Turn on the power and open the cover of the automated instrument. Place the sequencing chip, chip adapter, enrichment cartridge, tip cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge to the appropriate position of the automated instrument.Touch set up run and step by step on the screen. Close the cover and touch start check on the screen. After the deck scan process, touch next.Check the display contents, kit type, chip ID, sample ID, chip type, plans. Set the time and touch OK.After finishing the chip loading, touch next and open the cover. Unload the sequencing chip from the chip adapter with gloved hands.Place the chip into the chip container, wrap it with parafilm, and store the chip at four degrees Celsius until the sequencing reaction. Then remove the enrichment cartridge, PCR plate, PCR frame seal, recovery tube, solution cartridge, and reagent cartridge from the appropriate position of the automated instrument with gloved hands. Transfer an empty tip cartridge to the waste tip position of the automated instrument with gloved hands.Touch next and close the cover. Then touch start and clean the automated instrument with ultraviolet rays for four minutes. Dissolve a sodium chlorite tablet in 1, 000 milliliters of ultra-pure water and filter the solution with a 0.22 micron filter.Once the sequencing instrument has warmed up, touch clean and next on the screen of the sequencing instrument. Clean the sequencing instrument with 250 milliliters of filter-sterilized sodium chlorite solution, then 250 milliliters of ultra-pure water. Then touch initialize and select the appropriate sequencing kit on the screen.Install a gray shipper with gloved hands. Initialize the sequencing instrument with the wash solution, the pH adjustment solution, and the pH standard solution. Next, add 20 microliters of DATP, DGTP, DCTP, and DTTP nucleotides in 50 milliliter tubes with a 100 microliter pipette.Install a gray shipper at the appropriate location with gloved hands. Load the 50 milliliter tube and screw it onto the sequencing instrument. Select next on the screen to start the initialization step.After completing the initialization step, touch run on the screen and select the appropriate library preparation instrument. Scan the two-dimensional barcode of the chip. Insert the sequencing chip in the appropriate position.Close the chip clamp and instrument door and touch chip check, next, and OK on the screen to start the sequencing run. After the sequencing reaction, transfer the data and perform data analyzing pipeline on the sequencing server. The absolute DNA quantities and the RQ value, an indicator of the degradation level of genomic DNA, show that a higher DNA yield is achieved using the TIC specimens compared with the FFPE specimens.In addition, the RQ values from the TIC DNA were significantly higher compared with that of the FFPE DNA across different tumor types. APC Q1367*was identified in both FFPE and TIC DNA extracted from site one and APC S1356*KRAS G12D, and TP53 M237i were detected in both FFPE and TIC DNA extracted from site two, three, and four suggesting that the identical somatic mutations were identified in paired FFPE and TIC DNA samples prepared from the same tumor site. Notably, the same somatic mutations were detected between primary colorectal cancer and two metastatic liver cancer samples suggesting that the tumor clones from site two metastasized to liver.After its development, this technique paved the way for researchers in the field of molecular and medical research to identify genetic cancers.

Research

JoVE Journal

Cancer Research

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Tümör mikroçevresinin A Mimik: Hücreler arası İletişim Zenginleştirilmiş hücre popülasyonları oluşturma ve incelenmesi için Basit Bir Yöntem

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

Kanser Araştırmaları

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Tümör hücrelerinin tümör hücreleri dolaşan yakalamak için tıbbi bir tel kullanarak karakterizasyonu: 3D bir yaklaşım tabanlı ayirt ve DNA balık

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

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

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 ...

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

Dijital polimeraz zincir reaksiyonu tahlil için tüp içinde çip biçimini kullanarak bir sporadik ailesel Adenomatous poliposis hastada genetik varyasyon

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

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Hasta Biofluidinde tümör Ilişkili sirkülasyon DNA 'Sı tespiti ve Izlenmesi

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

Rapid Optical Clearing for Semi-High-Throughput Analysis of Tumor Spheroids

Tümör Sferoidlerinin Yarı Yüksek Verimli Analizi için Hızlı Optik Temizleme

Tumor spheroids are fast becoming commonplace in basic cancer research and drug development. Obtaining data regarding protein expression within the ...

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Culture and Imaging of Ex Vivo Organotypic Pseudomyxoma Peritonei Tumor Slices from Resected Human Tumor Specimens

Rezeke Edilen İnsan Tümör Örneklerinden Ex Vivo Organotipik Psödomiksoma Peritonei Tümör Dilimlerinin Kültürü ve Görüntülenmesi

Pseudomyxoma peritonei (PMP) is a rare condition that results from the dissemination of a mucinous primary tumor and the resultant accumulation of ...

Radyoaktif Olmayan Kemilüminesans Tespiti Kullanılarak Telomer Uzunluğunun Ölçülmesi

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Yazar Spotlight: TAGGG Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu  

TAGGG Telomer Uzunluk Testi'nin Performans Parametrelerinin Optimizasyonu

Telomeres are repetitive sequences which are present at chromosomal ends; their shortening is a characteristic feature of human somatic cells. Shortening ...