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

Agradecemos a todo o pessoal médico e auxiliar do hospital e os pacientes para consentir a participar. Agradecemos a Gabrielle White Wolf, PhD, do grupo de Emilia (www.edanzediting.com/ac) para a edição de um projecto deste relatório. Este estudo foi suportado por um subsídio para o projeto de pesquisa do genoma da província de Yamanashi (YH e modus operandi) e uma subvenção da Fundação médica de The YASUDA (YH).

1. TIC preparação para avaliação microscópica rápida usando o vidro Normal desliza
<ol><li>Execute a preparação de TIC logo que possível após materiais clínicos tecido patológico estão disponíveis. Se espécimes TIC não podem imediatamente ser preparados, mantenha os materiais de tecido cobertos com solução salina umedecida gaze estéril e guarde na geladeira para evitar a secagem de tecidos.</li>
	<li>Prepare-se 5 mm3 tecido material tais como tumores sólidos (por exemplo, fígado...

<ol>
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Genet. 45 (10), 1113-1120 (2013).</li><li>Nagasaki, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rare+variant+discovery+by+deep+whole-genome+sequencing+of+1,070+Japanese+individuals.">Rare variant discovery by deep whole-genome sequencing of 1,070 Japanese individuals.</a> Nat Commun. 6 (8018), (2015).</li><li>Vogelstein, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+genome+landscapes.">Cancer genome landscapes.</a> Science. 339 (6127), 1546-1558 (2013).</li><li>Zehir, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+landscape+of+metastatic+cancer+revealed+from+prospective+clinical+sequencing+of+10,000+patients.">Mutational landscape of metastatic cancer revealed from prospective clinical sequencing of 10,000 patients.</a> Nat Med. 23 (6), 703-713 (2017).</li><li>Garraway, L. 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Neste estudo, apresentamos um método alternativo para obtenção de tumor DNA de amostras clínicas patológicas, usando TIC. Preparação de TIC é muito simples e precisa de menos tempo em comparação com métodos FFPE, sem a exigência de instrumentos especiais10. Todos os procedimentos de preparação da TIC para extração de DNA podem ser concluídos dentro de dois dias (Figura 1). Esse método, portanto, encurta o tempo de retorno para a realização de teste...

Próxima geração tecnologia de sequenciamento forneceu pesquisadores avanços significativos na análise da informação do genoma em variações genéticas, doença mendeliana, predisposição hereditária e câncer 1,2,3 . O Atlas de genoma do câncer (TCGA) e International Cancer Genome Consortium (ICGC) têm perseguido a identificação de alterações genéticas em diversos tipos de cancros comuns4. Centenas de genes de condutor essencial do câncer foram identificadas com êxito, e algumas destas moléculas são sendo direcionadas para o desenvolvimento de drogas a1,5,6.
Na prática clínica, espécimes FFPE são comumente usados para diagnóstico patológico e testes moleculares para várias doenças, incluindo câncer. No entanto, durante o processo de fixação com formalina, cross-linking da ADN-proteína ou DNA-DNA ocorre e fragmentação de DNA é induzida. Assim, amostras de DNA FFPE nem sempre são apropriadas para a análise genética por causa da baixa qualidade e quantidade de DNA7,8,9. Além disso, leva vários dias para preparar espécimes FFPE e habilidade técnica é necessária preparar com precisão as seções. Portanto, é desejável desenvolver um método simples e rápido para a obtenção de DNA intacto de alta qualidade.
Citologia é um método alternativo para diagnóstico patológico. Preparação das amostras citológicas é um mais simples, menos cara e mais rápida abordagem comparada com FFPE preparação10. A técnica TIC tem sido realizada em linfonodos sentinela e os tecidos marginais de doentes com cancro da mama para diagnóstico rápido no intra-operatório por alguns anos11,12. No entanto, existem alguns relatórios que têm examinado se o DNA genômico de alta qualidade pode ser extraído amostras TIC e usados para posterior análise genética. Amostras citológicas comumente estão manchadas de Papanicolaou (Pap) ou coloração Giemsa, e informamos anteriormente que a quantidade e a qualidade do DNA extraído de amostras de tique (especialmente amostras manchadas de Giemsa) são superiores para as amostras obtidas da FFPE tecidos13. Em comparação com a coloração de Papanicolau, coloração Giemsa tem uma vantagem em que exigem menos procedimentos de coloração. Na coloração de Papanicolau, depois que as amostras foram fixadas e coradas, eles devem ser montados com montagem médio (por exemplo, Malinol) para distinguir o conteúdo de amostra, tais como as células do tumor, as células normais e células inflamatórias sob um microscópio. Se o espécime de Pap é preparado sem a etapa de montagem, é quase impossível observar as células sob um microscópio, porque a amostra é secada. Em comparação, coloração Giemsa pode ser observado no estado seco, portanto, a etapa de montagem não é necessária para avaliação celular rápida. Por microdissection, coloração Giemsa é mais adequado porque requer espécimes secos.
Neste relatório, apresentamos um método simples e rápido para preparar espécimes TIC com coloração Giemsa e demonstrar que a TIC é uma fonte melhor para DNA, em comparação com espécimes FFPE.

<table border="0" cellpadding="0" cellspacing="0" width="796">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">FINE FROST white20 micro slide glass</td>
			<td style="width:191px;">Matsunami Glass ind, Ltd</td>
			<td style="width:143px;">SFF-011</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Arcturus PEN Membrane Glass Slides&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>LCM0522</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Cyto Quick A solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20571</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Cyto Quick B solution</td>
			<td>Muto Pure Chemicals</td>
			<td>20581</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">May-Grunwald Solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15053</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Giemsa solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15002</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">QIAamp DNA FFPE tissue kit</td>
			<td>Qiagen</td>
			<td>56404</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444557</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">TaqMan RNase P Detection Reagents Kit&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4316831</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">TaqMan Assay from FFPE DNA QC Assay v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324034</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">MicroAmp Fast Optical 96-Well Reaction Plate&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>4346907</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp optical Adhesive Film</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4311971</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroMixer E36</td>
			<td style="width:191px;">TITEC</td>
			<td style="width:143px;">0027765-000</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">VIIA7-03</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Himac CF16RXII</td>
			<td style="width:191px;">Hitachi-koki</td>
			<td style="width:143px;">CF16RII</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion Library TaqMan Quantitation Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4468802</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Cancer Hotspot Panel v2</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4475346</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion AmpliSeq Library Kit 2.0</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4480442</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion Xpress Barcode Adapters 1-16 Kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4471250</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion PGM Hi-Q View Sequencing Kit (200 base)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">A30044</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion Chef System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4484177</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Veriti 96-well Thermal Cycler</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>Veriti200</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion 318 Chip Kit v2 BC</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>4488150</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion PGM System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>PGM11-001</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion PGM Wash 2 Bottle kit</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td>A25591</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;">16-position Magnetic Stand</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4457858</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM12450</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Nuclease-free water</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">AM9938</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Optical 96-well Reaction Plates</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
			<td style="width:143px;">4306737</td>
			<td style="width:180px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Clear Adhesive Film</td>
			<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>
		</tr>
		<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

É fundamental para determinar o status mutacional no câncer antes de administração e tratamento de drogas de alvo moleculares específicas para pacientes com câncer. Na prática clínica, fixada em formol parafina (FFPE) os tecidos são amplamente utilizados para testes genéticos. No entanto, FFPE DNA geralmente é danificado e fragmentada durante o processo de fixação com formol. Portanto, FFPE DNA não às vezes é adequada para testes genéticos por causa de baixa qualidade e quantidade de DNA. Aqui nós apresentamos um método de citologia de impressão de toque (TIC) para obter o DNA genômico de células de câncer, que pode ser observado sob um microscópio. Número de células de morfologia e câncer de células pode ser avaliado usando amostras TIC. Além disso, a extração de DNA genômico de amostras TIC pode ser concluída dentro de dois dias. O montante total e qualidade de TIC DNA obtido usando esse método foi maior do que a da FFPE DNA. Este método rápido e simples permite que os pesquisadores para obter DNA de qualidade para testes genéticos (por exemplo, análise de sequenciamento na próxima geração, digital PCR e PCR quantitativo em tempo real) e para encurtar o tempo de retorno para relatar os resultados.

A Figura 1 mostra todo o processo de preparar espécimes TIC para extração de DNA. Notavelmente, o procedimento leva apenas dois dias para obter o DNA genômico de amostras TIC. Nós avaliamos quaisquer efeitos do armazenamento do tumor antes do processamento do slide. Nós achamos que as células tumorais foram anexadas a lâmina de vidro quando as amostras de tecido foram tocadas imediatamente para o slide, e quando os tecidos foram mantidos em salina ume...

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

Obtenção de DNA genômico de alta qualidade dos tecidos de tumor é um primeiro passo essencial para a análise de alterações genéticas, utilizando a próxima geração de sequenciamento. Neste artigo, apresentamos um método simples e rápido para enriquecer as células do tumor e obter DNA intacto do toque espécimes de citologia de impressão.

Método simples e rápido para obter alta qualidade Tumor DNA de espécimes clínico-patológica usando toque citologia de impressão

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

A Mimic do microambiente do tumor: um método simples para geração de populações celulares enriquecidas e Investigando intercelular Comunicação

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

Pesquisa do câncer

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

Caracterização de células tumorais, usando um fio de médico para a captura de células tumorais de circulação: uma abordagem 3D baseado no DNA peixe e imunofluorescência

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

Usando 22 3 anticorpo Anti-PD-L1 concentrar-se na biópsia e citologia amostras de pacientes de câncer de pulmão não-pequenas células

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

Ensaio de reação em cadeia de polimerase digital para a variação genética em paciente esporádicos polipose adenomatosa familiar usando o formato de microplaqueta-em-um-tubo

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

Deteção e monitoração do ADN de circulação associado do tumor em Biofluids pacientes

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

Limpeza óptica rápida para análise semi-de alto rendimento de esferoides tumorais

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

Cultura e Imagem de Cortes Extraviais de Tumor Organotípico de Pseudomixoma Peritonei Ex Vivo de Espécimes Tumorais Humanos Ressecados

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

Quantificando o comprimento dos telômeros usando detecção de quimioluminescência não radioativa

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Autor em destaque: Otimização dos parâmetros de desempenho do ensaio de comprimento dos telômeros TAGGG  

Otimização dos parâmetros de desempenho do ensaio de comprimento telomérico TAGGG

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