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

Wir danken allen medizinischen und Dienstpersonal des Krankenhauses und die Patienten für die Zustimmung zur Teilnahme. Wir bedanken uns bei Gabrielle White Wolf, PhD, Edanz-Gruppe (www.edanzediting.com/ac) für einen Entwurf des Berichts bearbeiten. Diese Studie wurde unterstützt durch eine Beihilfe für Genome Research Project aus der Präfektur Yamanashi (YH und M.O.) und einen Zuschuss von der YASUDA Medical Foundation (YH).

(1) TIC Vorbereitung für schnelle mikroskopische Beurteilung mit normalem Glas gleitet
<ol><li>Nach klinischen pathologischen Gewebe Materialien zur Verfügung stehen durchführen Sie die TIC-Vorbereitung so bald wie möglich. Wenn TIC Proben sofort vorbereitet werden können, halten Sie die Gewebe Materialien bedeckt mit Kochsalzlösung befeuchtet steriler Gaze und lagern im Kühlschrank Trocknen des Gewebes zu verhindern.</li>
	<li>Bereiten Sie 5 mm3 Gewebematerial wie soliden Tumoren (z.B.Leber,...

<ol>
	<li>Vogelstein, B., Kinzler, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Path+to+Cancer+-+Three+Strikes+and+You're+Out.">The Path to Cancer - Three Strikes and You're Out.</a> N Engl J Med. 373 (20), 1895-1898 (2015).</li><li>Weinstein, J. N., 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=The+cancer+genome+atlas+pan-cancer+analysis+project.">The cancer genome atlas pan-cancer analysis project.</a> Nat. 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. A., Lander, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lessons+from+the+cancer+genome.">Lessons from the cancer genome.</a> Cell. 53 (1), 17-37 (2013).</li><li>Chalkley, R., Hunter, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone-histone+propinquity+by+aldehyde+fixation+of+chromatin.">Histone-histone propinquity by aldehyde fixation of chromatin.</a> Proc Natl Acad Sci U S A. 72 (4), 1304-1308 (1975).</li><li>Ben-Ezra, J., Johnson, D. A., Rossi, J., Cook, N., Wu, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixation+on+the+amplification+of+nucleic+acids+from+paraffin-embedded+material+by+the+polymerase+chain+reaction.">Effect of fixation on the amplification of nucleic acids from paraffin-embedded material by the polymerase chain reaction.</a> J Histochem Cytochem. 39 (3), 351-354 (1991).</li><li>Srinivasan, M., Sedmak, D., Jewell, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+fixatives+and+tissue+processing+on+the+content+and+integrity+of+nucleic+acids.">Effect of fixatives and tissue processing on the content and integrity of nucleic acids.</a> Am.J.Pathol. 161 (6), 1961-1971 (2002).</li><li>Adhya, A. K., Mohanty, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Utility+of+touch+imprint+cytology+in+the+preoperative+diagnosis+of+malignancy+in+low+resource+setting.">Utility of touch imprint cytology in the preoperative diagnosis of malignancy in low resource setting.</a> Diagn Cytopathol. 45 (6), 507-512 (2017).</li><li>Lumachi, F., Marino, F., Zanella, S., Chiara, G. B., Basso, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Touch+Imprint+Cytology+and+Frozen-section+Analysis+for+Intraoperative+Evaluation+of+Sentinel+Nodes+in+Early+Breast+Cancer.">Touch Imprint Cytology and Frozen-section Analysis for Intraoperative Evaluation of Sentinel Nodes in Early Breast Cancer.</a> Anticancer Research. 32 (8), 3523-3526 (2012).</li><li>Sumiyoshi, K., 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=Usefulness+of+intraoperative+touch+smear+cytology+in+breast-conserving+surgery.">Usefulness of intraoperative touch smear cytology in breast-conserving surgery.</a> Exp Ther Med. 1 (4), 641-645 (2012).</li><li>Amemiya, K., 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=Touch+imprint+cytology+with+massively+parallel+sequencing+(TIC-seq):+a+simple+and+rapid+method+to+snapshot+genetic+alterations+in+tumors.">Touch imprint cytology with massively parallel sequencing (TIC-seq): a simple and rapid method to snapshot genetic alterations in tumors.</a> Cancer Med. 5 (12), 3426-3436 (2016).</li><li>Goto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutational+analysis+of+multiple+lung+cancers:+Discrimination+between+primary+and+metastatic+lung+cancers+by+genomic+profile.">Mutational analysis of multiple lung cancers: Discrimination between primary and metastatic lung cancers by genomic profile.</a> Oncotarget. 8 (19), 31133-31143 (2017).</li><li>Goto, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+tumor-derived+DNA+dispersed+in+the+airway+improves+the+diagnostic+accuracy+of+bronchoscopy+for+lung+cancer.">Detection of tumor-derived DNA dispersed in the airway improves the diagnostic accuracy of bronchoscopy for lung cancer.</a> Oncotarget. 8 (45), 79404-79413 (2017).</li><li>Hirotsu, Y., 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=Targeted+and+exome+sequencing+identified+somatic+mutations+in+hepatocellular+carcinoma.">Targeted and exome sequencing identified somatic mutations in hepatocellular carcinoma.</a> Hepatol Res. 46 (11), 1145-1151 (2016).</li><li>Hirotsu, Y., 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=Comparison+between+two+amplicon-based+sequencing+panels+of+different+scales+in+the+detection+of+somatic+mutations+associated+with+gastric+cancer.">Comparison between two amplicon-based sequencing panels of different scales in the detection of somatic mutations associated with gastric cancer.</a> BMC Genomics. 17 (1), 833 (2016).</li><li>Hirotsu, Y., 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=Intrinsic+HER2+V777L+mutation+mediates+resistance+to+trastuzumab+in+a+breast+cancer+patient.">Intrinsic HER2 V777L mutation mediates resistance to trastuzumab in a breast cancer patient.</a> Med Oncol. 34 (1), 3 (2017).</li><li>Hirotsu, Y., 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=Detection+of+BRCA1+and+BRCA2+germline+mutations+in+Japanese+population+using+next-generation+sequencing.">Detection of BRCA1 and BRCA2 germline mutations in Japanese population using next-generation sequencing.</a> Mol Genet Genomic Med. 3 (2), 121-129 (2015).</li><li>Hirotsu, Y., 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=Multigene+panel+analysis+identified+germline+mutations+of+DNA+repair+genes+in+breast+and+ovarian+cancer.">Multigene panel analysis identified germline mutations of DNA repair genes in breast and ovarian cancer.</a> Mol Genet Genomic Med. 3 (5), 459-466 (2015).</li><li>Li&#232;vre, 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=KRAS+mutation+status+is+predictive+of+response+to+cetuximab+therapy+in+colorectal+cancer.">KRAS mutation status is predictive of response to cetuximab therapy in colorectal cancer.</a> Cancer Res. 66 (8), 3992-3995 (2006).</li><li>Mok, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osimertinib+or+Platinum-Pemetrexed+in+EGFR+T790M-Positive+Lung+Cancer.">Osimertinib or Platinum-Pemetrexed in EGFR T790M-Positive Lung Cancer.</a> N Engl J Med. 376 (7), 629-640 (2017).</li><li>Wong, S. Q., 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=Targeted-capture+massively-parallel+sequencing+enables+robust+detection+of+clinically+informative+mutations+from+formalin-fixed+tumours.">Targeted-capture massively-parallel sequencing enables robust detection of clinically informative mutations from formalin-fixed tumours.</a> Sci rep. 13 (3), 3493 (2013).</li><li>Wong, S. Q., 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=Sequence+artefacts+in+a+prospective+series+of+formalin-fixed+tumours+tested+for+mutations+in+hotspot+regions+by+massively+parallel+sequencing.">Sequence artefacts in a prospective series of formalin-fixed tumours tested for mutations in hotspot regions by massively parallel sequencing.</a> BMC Med Genomics. 13 (7), 23 (2014).</li></ol>

In dieser Studie haben wir eine alternative Methode für den Erhalt der Tumor DNA aus klinischen pathologischen Proben mit TIC vorgestellt. TIC-Zubereitung ist sehr einfach und benötigt weniger Zeit im Vergleich zu FFPE Methoden, ohne die Forderung nach speziellen Instrumenten10. Alle Verfahren von der TIC-Vorbereitung zur DNA-Extraktion können innerhalb von zwei Tagen (Abbildung 1) abgeschlossen werden. Diese Methode verkürzt somit die Turnaround-Zeit für die Dur...

Nächste Generation Sequencing Technologie hat Forscher erhebliche Fortschritte bei der Analyse von Genominformationen in genetischen Variationen, Mendelian Krankheit, erbliche Veranlagung und Krebs 1,2,3 bereitgestellt . Die Cancer Genome Atlas (TCGA) und internationalen Krebs Genom Konsortium (ICGC) haben die Identifizierung der genetischen Veränderungen, die in verschiedenen Arten von gemeinsamen Krebserkrankungen4verfolgt. Hunderte von wesentlicher Treiber Krebsgene erfolgreich identifiziert worden, und einige dieser Moleküle sind für Droge-Entwicklung1,5,6angestrebt.
In der klinischen Einstellung sind FFPE-Proben für die pathologische Diagnose und molekulare Tests für verschiedene Krankheiten, einschließlich Krebs gebräuchlich. Jedoch bei der Fixierung mit Formalin, tritt Vernetzung der DNA-Protein oder DNA-DNA und DNA-Fragmentierung wird induziert. Somit eignen sich FFPE DNA-Proben nicht immer zur genetischen Analyse wegen der geringen Qualität und Quantität der DNA-7,8,9. Darüber hinaus dauert es mehrere Tage FFPE-Proben vorbereiten und technisches Geschick ist notwendig, um die Abschnitte genau vorbereitet. Daher ist es wünschenswert, eine einfache und schnelle Methode zur Gewinnung qualitativ hochwertige intakte DNA zu entwickeln.
Zytologie ist eine alternative Methode für die pathologische Diagnose. Zytologische Probenvorbereitung ist eine einfachere, weniger teuer und schneller Ansatz im Vergleich zu FFPE Vorbereitung10. Die TIC-Technik wurde auf Sentinel-Lymphknoten und marginale Gewebe von Patientinnen mit Brustkrebs für intraoperative Schnelldiagnose für einige Jahre11,12durchgeführt. Allerdings gibt es einige Berichte, die untersucht, ob qualitativ hochwertige genomischer DNA aus TIC Proben extrahiert werden kann und für spätere genetische Analyse verwendet. Zytologische Präparate sind häufig mit Papanicolaou (Pap) oder Giemsa Färbung gefärbt, und wir haben bereits berichtet, dass die Menge und Qualität der von TIC Proben (vor allem Giemsa gefärbten Proben) extrahierte DNA Proben aus FFPE überlegen sind Gewebe-13. Verglichen mit PAP-Färbung, hat Giemsa Färbung einen Vorteil in weniger befleckenden Verfahren erfordern. Pap-Färbung, nach die Proben wurden behoben, und gebeizt, müssen sie montiert werden mit Montage Medium (z. B.Malinol) Probe Inhalt, z. B. Tumorzellen normalen Zellen und entzündlichen Zellen unter dem Mikroskop zu unterscheiden. Wenn die Pap-Probe ohne Montage Schritt bereit ist, ist es fast unmöglich, die Zellen unter dem Mikroskop zu beobachten, weil die Probe getrocknet ist. Zum Vergleich: Giemsa Färbung im getrockneten Zustand beobachtet werden, daher die Montage-Schritt ist nicht erforderlich für schnelle zelluläre Bewertung. Für Mikrodissektion ist die Giemsa Färbung besser geeignet, da trockene Proben erforderlich.
In diesem Bericht wir stellen eine einfache und schnelle Methode für die Zubereitung von TIC Exemplare mit Giemsa Färbung und zeigen, dass TIC eine bessere Quelle für DNA verglichen mit FFPE-Proben.

<table border="0" cellpadding="0" cellspacing="0" width="796">
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			<td height="41" style="height:41px;">Arcturus PEN Membrane Glass Slides&#160;</td>
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			<td>20581</td>
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			<td>Muto Pure Chemicals</td>
			<td>15053</td>
			<td></td>
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			<td height="41" style="height:41px;">Giemsa solution</td>
			<td>Muto Pure Chemicals</td>
			<td>15002</td>
			<td></td>
		</tr>
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			<td height="41" style="height:41px;">QIAamp DNA FFPE tissue kit</td>
			<td>Qiagen</td>
			<td>56404</td>
			<td></td>
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			<td height="41" style="height:41px;">TaqMan Fast Advanced Master Mix</td>
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			<td height="41" style="height:41px;">TaqMan RNase P Detection Reagents Kit&#160;</td>
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			<td>4316831</td>
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			<td height="41" style="height:41px;">TaqMan Assay from FFPE DNA QC Assay v2</td>
			<td>Thermo Fisher Scientific</td>
			<td>4324034</td>
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			<td height="41" style="height:41px;">MicroAmp Fast Optical 96-Well Reaction Plate&#160;</td>
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			<td height="41" style="height:41px;width:283px;">MicroMixer E36</td>
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			<td height="41" style="height:41px;width:283px;">ViiA 7 Real-Time PCR System</td>
			<td style="width:191px;">Thermo Fisher Scientific</td>
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			<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<tr height="41">
			<td height="41" style="height:41px;width:283px;">Ion Chef System</td>
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			<td>4484177</td>
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			<td height="41" style="height:41px;width:283px;">Veriti 96-well Thermal Cycler</td>
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			<td>Veriti200</td>
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			<td height="41" style="height:41px;width:283px;">Ion 318 Chip Kit v2 BC</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM System</td>
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			<td>PGM11-001</td>
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			<td height="41" style="height:41px;width:283px;">Ion PGM Wash 2 Bottle kit</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
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			<td height="41" style="height:41px;width:283px;">16-position Magnetic Stand</td>
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			<td height="41" style="height:41px;width:283px;">Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td>
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			<td height="41" style="height:41px;width:283px;">MicroAmp&#8482; Optical 96-well Reaction Plates</td>
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			<td height="41" style="height:41px;width:283px;">Agencourt&#8482; AMPure&#8482; XP Kit</td>
			<td style="width:191px;">Beckman Coulter</td>
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		</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

Es ist entscheidend für die Mutationszucht Status bei Krebs vor Verwaltung und Behandlung der spezifische molekulare zielgerichtete Medikamente für Krebspatienten zu bestimmen. In der klinischen Einstellung sind Formalin fixierten Paraffin-eingebetteten (FFPE) Gewebe für Gentests verbreitet. Proteinkinase DNA ist jedoch in der Regel beschädigt und bei der Fixierung mit Formalin fragmentiert. Proteinkinase DNA ist daher manchmal nicht ausreichend für die genetische Untersuchung wegen geringer Qualität und Quantität der DNA. Hier präsentieren wir eine Methode der Touch Impressum Zytologie (TIC), genomischen DNA von Krebszellen zu erhalten, die unter dem Mikroskop beobachtet werden kann. Zelle Morphologie und Krebs-Zell-Zahlen können mit TIC Proben ausgewertet werden. Darüber hinaus kann die Extraktion von genomischer DNA aus TIC Proben innerhalb von zwei Tagen abgeschlossen werden. Den Gesamtbetrag und die Qualität der TIC DNA erhalten mit dieser Methode war höher als die der Proteinkinase DNA. Diese schnelle und einfache Methode erlaubt Forschern, qualitativ hochwertige DNA für genetische Untersuchungen (z.B., nächste Generation Sequenzierung Analyse, digitale PCR und quantitative Real-Time PCR) zu erhalten und um die Bearbeitungszeit für die Berichterstattung über Ergebnisse zu verkürzen.

Abbildung 1 zeigt den gesamten Prozess von der Vorbereitung TIC Proben zur DNA-Extraktion. Insbesondere übernimmt die Prozedur nur zwei Tage genomischen DNA von TIC Proben zu erhalten. Wir bewerten alle Effekte der Tumor Lagerung vor der Verarbeitung der Folie. Wir fanden, dass Tumorzellen auf dem Objektträger befestigt wurden, wenn Gewebeproben sofort auf die Folie berührt wurden und Geweben in Kochsalzlösung befeuchtet steril-Gaze für 1 h (<strong clas...

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

Erhalten qualitativ hochwertige genomischen DNA von Tumorgewebe ist ein wichtiger erster Schritt für die Analyse von genetischer Veränderungen, die Sequenzierung der nächsten Generation. In diesem Artikel präsentieren wir Ihnen eine einfache und schnelle Methode, um Tumorzellen zu bereichern und intakte DNA von Touch Impressum Zytologische Proben erhalten.

Einfache und schnelle Methode, um qualitativ hochwertige Tumor DNA von klinischen pathologischen Proben mit Touch Impressum Zytologie zu erhalten

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

Ein Mimic des Tumor-Microenvironment: Eine einfache Methode für Enriched Zellpopulationen Generierung und Untersuchung der interzellulären Kommunikation

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

Forschung

Krebsforschung

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

Charakterisierung von Tumorzellen mit einem medizinischen Draht zur Erfassung von zirkulierenden Tumorzellen: 3D Ansatz basierend auf Immunfluoreszenz und DNA-Fisch

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

Mit 22 3 Anti-PD-L1-Antikörper konzentrieren sich auf Biopsie und Zytologie Proben von Patienten mit nicht-kleinzelligem Lungenkrebs

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

Digitalen Polymerase-Kettenreaktion-Assay für die genetische Variation bei einem sporadischen familiäre adenomatöse Polyposis Patienten mit dem Chip-in-Rohr-Format

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

Nachweis und Überwachung von tumorassoziierter zirkulierender DNA in Biofluiden von Patienten

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

Rapid Optical Clearing für die Semi-Hochdurchsatzanalyse von Tumorsphäroiden

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

Kultivierung und Bildgebung von ex vivo organotypischen Pseudomyxoma-Peritonei-Tumorschnitten aus resezierten humanen Tumorproben

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

Quantifizierung der Telomerlänge mittels nicht-radioaktiver Chemilumineszenzdetektion

Optimization of Performance Parameters of the TAGGG Telomere Length Assay

Autor im Rampenlicht: Optimierung der Leistungsparameter des TAGGG Telomerlängen-Assays  

Optimierung der Leistungsparameter des TAGGG Telomerlängen-Assays

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