우리는 병원과 환자 들의 의료 및 보조 직원 참여 동의 감사. 우리는 가브리엘 화이트 울프,이 보고서의 초안을 편집 하기 위해 Edanz 그룹 (www.edanzediting.com/ac)에서 박사 학위를 감사 합니다. 이 연구는 야마나시 현 (Y.H. 고 지)에서 게놈 연구 프로젝트와 부여에서는 야스다 의료 재단 (Y.H.)는 특정에 의해 지원 되었다.

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저자는 공개 없다.

이 연구에서 우리는 콩을 사용 하 여 임상 병리학 표본에서 종양 DNA를 얻기 위해 다른 방법을 제시. TIC 준비는 매우 간단 하 고 특별 한 악기10에 대 한 요구 사항 없이 FFPE 방법에 비해 더 적은 시간이 필요. (그림 1) 2 이내 TIC 준비에서 DNA 추출에 모든 절차를 완료할 수 있습니다. 이 메서드는 따라서 유전자 검사를 수행 하기 위한 처리 시간이 줄어듭니다. 특...

차세대 시퀀싱 기술 제공 하고있다 연구팀은 유전자 변형, Mendelian 질병, 유전 경향, 및 암 1,2,3 게놈 정보 분석에 중요 한 발전 . 암 게놈 아틀라스 (TCGA) 및 국제 암 게놈 컨소시엄 (ICGC) 유전 변경 일반적인 암4의 여러 종류의 식별을 추구 했습니다. 수백 개의 필수 암 드라이버 유전자 성공적으로 확인 되었습니다, 그리고 이러한 분자의 일부 약물 개발1,,56타겟이 되고있다.
임상 설정에서 FFPE 표본 병 적인 진단과 분자 암 등 각종 질병에 대 한 테스트를 위해 일반적으로 사용 됩니다. 그러나, 포 르 말린으로 고정 과정 DNA-단백질 또는 DNA-DNA cross-linking과 DNA 파편을 유도. 따라서, FFPE DNA 샘플 적합 하지 않습니다 항상 유전자 분석에 대 한 낮은 질 및 양의 DNA7,,89. 또한, 고 기술 섹션을 정확 하 게 준비 해야 하는 FFPE 표본 준비를 몇 일 걸립니다. 따라서, 그것은 높은-품질 그대로 DNA를 얻기 위한 간단 하 고 빠른 방법을 개발 하는 것이 좋습니다.
세포학 병 적인 진단 위한 대체 방법입니다. Cytological 샘플 준비는 간단 하 게, 덜 FFPE 준비10에 비해 비싸고 더 빠른 접근 이다. TIC 기술 센 티 넬 림프절과 몇 년11,12자가 빠른 진단을 위해 유방암 환자에서 한계 조직에 수행 되었습니다. 그러나, TIC 표본에서 높은-품질 게놈 DNA를 추출할 수 있습니다 여부를 조사 하 고 후속 유전자 분석에 사용 되는 몇 가지 보고 있다. Cytological 표본은 일반적으로 물 들일 Papanicolaou (Pap) 또는 Giemsa 얼룩, 그리고 우리 이전 TIC 표본 (특히 Giemsa 얼룩이 샘플)에서 추출한 DNA의 품질과 양을 FFPE에서 얻은 샘플을 우수 하다는 것을 보고 조직13. Pap 얼룩과 비교, Giemsa 얼룩 장점이 덜 착 절차 요구에 있습니다. Pap 얼룩에 샘플 수정 되 고 스테인드, 후 그들은 해야 합니다 탑재할 종양 세포, 정상 세포를 현미경으로 염증 세포 등 샘플 내용을 구별을 위한 매체 (예, Malinol)를 장착. Pap 견본 설치 단계 없이, 준비 하는 경우 거의 불가능 한 표본은 건조 하기 때문에 현미경으로 세포를 관찰 하는 것 아니다. 비교에서는, Giemsa 얼룩 말린된 상태에서 관찰 될 수 있다, 그러므로, 설치 단계 빠른 세포 평가 위해 필요 하지 않습니다. 서, 대 한 Giemsa 얼룩은 더 적당 한 건조 표본 필요 하기 때문 에입니다.
이 보고서에서 우리 TIC 표본을 Giemsa 얼룩과 준비에 대 한 간단 하 고 빠른 방법을 소개 하 고 TIC가 더 나은 FFPE 표본에 비해 DNA에 대 한 설명.

<table><tbody><tr><td>FINE FROST white20 micro slide glass</td><td>Matsunami Glass ind, Ltd</td><td>SFF-011</td><td></td></tr><tr><td>Arcturus PEN Membrane Glass Slides</td><td>Thermo Fisher Scientific</td><td>LCM0522</td><td></td></tr><tr><td>Cyto Quick A solution</td><td>Muto Pure Chemicals</td><td>20571</td><td></td></tr><tr><td>Cyto Quick B solution</td><td>Muto Pure Chemicals</td><td>20581</td><td></td></tr><tr><td>May-Grunwald Solution</td><td>Muto Pure Chemicals</td><td>15053</td><td></td></tr><tr><td>Giemsa solution</td><td>Muto Pure Chemicals</td><td>15002</td><td></td></tr><tr><td>QIAamp DNA FFPE tissue kit</td><td>Qiagen</td><td>56404</td><td></td></tr><tr><td>TaqMan Fast Advanced Master Mix</td><td>Thermo Fisher Scientific</td><td>4444557</td><td></td></tr><tr><td>TaqMan RNase P Detection Reagents Kit</td><td>Thermo Fisher Scientific</td><td>4316831</td><td></td></tr><tr><td>TaqMan Assay from FFPE DNA QC Assay v2</td><td>Thermo Fisher Scientific</td><td>4324034</td><td></td></tr><tr><td>MicroAmp Fast Optical 96-Well Reaction Plate&amp;nbsp;</td><td>Thermo Fisher Scientific</td><td>4346907</td><td></td></tr><tr><td>MicroAmp optical Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4311971</td><td></td></tr><tr><td>MicroMixer E36</td><td>TITEC</td><td>0027765-000</td><td></td></tr><tr><td>ViiA 7 Real-Time PCR System</td><td>Thermo Fisher Scientific</td><td>VIIA7-03</td><td></td></tr><tr><td>Himac CF16RXII</td><td>Hitachi-koki</td><td>CF16RII</td><td></td></tr><tr><td>Ion Library TaqMan Quantitation Kit</td><td>Thermo Fisher Scientific</td><td>4468802</td><td></td></tr><tr><td>Ion AmpliSeq Cancer Hotspot Panel v2</td><td>Thermo Fisher Scientific</td><td>4475346</td><td></td></tr><tr><td>Ion AmpliSeq Library Kit 2.0</td><td>Thermo Fisher Scientific</td><td>4480442</td><td></td></tr><tr><td>Ion Xpress Barcode Adapters 1-16 Kit</td><td>Thermo Fisher Scientific</td><td>4471250</td><td></td></tr><tr><td>Ion PGM Hi-Q View Sequencing Kit (200 base)</td><td>Thermo Fisher Scientific</td><td>A30044</td><td></td></tr><tr><td>Ion Chef System</td><td>Thermo Fisher Scientific</td><td>4484177</td><td></td></tr><tr><td>Veriti 96-well Thermal Cycler</td><td>Thermo Fisher Scientific</td><td>Veriti200</td><td></td></tr><tr><td>Ion 318 Chip Kit v2 BC</td><td>Thermo Fisher Scientific</td><td>4488150</td><td></td></tr><tr><td>Ion PGM System</td><td>Thermo Fisher Scientific</td><td>PGM11-001</td><td></td></tr><tr><td>Ion PGM Wash 2 Bottle kit</td><td>Thermo Fisher Scientific</td><td>A25591</td><td></td></tr><tr><td>Agencourt&amp;trade; AMPure&amp;trade; XP Kit</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>16-position Magnetic Stand</td><td>Thermo Fisher Scientific</td><td>4457858</td><td></td></tr><tr><td>Nonstick, RNase-free Microfuge Tubes, 1.5 mL (Low binding tube)</td><td>Thermo Fisher Scientific</td><td>AM12450</td><td></td></tr><tr><td>Nuclease-free water</td><td>Thermo Fisher Scientific</td><td>AM9938</td><td></td></tr><tr><td>MicroAmp&amp;trade; Optical 96-well Reaction Plates</td><td>Thermo Fisher Scientific</td><td>4306737</td><td></td></tr><tr><td>MicroAmp&amp;trade; Clear Adhesive Film</td><td>Thermo Fisher Scientific</td><td>4306311</td><td></td></tr><tr><td>Agencourt&amp;trade; AMPure&amp;trade; XP Kit</td><td>Beckman Coulter</td><td>A63881</td><td></td></tr><tr><td>Ethanol(99.5)</td><td>Nacalai Tesque</td><td>08948-25</td><td></td></tr><tr><td>Sodium hydroxide (10M)</td><td>Sigma</td><td>72068</td><td></td></tr><tr><td>DTU-Neo</td><td>TAITEC</td><td>0063286-000</td><td></td></tr><tr><td>E-36</td><td>TAITEC</td><td>0027765-000</td><td></td></tr><tr><td>ECLIPSE Ci-L</td><td>Nikon</td><td>704354</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-2XLS+</td><td>METTLER TOLEDO</td><td>17014393</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-10XLS+</td><td>METTLER TOLEDO</td><td>17014388</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-20XLS+</td><td>METTLER TOLEDO</td><td>17014392</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-100XLS+</td><td>METTLER TOLEDO</td><td>17014384</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-200XLS+</td><td>METTLER TOLEDO</td><td>17014391</td><td></td></tr><tr><td>Pipet-Lite LTS Pipette L-1000XLS+</td><td>METTLER TOLEDO</td><td>17014382</td><td></td></tr><tr><td>petit-change</td><td>WAKEN</td><td>MODEL8864</td><td>Mini centrifuge</td></tr><tr><td>petit-incubator</td><td>WAKEN</td><td>WKN-2290</td><td>Air incubator</td></tr><tr><td>SensiCare Powder-free Nitrile Exam Gloves</td><td>MEDLINE</td><td>SEM486802</td><td></td></tr><tr><td>Sterile gauze</td><td>Osaki</td><td>11138</td><td></td></tr><tr><td>Refrigerator MediCool</td><td>SANYO</td><td>MPR-312DCN-PJ</td><td></td></tr><tr><td>FEATHER TRIMMING BLAD</td><td>FEATHER</td><td>No.130</td><td></td></tr><tr><td>FEATHER TRIMMING BLAD</td><td>FEATHER</td><td>No.260</td><td></td></tr><tr><td>FEATHER S</td><td>FEATHER</td><td>FA-10</td><td></td></tr><tr><td>Vortex Genius 3</td><td>IKA</td><td>41-0458</td><td>Vortex mixer</td></tr><tr><td>Pincette</td><td>NATSUME</td><td>A-5</td><td></td></tr><tr><td>1.5 mL microtube</td><td>BIOBIK</td><td>RC-0150</td><td></td></tr></tbody></table>

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

그것은 관리와 암 환자에 대 한 특정 분자 표적된 약의 치료 전에 암에서 mutational 상태를 결정 하는 중요 한입니다. 임상 설정에서 포 르 말린 고정 파라핀 포함 (FFPE) 조직 유전자 테스트를 위한 널리 사용 됩니다. 그러나, FFPE DNA는 일반적으로 손상 되 고 포 르 말린으로 고정 과정 조각. 따라서, FFPE DNA는 때로는 낮은 품질 및 수량 DNA의 유전자 검사에 적합 하지. 여기 우리가 터치 인쇄물 세포학 (TIC), 암 세포에서 게놈 DNA를 현미경으로 관찰 될 수 있는 방법 제시. 셀 형태 및 암 세포 숫자는 TIC 견본을 사용 하 여 평가할 수 있습니다. 또한, TIC 샘플에서 게놈 DNA 추출 2 이내에 완료할 수 있습니다. 이 메서드를 사용 하 여 얻은 TIC DNA의 품질과 총 FFPE DNA 보다 높았다. 이 신속 하 고 간단한 방법 연구를 결과 보고에 대 한 처리 시간을 단축 하 고 유전자 검사 (예를 들어, 다음 세대 시퀀싱 분석, 디지털 PCR 및 양적 실시간 PCR)에 대 한 높은-품질 DNA를 수 있습니다.

그림 1 에서 DNA 추출 TIC 표본을 준비 전체 프로세스를 보여 줍니다. 특히, 절차 TIC 샘플에서 게놈 DNA를만 2 일 걸립니다. 우리는 슬라이드 처리 하기 전에 종양 스토리지의 어떤 효과 평가합니다. 우리는 조직 표본을 즉시 슬라이드에 감동 했다 그리고 염 분 moistened 살 균-거 즈에 1 h (그림 2)에 대 한 조직을 유지 했다 종양 세포를...

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

종양 조직에서 높은-품질 게놈 DNA를 얻는 유전 변경 다음 세대 시퀀싱을 사용 하 여 분석 하기 위한 필수적인 첫 번째 단계입니다. 이 문서에서는, 우리는 종양 세포를 풍부 하 게 하 고 그대로 DNA 터치에서 인쇄물 세포학 표본 하는 간단 하 고 빠른 방법을 제시.

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

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

simple-rapid-method-to-obtain-high-quality-tumor-dna-from-clinical

Research

JoVE Journal

Cancer Research

10.8K 조회수.  Yamanashi Central Hospital. 종양 조직에서 높은-품질 게놈 DNA를 얻는 유전 변경 다음 세대 시퀀싱을 사용 하 여 분석 하기 위한 필수적인 첫 번째 단계입니다. 이 문서에서는, 우리는 종양 세포를 풍부 하 게 하 고 그대로 DNA 터치에서 인쇄물 세포학 표본 하는 간단 하 고 빠른 방법을 제시.

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

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

고체 및 액체 종양에서 실행 가능한 돌연변이의 검출을위한 차세대 시퀀싱

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

연구

암 연구학

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

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

순환 종양 세포를 캡처에 대 한 의료 와이어를 사용 하 여 종양 세포의 특성: 면역 형광 DNA 물고기 기반으로 3 차원 접근

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

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

환자 생체 유체에서 종양 관련 순환 DNA의 검출 및 모니터링

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

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

낮은 종양 함량 조직에서 종양 농축을 위한 자동화된 해부 프로토콜

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

표적 시퀀싱 접근법을 통한 비교 병변 분석

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

행동분석학

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

면역학 및 감염병학

Isolation of nuclei for multiome sequencing in solid tumor specimen(고형 종양 표본에서 멀티옴 염기서열분석을 위한 핵 분리)

Optimized Nuclei Isolation from Fresh and Frozen Solid Tumor Specimens for Multiome Sequencing

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) data, represents a breakthrough in our ability to discern tumor cell heterogeneity-a primary focus of translational cancer research at this time. However, the quality of sequencing data acquired using this advanced modality is highly dependent on the quality of the input material. 
Digestion conditions need to be optimized to maximize cell yield without sacrificing quality. This is particularly challenging in the context of solid tumors with dense desmoplastic matrices that must be gently broken down for cell release. Freshly isolated cells from solid tumor tissue are more fragile than those isolated from cell lines. Additionally, as the cell types isolated are heterogeneous, conditions should be selected to support the total cell population. 
Finally, nuclear isolation conditions must be optimized based on these qualities in terms of lysis times and reagent types/ratios. In this article, we describe our experience with nuclear isolation for the 10x Genomics multiome sequencing platform from solid tumor specimens. We provide recommendations for tissue digestion, storage of single-cell suspensions (if desired), and nuclear isolation and assessment.

저자 스포트라이트: Enhancing Nuclei Isolation for Multiome Sequencing in Challenging Tumor Microenvironments

The protocol provides a reliable and optimized approach to the isolation of nuclei from solid tumor specimens for multiome sequencing using the 10x Genomics platform, including recommendations for tissue dissociation conditions, cryopreservation of single-cell suspensions, and assessment of isolated nuclei.

Multiome Sequencing을 위한 신선 및 냉동 고형 종양 검체에서 최적화된 핵 분리

Multiome sequencing, which provides same-cell/paired single-cell RNA- and the assay for transposase-accessible chromatin with sequencing (ATAC-sequencing) ...

Integration of Wet and Dry Bench Processes Optimizes Targeted Next-generation Sequencing of Low-quality and Low-quantity Tumor Biopsies

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using bioinformatics pipelines (&#34;dry bench&#34;). Both elements are essential to produce accurate and reliable results, which are particularly critical for clinical laboratories. Targeted NGS technologies have increasingly found favor in oncology applications to help advance precision medicine objectives, yet the methods often involve disconnected and variable wet and dry bench workflows and uncoordinated reagent sets. In this report, we describe a method for sequencing challenging cancer specimens with a 21-gene panel as an example of a comprehensive targeted NGS system. The system integrates functional DNA quantification and qualification, single-tube multiplexed PCR enrichment, and library purification and normalization using analytically-verified, single-source reagents with a standalone bioinformatics suite. As a result, accurate variant calls from low-quality and low-quantity formalin-fixed, paraffin-embedded (FFPE) and fine-needle aspiration (FNA) tumor biopsies can be achieved. The method can routinely assess cancer-associated variants from an input of 400 amplifiable DNA copies, and is modular in design to accommodate new gene content. Two different types of analytically-defined controls provide quality assurance and help safeguard call accuracy with clinically-relevant samples. A flexible &#34;tag&#34; PCR step embeds platform-specific adaptors and index codes to allow sample barcoding and compatibility with common benchtop NGS instruments. Importantly, the protocol is streamlined and can produce 24 sequence-ready libraries in a single day. Finally, the approach links wet and dry bench processes by incorporating pre-analytical sample quality control results directly into the variant calling algorithms to improve mutation detection accuracy and differentiate false-negative and indeterminate calls. This targeted NGS method uses advances in both wetware and software to achieve high-depth, multiplexed sequencing and sensitive analysis of heterogeneous cancer samples for diagnostic applications.

An integrated system for targeted next-generation sequencing of oncology specimens is described. This cross-platform system is optimized for low-quality and low-quantity tumor biopsies, accommodates low DNA inputs, includes well-characterized multi-variant controls, and features a novel variant caller that is informed by quantitative pre-analytical quality control measures.

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱

All next-generation sequencing (NGS) procedures include assays performed at the laboratory bench (&#34;wet bench&#34;) and data analyses conducted using ...

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

요약

더 많은 비디오 탐색

이 비디오의 챕터

고체 및 액체 종양에서 실행 가능한 돌연변이의 검출을위한 차세대 시퀀싱

순환 종양 세포를 캡처에 대 한 의료 와이어를 사용 하 여 종양 세포의 특성: 면역 형광 DNA 물고기 기반으로 3 차원 접근

환자 생체 유체에서 종양 관련 순환 DNA의 검출 및 모니터링

낮은 종양 함량 조직에서 종양 농축을 위한 자동화된 해부 프로토콜

표적 시퀀싱 접근법을 통한 비교 병변 분석

침투 림프구의 분석을위한 비 효소 해리 신선한 인간의 조직에 대한 간단하고 빠른 프로토콜

Multiome Sequencing을 위한 신선 및 냉동 고형 종양 검체에서 최적화된 핵 분리

Multiome Sequencing을 위한 신선 및 냉동 고형 종양 검체에서 최적화된 핵 분리

Multiome Sequencing을 위한 신선 및 냉동 고형 종양 검체에서 최적화된 핵 분리

낮은 품질과 낮은 수량 종양 생검의 습식 및 건식 벤치 프로세스의 통합을 대상으로 최적화하여 차세대 시퀀싱