이 작품은 캐나다 암 학회 연구원 추진하는 사업, 아무도 부여 없습니다. 019,038.

<html> 1. 전립선 샘플 모음집 <ol><li> prostatectomy시 전립선 샘플을 수집합니다. 시료는 해부 랜드마크를 사용하여 지향하고 있습니다. 전립선과 정액 vesicles은 다음과 같이 색칠하고 있습니다 : 오른쪽 녹색, 왼쪽 파란색. </li><li> 전립선의 임의의 가로 midsection은 직장 표면에 수직 이동 액화 질소에 냉동 및 -80 ° C ~ 9시 저장됩니다. </li><li> 표본의 틀었 조각은 (후부, 앞부분은 오른쪽과 왼쪽) 지향, photocopied quadrisected 있습니다. 섹션은 그라 이오 스탯을 사용하여 절단하고 있습니다. </li><li> 섹션은 H &amp; E 물들일과 스테인드 슬라이드와 해당 이미지에 종양이 대 정상적인 영역을 결정하고 윤곽을 그리다하는 병리학자에 의해 검토됩니다. 표시된 영역은 RNA가 후속 단계에서 추출됩니다있는 종양 조직을 추출할 수있는 영역을 표시하기위한 가이드로 사용됩니다. </li></ol>&#39;하다&gt; 2. 샘플에서 miRNA 포함하여 총 RNA를, 분리 <ol><li> 드라이 아이스와 delineated 복사를 참조에 대한 장소 냉동 전립선 샘플, 전립선 종양의 일부를 (50-100 밀리그램 사이) 그만. </li><li> TRIzol 시약 1 ML에서 전립선 종양 조직을 Homogenize. 다음 단계에서 수량은 TRIzol 시약 1 ML의 사용을 기반으로합니다. </li></ol> 참고 : 다음은 소규모 고립 그러나 다른 키트 RNA를 포함하는 총 RNA도 사용할 수 추출 RNA에 대한 TRIzol 시약을 사용 하였다. <ol start="3"><li> 상온에서 5 분간 균질 견본을 품어. </li><li> 샘플로 클로로포름의 0.2 ML을 추가하고 15 초 동안 풍성히 악수. 실온에서 3 분간 샘플을 부화 후 4에서 15 분 동안 12,000 XG에 원심 분리기 ° C. </li><li> 새로운 튜브로 무색 위 수성 위상을 전송하고, 이소 프로필 알코올 0.5 ML을 추가합니다.상온에서 10 분 샘플을 부화 후 4에서 10 분 동안 12,000 XG에 원심 분리기 ° C. </li><li> RNA를 포함하는 펠렛을 방해하지 않고 뜨는을 자세히 기음. 75 % 에탄올 1 ML과 함께 RNA 펠릿 씻으십시오. 와동 4에 7500의 XG에서 5 분간 원심 분리하여 샘플을 다시 침전 ° C. </li><li> 조심스럽게 RNA 펠릿가 완전히 건조되지 못하게하기 위해 5-10 분 동안 표면에 뜨는 건조 RNA 펠렛을 기음. 펠렛의 크기에 맞는 Nuclease없는 물에 다시 디졸브. NanoDrop 1000 분광 광도계 (260 nm의 및 280 nm의 흡광도에서 측정)를 사용하여 RNA의 농도를 측정합니다. </li><li> 애질런트 Bioanalyzer를 사용하여 RNA 샘플의 품질과 무결성을 확인합니다. </li></ol> 3. RNA의 역방향 스크립트 작성 <ol><li> RNA의 역방향 전사는 제조 업체의 지침 (Qiagen)에 따라 miScript 역방향 전송 키트를 사용하여 수행되었다. 이 키트를 포함transcriptase와 폴리 (A) 중합 효소를 반대. miScript RT 버퍼는 MG 2를 포함 +, dNTPs, oligo-DT primers, 그리고 무작위 primers. </li><li> 10 PG 및 cDNA 합성하는 RNA 1 μg 사이에 사용하십시오. RNA의 1 이상 μg를 사용하는 경우 해당 볼륨에 선형적으로 반응을 확장할. </li><li> 20 μl의 최종 볼륨에 대한 반응을 가져올 수있는 5 배 miScript RT 버퍼 (4 μl), miScript 역방향 전사 믹스 (1 μl)와 RNase없는 물이 들어있는 마스터 믹스를 준비합니다. 또한 마스터 믹스의 템플릿 RNA가 (최대 1 μg)가 있습니다. </li><li> 37시 60 분 샘플을 품어 ° C ~ 95 ° C.에서 5 분간 배양하여 바로 다음에 이 단계 블록, 또는 물에 목욕을 가열, PCR 기계에서 수행할 수 있습니다. Thermocyclers는 가장 적합하고 정확한 방법입니다. 단기 얼음 cDNA를 저장하고, -20 ° C에서 장기 보관을위한. </li></ol> 4. 표준 곡선을 생성 <ol><li> exper 이전대상 miRNAs와 iment, 표준 곡선들은 건너 포인트 (CP) (그림 2)에 대해 알려진 농도의 cDNAs를 사용하여 생성됩니다. </li><li> 2 배, 10 배, 50 배, 250 배, 그리고 관심의 유전자의 실질적인 표현을하는 것으로 알려져 샘플의 1,250 배 원래 cDNA의 dilutions 일련의를 준비합니다. </li><li> 5 항 &quot;miRNA의 탐지를위한 실시간 PCR&quot;에 명시된 바와 같이 cDNA 시리얼 dilutions없는 정적 40x 희석에있는 것을 수정하여, PCR을 실행합니다. </li><li> 당신의 표준 곡선을 생성하는 RelQuant 소프트웨어 (로슈)를 사용하여 분석을 수행합니다. </li></ol> 참고 : 새로운 표준 곡선은 관심있는 각각의 유전자가 생성되어야합니다. 5. miRNA의 탐지를위한 실시간 PCR <ol><li> miRNAs를위한 실시간 PCR은 제조 업체의 지침 (Qiagen)에 따라 miScript SYBR 녹색 PCR Kit 및 miScript의 프라이머 분석을 사용하여 수행되었다. 포함하는 마스터 믹스를 준비2X QuantiTect SYBR 녹색 PCR 마스터 믹스, 10X miScript 유니버셜 프라이머, 10X miScript 프라이머 분석 및 RNase없는 물. 20 μl 볼륨 반응을위한 마스터 믹스를 준비합니다. </li><li> 프라이머 분석은 관심 miRNA에만 적용됩니다. reconstitute 10X miScript의 프라이머 분석하려면, 간단히 병을 원심 분리기와 550 μl TE 버퍼, 산도 8.0을 추가합니다. -20 ° C.의 소용돌이 섞어 짧게 유리병, 작은 볼륨으로 나누어지는의 primers, 및 매장 두 primers이 필요합니다. 대상 유전자와 기준 유전자에 대한 primers RNU6B은 레퍼런스 유전자 usedas입니다. </li><li> -20 ° C.의 cDNA 40x 및 매장 추가 aliquots를 희석 </li><li> cDNA는 PCR을위한 템플릿 역할을합니다. 40x 희석 cDNA 2 μl를 사용하여 20 μl 가벼운 자전거 타는 사람 모세 혈관 (로슈)로 분배. </li><li> 각각의 모세관에 마스터 믹스 18 μl를 추가하고, 모세관 어댑터를 사용하여 원심 분리기. </li><li> 같은 LightCycler 세와 같은 모세관 기반의 리얼 타임 자전거 타는 사람에 모세 혈관을 놓습니다.32 모세 회전 목마 형식 5 리얼 타임 PCR 시스템. </li><li> 다음과 같이 PCR 사이클 프로그램을 실행합니다 : 2X QuantiTect SYBR 녹색 PCR 마스터 믹스에 HotStarTaq 효소를 활성화하기 위해, 95의 사전 품어 15 분 동안 ° C에서. 50주기를 얹는 : 변성, 15 초, 94 ° C; 어닐링, 30의, 55 ° C; 확장, 30 초, 70 ° C. </li><li> 캘리 브 레이터로 샘플을 선택하고 1의 표준 목표 금액을 설정합니다. 캘리 브 레이터에 대한 모든 다른 샘플에 miRNA의 상대적 표현을 비교합니다. </li></ol> 참고 : 공부 내에서 동일한 보정 샘플 결과의 일관성을 유지하는 데 사용해야합니다. 6. 데이터 분석 <ol><li> PCR 반응을위한 증폭 곡선 분자 Biochemicals의 LightCycler 소프트웨어 버전 3.5 (로슈)의 그래픽과 수치로 표시됩니다. , &quot;부량&quot;탭에 반응을 수량ND는 텍스트 파일로 데이터를 내보낼 수 있습니다. </li><li> 부량 결과를 생성하는 RelQuant 분석 소프트웨어 (로슈)로 데이터를 가져옵니다. 대상 유전자 참조 유전자와 표준 곡선 데이터를위한 별도의 파일을 가져옵니다. </li><li> 대상 및 참조 유전자 모두 캘리 브 레이터의 위치를​​ 지정합니다. 또한 샘플의 위치를​​ 지정합니다. 데이터는 캘리 브 레이터의 기준 비율을 대상으로 나누어 서로 다른 샘플 비율을 참조하는 대상으로 표시됩니다. 이전에 특정 miRNA와 가사 유전자에 대해 생성된 표준 곡선은 미지 농도의 miRNA 목표에 대한 양적 데이터를 바탕위한 참조 표준으로 사용됩니다. </li><li> 샘플 그룹으로 분석 농도와 세중의 표준 편차를 계산 의미하고 세 복제합니다. triplicates 중 하나가 집합의 나머지 부분과 일관성이있다면, 그것은 프로그램에 의해 제외됩니다. </li></ol> 7. 대표 결과 &lt;P 클래스 = &quot;jove_content&quot;&gt; 전립선 샘플에 qPCR 분석의 예제는 그림 3에 표시됩니다. 결과뿐만 아니라 그래픽, 수치 표시됩니다. 대상 유전자 미르-98, 표현 사이클 25 정도 지연될 증폭을 보여주면서 레퍼런스 유전자, U6의 표현 수준을 보여주는 그래프, 대한 사이클 20 지수 증폭을 시작합니다. 본 실험에서 데이터를 텍스트 파일로 내보낸 및 RelQuant 분석 소프트웨어에 의해 분석되었다. 캘리 브 레이터 및 샘플을 포함하는 모세 혈관의 위치가 지정됩니다. 그림 4는 캘리 브 레이터가 1로 설정하고, 캘리 브 레이터에 상대적으로 다른 샘플을 표현하는 방법을 보여줍니다. <img src="/files/ftp_upload/3874/3874fig1.jpg" alt="그림 1"/> 그림 1. miScript 리버스 녹음 및 실시간 PCR의 여러 단계. <img src="/files/ftp_upload/3874/3874fig2.jpg" alt="그림 2"/> 그림 2. 표준 곡선은 2 배, 10 배, 50 배, 250 배, 그리고 1250 배 원래 cDNA 샘플의 dilutions 일련를 사용하여 생성됩니다. <img src="/files/ftp_upload/3874/3874fig3.jpg" alt="그림 3"/> 그림 3. 로슈 분자 Biochemicals의 LightCycler 소프트웨어는 그래픽의 실험과 텍스트에 의한 전체 정보를 보여줍니다. 정량 실시간 PCR의 증폭 플롯 다른 샘플에서 형광 증가 보여줍니다. <img src="/files/ftp_upload/3874/3874fig4.jpg" alt="그림 4"/> 그림 4. 데이터 RelQuant LightCycler 분석 소프트웨어를 사용하여 계량했다. 보통 세 그룹과 분명히 일관성없는 결과는 제외됩니다 생산 및 농도와 세중의 표준 편차를 계산 뜻 샘플로 분석되는 샘플 복제합니다.

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관심의 어떠한 충돌 선언 없습니다.

일부 miRNAs의 탈선 표현은 일관되게 정상 조직 10, 그리고 이러한 miRNAs 중 일부는 전립선 암의 11에 대한 잠재적인 새로운 치료 대리인으로 명명되었습니다에 비해 전립선 종양에서 발견되었습니다. 따라서 miRNAs의 탈선 표현 수준은 유용한 진단 및 / 또는 전조 생체 자료가 될 수 있습니다. 여기서 제시 실시​​간 qPCR 방법론은 전립선 종양 조직에서 miRNA 수준의 정확한 양을 정함 ?...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> TRIzol 시약 </td><td> Invitrogen </td><td> 15,596 </td></tr><tr><td> miScript 역방향 전송 키트 </td><td> Qiagen </td><td> 218,061 </td></tr><tr><td> miScript 프라이머의 Assays </td><td> Qiagen </td><td> 구체적인 실험 </td></tr><tr><td> miScript SYBR 녹색 PCR 키트 </td><td> Qiagen </td><td> 218,073 </td></tr><tr><td> LightCycler 3.5 리얼 타임 PCR 시스템 </td><td> 로슈 </td><td></td></tr><tr><td> 라이트 자전거 타는 사람 모세 혈관 </td><td> 로슈 </td><td> 04929292001 </td></tr><tr><td> NanoDrop 1000 분광 광도계 </td><td> 써모 과학 </td><td> 2538 </td></tr><tr><td> 애질런트 2100 Bioanalyzer </td><td> G2943CA </td><td></td></tr></tbody></table>

microrna detection in prostate tumors by quantitative real-time pcr (qpcr)

MicroRNAs (miRNAs)는 단일 좌초된, 18-24 염기 길이 비 코딩 RNA 분자입니다. 그들은 개발 1, apoptosis 2 및 세포주기 조절 3를 포함해 거의 모든 세포 과정에 관여하고 있습니다. MiRNAs들은 목표 메신저 RNAs (mRNAs) 5에 바인딩하여 4 인간 유전자의 90 %에 30 %의 표현을 조절하는 것으로 추정된다. miRNAs의 전역 dysregulation는 다양한 질환 및 암 subtypes 6에서보고되었습니다. 그들의 유행과 독특한 구조로 인해,이 작은 분자 생체 치료, 대리인 및 / 또는 타겟의 차세대 될 것 같다. miRNA 발현을 조사하는 데 사용되는 방법은 SYBR 녹색 나는 염료 기반뿐만 아니라 Taqman-탐침 기반 qPCR을 포함합니다. miRNAs 효과적으로 임상 환경에서 사용할 수있다면 그것은 신선 및 / 또는 보관된 임상 시료에서의 검색이 정확하고 재현성 및 SP가 될 게 급선무다ecific. qPCR 널리 같은 microarray 연구 7과 전체 게놈 분석의 miRNAs의 표현 검증에 사용되고 있습니다. 이 프로토콜에 사용되는 샘플은 임상화된 전립선암에 대한 급진적인 prostatectomy을 받았습니다 환자에서 있었다, 그러나 다른 조직과 세포 라인 스냅인 냉동는 절제술 후 액체 질소에 있던 전립선 표본 들여 대체하실 수 있습니다. 임상 변수와 각각의 환자에 대한 후속 정보는 이후의 분석 8시 수집되었다. 전립선 종양 샘플에 miRNA 수준의 부량. 종양의 qPCR 분석의 주요 단계는 다음과 같습니다 miRNA 특정 primers를 사용하여 총 RNA 추출, cDNA 합성 및 qPCR 제품의 탐지. mRNA, miRNA 및 기타 작은 RNAs를 포함 총 RNA는 TRIzol 시약을 사용하여 표본에서 추출되었다. Qiagen의 miScript 시스템은 cDNA 합성 및 qPCR를 (그림 1)를 수행하는 데 사용되었다. 내생 miRNAs는 PO 없습니다역방향 전송 과정에서 따라서 lyadenylated, 폴리 (A) 중합 효소는 miRNA를 polyadenylates. miRNA는 oligo-DT 및 역방향 Transcriptase를 사용하여 cDNA 합성하는 템플릿으로 사용됩니다. oligo-DT primers의 5 &#39;끝에 보편 태그 시퀀스가​​ PCR 단계에서 cDNA의 증폭을 용이하게합니다. PCR 제품의 증폭은 SYBR 녹색, 이중 좌초된 DNA에 intercalates 염료에 의해 방출되는 형광의 수준에 의해 감지된다. 유니버설 태그 시퀀스에 바인딩 범용 프라이머와 함께 특정 miRNA의 primers는 특정 miRNA 시퀀스를 증폭합니다. miScript 프라이머의 Assays는 만 이상의 사람이 특정 miRNAs와 murine 특정 miRNAs 수백 수 있습니다. 상대 부량 방법은 miRNAs의 표현을 수량 여기에 사용되었습니다. 다른 샘플 사이 다양성에 대한 해결하려면 대상 miRNA의 발현 수준은 참조 유전자의 발현 수준으로 정규화됩니다. GE의 선택NE 분석의 상대적인 양을 정함 방식의 중요한 목표의 표현을 정상화하는하는입니다. 일반적으로이 용량에 사용된 참조 유전자의 예로는 그들이 안정적으로 대부분의 샘플을 통해 표현되는 것으로 간주되는대로 작은 RNAs의 RNU6B, RNU44 및 RNU48 있습니다. 이 프로토콜에서는 RNU6B는 참조 유전자로 사용됩니다.

Watch this Scientific Journal Video about MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR at JoVE.com

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

양적 실시간 중합 효소 사슬 반응 (qPCR)는 종양 샘플에서 여러 microRNA의 표현 수준 (miRNA) 분자를 조사하기 위해 신속하고 민감한 방법입니다. 다른 miRNA 분자의 수백이 메서드 표현식을 사용하면, 증폭 계량, 같은 cDNA 템플릿에서 분석할 수 있습니다.

정량 실시간 PCR (qPCR)에 의한 전립선 종양에 MicroRNA 감지

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

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Research

JoVE Journal

Medicine

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

24.4K 조회수.  University of Toronto. 양적 실시간 중합 효소 사슬 반응 (qPCR)는 종양 샘플에서 여러 microRNA의 표현 수준 (miRNA) 분자를 조사하기 위해 신속하고 민감한 방법입니다. 다른 miRNA 분자의 수백이 메서드 표현식을 사용하면, 증폭 계량, 같은 cDNA 템플릿에서 분석할 수 있습니다.

비디오: MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Gynecologic 종양학의 센티널 림프절의 Intraoperative 감지 Multispectral 실시간 형광 이미징

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

연구

의학

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

암 세포에 의해 내피 세포 단층의 침략을 측정하는 실시간 전기 임피던스 기반 기술

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Intravital 이미징을 사용하여 실시간으로 종양의 Nanoparticle의 이해 평가

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Taqman함으로써 어린이 Stools에서 Noroviruses의 감지 및 Genogrouping 한 단계 RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

양적 Immunofluorescence를 사용하여 종양의 단백질 표현의 이질 매핑

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

의 식별<em&gt; 잠자는 숲속의 미녀</em링커로 인한 PCR을 사용하여 고체 종양의&gt; Transposon의 삽입

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

운동은 성능의 실시간 피드백을 사용하여 재교육

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

경쟁 손쥐 골수 이식 모델에서 기증자 세포 Engraftment를 확인하려면 수량 실시간 PCR을 사용하여

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

정량 실시간 PCR을 사용하여 뇌척수액의 마이크로 RNA 프로파일

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

인간 전혈에서 실시간 세포 독성 분석 실험

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...