وقد تم تمويل هذا العمل من قبل المعهد الكندي لابحاث السرطان في المجتمع، لا تمنح. 019038.

<html> 1. البروستاتا جمع العينات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> جمع عينات من البروستاتا في وقت استئصال البروستاتا. توجه العينة باستخدام معالم التشريحية. ورسمت على البروستاتا والحويصلات المنوية على النحو التالي: حق أخضر الجانب، جانب اليسار الزرقاء. </li><li style=";text-align:right;direction:rtl"> يتم أخذ القسم الوسطي عشوائية عرضية من البروستاتا عمودي على سطح المستقيم، مجمدة في النيتروجين السائل، وتخزينها في -80 درجة مئوية 9. </li><li style=";text-align:right;direction:rtl"> شرائح من العينات المخزنة المصورة، الموجهة (الأمامية، الخلفية، اليمين واليسار)، quadrisected. يتم قطع المقاطع باستخدام ناظم البرد. </li><li style=";text-align:right;direction:rtl"> وملطخة المقاطع مع H &amp; E، واستعرض من قبل الطبيب الشرعي لتحديد وترسيم المناطق ورم مقابل طبيعي على الشرائح ملون وصورة المقابلة. وتستخدم المناطق ملحوظ كدليل للإشارة إلى المناطق التي لاستخراج أنسجة الورم من الذي سيتم استخراج الحمض النووي الريبي في الخطوات اللاحقة. </li></ol>كسر &quot;&gt; 2. عزل الحمض النووي الريبي الإجمالية، بما فيها ميرنا، من العينات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> عينات من مكان البروستات المجمدة على الجليد الجافة ويشير الى صورة المرسومة، قطع جزء صغير من الورم البروستات (ما بين 50 الى 100 ملغ). </li><li style=";text-align:right;direction:rtl"> تجانس الأنسجة السرطانية في البروستات 1 مل من الكاشف TRIzol. وتعتمد الكميات في الخطوات التالية على استعمال 1 مل من الكاشف TRIzol. </li></ol> ملاحظة: نحن هنا قد استخدمت الكاشف TRIzol لاستخراج الحمض النووي الريبي، ويمكن أيضا مجموعات أخرى إلا أن عزل الحمض النووي الريبي الصغيرة التي تحتوي على الحمض النووي الريبي مجموع استخدامها. <ol start="3" style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان العينات المتجانس لمدة 5 دقائق في درجة حرارة الغرفة. </li><li style=";text-align:right;direction:rtl"> إضافة 0.2 مل من الكلوروفورم إلى العينات وهزة بقوة لمدة 15 ثانية. احتضان العينات لمدة 3 دقائق في درجة حرارة الغرفة، ثم أجهزة الطرد المركزي في 12000 XG لمدة 15 دقيقة في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> نقل عديم اللون المرحلة المائية العليا لأنابيب جديدة، وإضافة 0.5 مل من الكحول الآيزوبروبيل.احتضان العينات لمدة 10 دقائق في درجة حرارة الغرفة، ثم أجهزة الطرد المركزي في 12000 XG لمدة 10 دقائق في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> بعناية نضح طاف من دون إزعاج بيليه الذي يحتوي على الحمض النووي الريبي. غسل بيليه RNA مع 1 مل من الإيثانول بنسبة 75٪. دوامة العينة وإعادة الرواسب عن طريق الطرد المركزي لمدة 5 دقائق في XG 7500 في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> نضح بعناية وطاف والجافة وبيليه الحمض النووي الريبي لمدة 5-10 دقائق، والتأكد من الحمض النووي الريبي بيليه لم يجف تماما. في Nuclease خالية من المياه الملائمة لحجم بيليه إعادة حل. قياس تركيز الحمض النووي الريبي باستخدام مقياس الطيف الضوئي 1000 NanoDrop (الامتصاصية التدبير في 260 نانومتر و 280). </li><li style=";text-align:right;direction:rtl"> التحقق من جودة وسلامة عينات الحمض النووي الريبي باستخدام اجيلنت Bioanalyzer. </li></ol> 3. عكس نسخ من الحمض النووي الريبي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم إجراء النسخ العكسي من الحمض النووي الريبي باستخدام miScript كيت النسخ العكسي وفقا لتعليمات الشركة الصانعة (QIAGEN). هذه المجموعة تضمعكس المنتسخة و(A) بوليميريز بولي. والواق RT miScript تشمل المغنيسيوم 2 +، dNTPs، بنسبة ضئيلة من DT الاشعال، والبادئات العشوائية. </li><li style=";text-align:right;direction:rtl"> استخدام ما بين 10 و ص 1 ميكروغرام من الحمض النووي الريبي لتجميع [كدنا]. في حالة استخدام أكثر من 1 ميكروغرام من الحمض النووي الريبي، توسيع نطاق رد الفعل خطيا إلى حجم مناسب. </li><li style=";text-align:right;direction:rtl"> إعداد مزيج الرئيسي الذي يحتوي على 5X miScript RT العازلة (4 ميكرولتر)، miScript ميكس النسخ العكسي (1 ميكرولتر)، والمياه ريبونوكلياز خالية لجلب ردود الفعل على الحجم النهائي من 20 ميكرولتر. وتشمل أيضا الحمض النووي الريبي قالب (حتى 1 ميكروغرام) في مزيج الرئيسي. </li><li style=";text-align:right;direction:rtl"> احتضان هذه العينات لمدة 60 دقيقة في درجة حرارة 37 مئوية تليها مباشرة عن طريق حضانة لمدة 5 دقائق عند 95 درجة مئوية. لا يمكن أن يؤديها هذه الخطوة في جهاز PCR، تسخين كتلة، أو ماء الاستحمام. Thermocyclers هي الطريقة الأنسب والأكثر دقة. تخزين [كدنا] على الجليد على المدى القصير، و -20 درجة مئوية لمدة التخزين على المدى الطويل. </li></ol> 4. توليد منحنى قياسي <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> قبل experiment مع miRNAs الهدف، يتم إنشاء منحنى قياسي باستخدام cDNAs تركيزات المعروفة ضد نقاط العبور (اف ب) (الشكل 2). </li><li style=";text-align:right;direction:rtl"> إعداد سلسلة من التخفيفات من 2 أضعاف و 10 ضعفا، و 50 أضعاف، أضعاف-250، و 1250 أضعاف [كدنا] الأصلي للنموذج الذي هو معروف لدينا تعبير كبير من الجينات اهتمامك. </li><li style=";text-align:right;direction:rtl"> تشغيل PCR على النحو المحدد في المادة 5 &quot;في الوقت الحقيقي PCR للكشف عن ميرنا&quot;، مع التعديل الذي كدنا] في التخفيفات المسلسل ليس تخفيف ثابت 40X. </li><li style=";text-align:right;direction:rtl"> لتحليل باستخدام البرمجيات RelQuant (روش) لإنشاء منحنى المعيار الخاص بك. </li></ol> ملاحظة: يجب إنشاء منحنى جديد موحدة لكل جينة من الفائدة. 5. في الوقت الحقيقي PCR للكشف من ميرنا <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> تم تنفيذ حقيقي PCR الوقت لmiRNAs باستخدام miScript SYBR الأخضر PCR كيت والفحص التمهيدي miScript وفقا لتعليمات الشركة الصانعة (QIAGEN). إعداد مزيج رئيسية تحتوي على2X QuantiTect SYBR الأخضر ميكس ماستر PCR، 10X miScript التمهيدي العالمي، 10X الفحص التمهيدي miScript، والمياه ريبونوكلياز خالية. إعداد مزيج الرئيسي لحجم رد فعل 20 ميكرولتر. </li><li style=";text-align:right;direction:rtl"> والفحص التمهيدي هو محدد لميرنا من الفائدة. الى 10x إعادة الفحص التمهيدي miScript، منبذة القارورة لفترة وجيزة، وإضافة 550 ميكروليتر TE العازلة، ودرجة الحموضة 8.0. دوامة القارورة لفترة وجيزة لخلط والشعائل قسامة إلى أصغر وحدات التخزين، وتخزينها عند درجة حرارة -20 درجة مئوية. ويلزم اثنان الاشعال: الاشعال للجينات الهدف وهذا الجين هو مرجع RNU6B usedas الجين مرجع. </li><li style=";text-align:right;direction:rtl"> يخفف من [كدنا] aliquots اضافية 40X وتخزين عند درجة حرارة -20 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> [كدنا] بمثابة قالب لPCR. استخدام 2 ميكرولتر من [كدنا] المخفف 40X والاستغناء إلى 20 ميكروليتر ضوء cycler الشعيرات الدموية (روش). </li><li style=";text-align:right;direction:rtl"> إضافة 18 ميكرولتر من مزيج الرئيسي إلى كل شعري، وأجهزة الطرد المركزي باستخدام محول الشعرية. </li><li style=";text-align:right;direction:rtl"> وضع الشعيرات الدموية في cycler في الوقت الحقيقي القائم الشعرية، مثل LightCycler 3.5 نظام PCR في الوقت الحقيقي مع شكل دائري 32-الشعرية. </li><li style=";text-align:right;direction:rtl"> تشغيل البرنامج الدراجات PCR على النحو التالي: لتفعيل البوليميراز HotStarTaq التي هي في 2X QuantiTect SYBR الأخضر ميكس ماستر PCR، قبل احتضان عند 95 درجة مئوية لمدة 15 دقيقة. تليها 50 دورة من: تمسخ، 15 ثانية و 94 درجة مئوية؛ الصلب، 30 ثانية و 55 درجة مئوية؛ تمديد، 30 ثانية، و 70 درجة مئوية. </li><li style=";text-align:right;direction:rtl"> اختيار عينة ليكون تدريج، وتعيين قيمته هدف تطبيع إلى 1. قارن التعبير النسبية لميرنا في جميع العينات الأخرى إلى تدريج. </li></ol> ملاحظة: ضمن دراسة، وينبغي استخدام نفس العينة معايرة للحفاظ على اتساق النتائج. 6. تحليل البيانات <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> وصفت منحنيات التضخيم لردود الفعل PCR بالرسوم والصور وعدديا بواسطة الجزيئية الكيميائية الحيوية LightCycler إصدار برامج 3.5 (روش). قياس ردود الفعل في علامة التبويب &quot;الكمي&quot;، وهوالثانية وتصدير البيانات إلى ملف نصي. </li><li style=";text-align:right;direction:rtl"> استيراد البيانات إلى برنامج التحليل RelQuant (روش) لتوليد نتائج القياس الكمي. استيراد ملفات منفصلة عن الجينات المستهدفة، والجينات المرجعية، والبيانات منحنى القياسية. </li><li style=";text-align:right;direction:rtl"> تحديد موقف من المعاير لكل من الهدف والجينات مرجع. أيضا تحديد المواقف من العينات. وأعرب عن البيانات كهدف للرجوع نسبة من عينات مختلفة مقسوما على نسبة إشارة إلى هدف للتدريج. يستخدم منحنى معيار ولدت من قبل لميرنا وخاصة الجينات التدبير المنزلي كمعيار مرجعي لاستقراء البيانات الكمية للأهداف ميرنا تركيزات غير معروف. </li><li style=";text-align:right;direction:rtl"> ثلاث نسخ مكررة من تحليل العينات كمجموعة ويعني يحسب تركيزات والانحرافات المعيارية من ثلاث نسخ. إذا كان أحد triplicates غير متناسقة مع بقية المجموعة، سوف يتم استبعاده من قبل البرنامج. </li></ol> 7. ممثل النتائج &lt;ويرد ف الطبقة = &quot;jove_content&quot;&gt; مثال لتحليل qPCR على عينات من البروستاتا في الشكل 3. وصفت النتائج عدديا، وكذلك بالرسوم والصور. الرسوم البيانية التي تبين مستويات التعبير عن الجينات إشارة، U6، تبدأ التضخيم الهائل في حوالي 20 دورة، في حين أن التعبير عن الجينات المستهدفة، ومير 98، وأظهرت تأخر ما يقرب من التضخيم في دورة 25. وقد تم تصدير البيانات من هذه التجربة في ملف نص وتحليلها بواسطة برنامج التحليل RelQuant. يتم تحديد المواقف من الشعيرات الدموية التي تحتوي على تدريج والعينات. الشكل 4 يوضح كيف يتم تعيين تدريج لكي تكون 1، والتعبير عن عينات أخرى بالنسبة للتدريج. <img src="/files/ftp_upload/3874/3874fig1.jpg" alt="الشكل 1"/> الشكل 1. الخطوات المختلفة في miScript العكسية النسخ والاسترداد في الوقت الحقيقي. <img src="/files/ftp_upload/3874/3874fig2.jpg" alt="الشكل 2"/> الشكل 2. يتم إنشاء منحنى قياسي باستخدام سلسلة من التخفيفات من 2 أضعاف و 10 ضعفا، و 50 أضعاف، أضعاف-250، و 1250 أضعاف العينة الأصلي [كدنا]. <img src="/files/ftp_upload/3874/3874fig3.jpg" alt="الشكل (3)"/> الشكل 3. روش LightCycler الكيميائية الحيوية الجزيئية برامج يظهر المعلومات الكاملة من التجربة بالرسوم والصور والنص من قبل. وتظهر زيادة كمية التضخيم PCR في الوقت الحقيقي والمؤامرات في مضان من عينات مختلفة. <img src="/files/ftp_upload/3874/3874fig4.jpg" alt="الشكل 4"/> وتم قياس كمية الشكل 4. البيانات باستخدام برامج التحليل RelQuant LightCycler. عادة، ثلاثة مكررات من عينات يتم تحليلها كمجموعة والعينات التي تنتج يتم استبعاد نتائج غير متناسقة بشكل واضح ويعني يحسب تركيزات والانحرافات المعيارية من ثلاث نسخ.

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الإعلان عن أي تضارب في المصالح.

وقد تم التعبير عن الشاذة من بعض miRNAs وجدت على الدوام في أورام البروستات بالمقارنة مع الأنسجة الطبيعية 10، والذين وردت أسماؤهم بعض هذه miRNAs كعوامل محتملة علاجية جديدة ضد سرطان البروستاتا 11. لذلك نجد أن مستويات التعبير المنحرف من miRNAs تكون مفيدة المؤشرات الحيو...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> اسم كاشف </td><td> شركة </td><td> فهرس العدد </td></tr><tr><td> TRIzol الكاشف </td><td> إينفيتروجن </td><td> 15596 </td></tr><tr><td> miScript كيت النسخ العكسي </td><td> QIAGEN </td><td> 218061 </td></tr><tr><td> فحوصات التمهيدي miScript </td><td> QIAGEN </td><td> تجربة محددة </td></tr><tr><td> miScript SYBR الأخضر PCR كيت </td><td> QIAGEN </td><td> 218073 </td></tr><tr><td> LightCycler 3.5 نظام PCR في الوقت الحقيقي </td><td> روش </td><td></td></tr><tr><td> ضوء Cycler الأوعية الشعرية </td><td> روش </td><td> 04929292001 </td></tr><tr><td> NanoDrop 1000 طيفي </td><td> الحرارية العلمية </td><td> 2538 </td></tr><tr><td> اجيلنت 2100 Bioanalyzer </td><td> G2943CA </td><td></td></tr></tbody></table>

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

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

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

الكمية بوليميريز سلسلة من ردود الفعل في الوقت الحقيقي (qPCR) هو وسيلة سريعة وحساسة للتحقيق في مستويات التعبير عن مختلف microRNA (ميرنا) الجزيئات في عينات الورم. ويمكن استخدام هذا التعبير أسلوب مئات من جزيئات ميرنا مختلفة يتم تضخيمها، كميا، وتحليلها من القالب نفسه [كدنا].

الكشف microRNA في أورام البروستات بواسطة PCR الوقت الحقيقي الكمية (qPCR)

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

24.4K Views.  University of Toronto. الكمية بوليميريز سلسلة من ردود الفعل في الوقت الحقيقي (qPCR) هو وسيلة سريعة وحساسة للتحقيق في مستويات التعبير عن مختلف microRNA (ميرنا) الجزيئات في عينات الورم. ويمكن استخدام هذا التعبير أسلوب مئات من جزيئات ميرنا مختلفة يتم تضخيمها، كميا، وتحليلها من القالب نفسه [كدن?...

Video: 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.

متعدد الأطياف في الوقت الحقيقي التصوير الإسفار عن الكشف عن جراحة داخلية من الحارس العقدة الليمفاوية في امراض النساء والاورام

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 ...

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.

في الوقت الحقيقي المعاوقة الكهربائية تقنية تستند إلى قياس غزو أحادي الطبقة الخلية البطانية من قبل خلايا السرطان

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.

تقييم امتصاص الجسيمات النانوية في الأورام في الوقت الحقيقي باستخدام التصوير Intravital

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.

كشف وGenogrouping من Noroviruses من براز الأطفال وذلك في خطوة واحدة Taqman 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.

تعيين تباين التعبير البروتين في الأورام باستخدام المناعي الكمي

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

تحديد<em&gt; الجمال النائم</em&gt; الإدراج ينقول في الأورام الصلبة باستخدام رابط بوساطة PCR

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.

حركة إعادة التدريب في الوقت الحقيقي باستخدام ملاحظات من الأداء

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.

باستخدام الكمي في الوقت الحقيقي PCR لتحديد الجهات المانحة Engraftment خلية في التنافسية نموذج الفئران زرع نخاع العظم

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.

النخاعي السوائل عن طريق الرنا الميكروي التنميط الكمي في الوقت الحقيقي PCR

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.

في الوقت الحقيقي السمسة فحوصات في الدم الكامل بالبشر

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