Bu çalışma Kanada Kanser Topluluğu Araştırma Enstitüsü tarafından finanse edildi, hiçbir vermek. 019.038.

<html> 1. Prostat Örnek Toplama <ol><li> Prostatektomi zamanda prostat örnekleri toplamak. Numune anatomik noktalar kullanılarak yönlendirilmelidir edilir. Prostat ve seminal veziküllerin aşağıdaki gibi boyanmıştır: sağ tarafında yeşil, sol tarafında mavi. </li><li> Prostatın rastgele enine midsection, rektal yüzeyine dik alınan sıvı nitrojen içinde dondurulmuş ve -80 ° C 9 saklanır. </li><li> Örneklerin banked dilimleri, (, arka, ön sağ ve sol) odaklı, fotokopi quadrisected vardır. Bölüm Kriyostat kullanılarak kesilir. </li><li> Bölüm H &amp; E ile boyandı ve lekeli slaytlar ve ilgili resmin üzerine tümör karşısında, normal alanlarını belirlemek ve tanımlamak için bir patolog tarafından incelenir. Işaretli alanların RNA sonraki adımlarda çıkartılacaktır hangi tümör dokusu ayıklamak için hangi bölgelerini göstermek için bir kılavuz olarak kullanılır. </li></ol>&quot;break&gt; 2. Örneklerden miRNA dahil RNA, Ayırma <ol><li> Kuru buz ve belirginlik fotokopi atıfta koyun donmuş prostat örnekleri, prostat tümörü küçük bir bölümünü (50 ila 100 mg arasında) kesip. </li><li> TRIzol Reaktif 1 mL prostat tümör dokusu homojenize edilir. Aşağıdaki adımlarla miktarlarda TRIzol Reaktif 1 mL kullanımına dayanmaktadır. </li></ol> Not: Burada küçük izole Ancak diğer kitleri RNA içeren toplam RNA da kullanılabilir, ayıklanması RNA TRIzol Reaktif kullanmışlardır. <ol start="3"><li> Oda sıcaklığında, 5 dakika süreyle homojenize örnekleri inkübe. </li><li> Numunelere kloroform 0.2 mL ve 15 saniye boyunca kuvvetlice çalkalanır. , Oda sıcaklığında 3 dakika süreyle inkübe örnekleri, daha sonra 4 azından 15 dakika süreyle 12,000 x g&#39;de santrifüj ° C. </li><li> Taze tüplere renksiz bir üst sulu faz transfer, ve izopropil alkol ve 0.5 ml ilave edilir.Oda sıcaklığında 10 dakika inkübe örnekleri, daha sonra 4 azından 10 dakika süreyle 12,000 x g&#39;de santrifüj ° C. </li><li> RNA içeren pelet bozmadan süpernatant dikkatlice aspire. % 75 Etanol ve 1 mL RNA pelet yıkanır. Vorteks 4 az 7500 x g&#39;de 5 dakika için santrifüjleme ile örnek ve tortu yeniden ° C. </li><li> Dikkatle RNA pelet tamamen kuru olmamasını sağlayarak, 5-10 dakika yüzer ve kuru RNA pelet aspire. Pelet boyutuna uygun Nükleaz içermeyen su içinde yeniden çözülür. NanoDrop 1000 spektrofotometre (260 nm ve 280 nm&#39;de absorbans ölçü) kullanılarak RNA konsantrasyonu ölçülür. </li><li> Agilent Bioanalyzer kullanarak RNA örnekleri kalitesini ve bütünlüğünü kontrol edin. </li></ol> 3. RNA ters transkripsiyon <ol><li> RNA ters transkripsiyon üreticinin talimatlarına (Qiagen) &#39;ye göre miScript Ters transkripsiyon Kit kullanılarak gerçekleştirildi. Bu kit içerirtranskriptaz ve bir poli (A) tersine polimeraz. MiScript RT Tampon Mg 2 + dahil, dNTP, oligo-dT primerler ve rastgele primerler. </li><li> 10 pg ve cDNA sentez RNA 1 mikrogram arasında kullanın. RNA fazla 1 mikrogram kullanıyorsanız, uygun hacimde doğrusal reaksiyon büyütmek. </li><li> 20 ul nihai hacmi tepkiler getirmek için 5X miScript RT Buffer (4 ul) miScript Ters Transkripsiyon Mix (1 ul) ve RNaz içermeyen su içeren bir ana karışım hazırlayın. Ayrıca master miks şablonu RNA (en fazla 1 mg) içerir. </li><li> 37 azından 60 dakika süreyle inkübe örnekleri ° C&#39;de 95 ° C&#39;de 5 dakika süreyle bir inkübasyon hemen sonra Bu adımda blok ya da su banyosu ısıtılması, bir PCR makinesinde gerçekleştirilebilir. Thermocyclers en uygun ve doğru yöntemdir. Kısa vadede buz üzerinde cDNA saklayın ve -20 ° C ile uzun süreli depolama için. </li></ol> 4. Standart Eğri oluşturma <ol><li> Exper öncesindeHedef miRNA&#39;lar ile iment, bir standart eğriden kesişme noktalarında (CP) (Şekil 2) karşı bilinen konsantrasyonlarının cDNAlann kullanılarak üretilir. </li><li> 2 kat, 10 kat, 50 kat, 250 kat ve ilgi gen önemli bir ifadesi olduğu bilinen bir örnek 1250 kat orijinal cDNA dilüsyonları bir dizi hazırlayın. </li><li> Bölüm 5 &quot;miRNA tespiti için real-time PCR&quot; belirtilen cDNA seri dilüsyonlar statik bir 40x seyreltme olduğunu değişiklik ile, PCR çalıştırın. </li><li> Standart eğrisi oluşturmak için RelQuant yazılımı (Roche) kullanılarak analiz gerçekleştirin. </li></ol> Not: yeni bir standart eğri ilgi her gen için oluşturulan edilmelidir. 5.. MiRNA tespiti için real-time PCR <ol><li> MiRNA&#39;lar için Real Time PCR üreticinin talimatlarına (Qiagen) göre miScript SYBR Green PCR Kiti ve miScript Astar Testi kullanılarak yapıldı. Içeren bir master karışımı hazırlayın2x QuantiTect SYBR Green PCR Master Mix, 10x miScript Universal Astar, 10x miScript Primer Testi ve RNaz içermeyen su. 20 ul hacim reaksiyon için bir ana karışım hazırlayın. </li><li> Primer Assay ilgi miRNA özgüdür. Sulandırmak 10x miScript Astar Testi için, kısaca flakon santrifüj ve 550 ul TE tamponu, pH 8.0 ekleyin. -20 ° C&#39;de Vorteks karıştırmak için kısaca şişe, küçük birimlere kısım astar ve mağaza Iki primer gereklidir. Hedef geni ve referans gen için primerler RNU6B referans gen usedas olup. </li><li> -20 ° C&#39;de cDNA 40x ve mağaza ekstra hacme seyreltilir </li><li> cDNA PCR için kalıp olarak hizmet eder. 40x seyreltilmiş cDNA 2 ul kullanın ve 20 ul ışık cycler kılcal (Roche) ile akıtın. </li><li> Her kılcal için ana karışım 18 ul ekleyin ve bir kılcal adaptörü kullanarak santrifüj. </li><li> Böyle LightCycler 3 gibi kılcal tabanlı Gerçek Zamanlı cycler, içinde kılcal yerleştirin.32-kapiller atlıkarınca formatı ile 5 Real-Time PCR Sistemi. </li><li> Aşağıdaki gibi PCR bisiklet programını çalıştırın: 2x QuantiTect SYBR Green PCR ana Karışım içinde HotStarTaq polimerazı aktive etmek için, 95 azından önceden 15 dakika süreyle inkübe ° C. 50 döngüleri Ardından: Denatürasyon, 15 s, 94 ° C; Tavlama, 30 s, 55 ° C; Eklenti, 30 s, 70 ° C </li><li> Kalibratör olmak için bir örnek seçin ve 1 olarak normalize hedef miktarını ayarlayın. Kalibratörü tüm diğer örnekleri arasında miRNA bağıl ifade karşılaştırın. </li></ol> Not: Çalışma kapsamında, aynı kalibre numune sonuçlarının tutarlılığı korumak için kullanılmalıdır. 6. Veri Analizi <ol><li> PCR reaksiyonlar için Amplifikasyon eğrileri Moleküler Biyokimyasallar LightCycler Yazılım sürümü 3.5 (Roche) ile grafiksel ve sayısal olarak tasvir edilmektedir. Bir, &quot;nicelik&quot; sekmesinde reaksiyonları ölçmeknd bir metin dosyasına verileri. </li><li> Ölçümü sonuçları üretmek için RelQuant analiz yazılımı (Roche) veri alma. Hedef gen, referans gen ve standart eğri veri için ayrı dosyalar alın. </li><li> Hedef ve referans gen hem kalibratör pozisyonunu belirtin. Ayrıca, örnek pozisyonları belirtir. Veri kalibratörü referans oranı hedef bölünmesiyle farklı örnekleri arasında oranı referans hedef olarak ifade edilir. Daha önce belirli bir miRNA ve temizlik gen için oluşturulan standart eğrisi bilinmeyen konsantrasyonlarının miRNA hedefleri için nicel veri extrapolating için bir referans standart olarak kullanılır. </li><li> Örnekleri bir grup olarak analiz ve konsantrasyonları ve üç nüsha standart sapmaları hesaplanır demek olan üç çoğaltır. Kopyaların bir dizi geri kalanı ile tutarsız ise, bu program tarafından çıkartılacaktır. </li></ol> 7. Temsilcisi Sonuçlar &lt;p = sınıfı &quot;jove_content&quot;&gt; prostat numuneler üzerinde qPCR analizi için bir örnek Şekil 3 &#39;de gösterilmiştir. Sonuçlar hem grafiksel olarak, sayısal olarak tasvir edilmektedir. Hedef gen, miR-98, ekspresyon döngü 25 azından yaklaşık gecikmeli amplifikasyon gösterirken referans gen, U6 ifade seviyelerini gösteren grafikleri, yaklaşık 20 döngü azından üstel amplifikasyon başlar. Bu deneyden elde edilen veriler metin dosyası olarak ihraç ve RelQuant analiz yazılımı ile analiz edildi. Kalibratör ve örnekler içeren kılcal Pozisyonlar belirtilir. Şekil 4 kalibratör 1 olarak ayarlayın ve kalibratör göre diğer örneklerin ifadesi nasıl göstermektedir. <img src="/files/ftp_upload/3874/3874fig1.jpg" alt="Şekil 1"/> Şekil 1. MiScript ters transkripsiyon ve Real-time PCR de çeşitli adımlar. <img src="/files/ftp_upload/3874/3874fig2.jpg" alt="Şekil 2"/> Şekil 2. Standart eğri 2 kat, 10 kat, 50 kat, 250 kat, 1250 kat orijinal cDNA örnek dilüsyonlarının bir dizi kullanılarak oluşturulur. <img src="/files/ftp_upload/3874/3874fig3.jpg" alt="Şekil 3"/> Şekil 3. Roche Molecular Biyokimyasallar LightCycler Yazılım grafiksel deney ve metin ile tüm bilgileri gösterir. Kantitatif Real-time PCR parsellerde çeşitli örneklerden floresans artış gösterirler. <img src="/files/ftp_upload/3874/3874fig4.jpg" alt="Şekil 4"/> Şekil 4. Veri RelQuant LightCycler analiz yazılımı kullanılarak ölçüldü. Genellikle, üç grup ve açıkça tutarsız sonuçlar dışlanan üretmek ve konsantrasyonları ve üç nüsha standart sapmaları hesaplanır anlamına örnekleri olarak analiz edilir örneklerin çoğaltır.

<ol>
	<li>Reinhart, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+plants.">MicroRNAs in plants.</a> Genes Dev. 16, 1616-1616 (2002).</li><li>Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+microRNA+Mir-14+suppresses+cell+death+and+is+required+for+normal+fat+metabolism.">The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism.</a> Curr. Biol. 13, 790 (2003).</li><li>Bueno, M. J., Perez, d. e. C. a. s. t. r. o., I, ., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation+pathways+by+microRNAs.">Control of cell proliferation pathways by microRNAs.</a> Cell Cycle. 7, 3143-3143 (2008).</li><li>Friedman, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92 (2009).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116, 281-281 (2004).</li><li>Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+microRNA+dysregulation+in+cancer.">Causes and consequences of microRNA dysregulation in cancer.</a> Nat. Rev. Genet. 10, 704 (2009).</li><li>Coppola, V., De, M. R., Bonci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+prostate+cancer.">MicroRNAs and prostate cancer.</a> Endocr. Relat. Cancer. 17, 1-1 (2010).</li><li>Gordanpour, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-221+Is+down-regulated+in+TMPRSS2:ERG+fusion-positive+prostate+cancer.">miR-221 Is down-regulated in TMPRSS2:ERG fusion-positive prostate cancer.</a> Anticancer Res. 31, 403-403 (2011).</li><li>Nam, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+TMPRSS2:ERG+fusion+gene+predicts+cancer+recurrence+after+surgery+for+localised+prostate+cancer.">Expression of the TMPRSS2:ERG fusion gene predicts cancer recurrence after surgery for localised prostate cancer.</a> Br. J. Cancer. 97, 1690 (2007).</li><li>Ambs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+of+microRNA+and+messenger+RNA+reveals+deregulated+microRNA+expression+in+prostate+cancer.">Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer.</a> Cancer Res. 68, 6162 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-34a+inhibits+prostate+cancer+stem+cells+and+metastasis+by+directly+repressing+CD44.">The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.</a> Nat. Med. 17, 211 (2011).</li></ol>

Çıkar çatışması ilan etti.

Bazı miRNA&#39;lar anormal ifadeler, sürekli normal doku 10, ve bu miRNA&#39;lar bazı prostat kanserine karşı 11 potansiyel yeni terapötik maddeler olarak adlandırılmış olan ile karşılaştırıldığında prostat tümörleri bulunmuştur. Dolayısıyla miRNA anormal ekspresyon düzeyleri yararlı tanısal ve / veya prognostik biyolojik olabilir. Burada sunulan Real-Time qPCR metodolojisi prostat tümör dokusunda miRNA düzeylerinin tayini için daha kesin bir ölçüm sağlar. Kullanı...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> TRIzol Reaktif </td><td> Invitrogen </td><td> 15596 </td></tr><tr><td> miScript Ters Transkripsiyon Kiti </td><td> Qiagen </td><td> 218061 </td></tr><tr><td> miScript Astar Tahliller </td><td> Qiagen </td><td> Belirli Deney </td></tr><tr><td> miScript SYBR Green PCR Kiti </td><td> Qiagen </td><td> 218073 </td></tr><tr><td> LightCycler 3.5 Real-Time PCR Sistemi </td><td> Roche </td><td></td></tr><tr><td> Işık Cycler kılcal </td><td> Roche </td><td> 04929292001 </td></tr><tr><td> NanoDrop 1000 spektrofotometre </td><td> Thermo Scientific </td><td> 2538 </td></tr><tr><td> Agilent 2100 Bioanalyzer </td><td> G2943CA </td><td></td></tr></tbody></table>

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

MicroRNA (miRNA&#39;lar) tek iplikçikli, 18-24 nükleotid uzunluğunda, kodlamayan RNA molekülleri vardır. Onlar geliştirme 1, 2 apoptoz ve hücre döngüsü regülasyon 3 olmak üzere hemen her türlü hücresel işlemi katılmaktadırlar. MiRNA&#39;lar hedef haberci RNA (mRNA) 5 bağlanarak 4 insan genlerinin% 90% 30 ifadesi düzenleyen tahmin edilmektedir. MiRNA&#39;lar yaygın düzensizliği çeşitli hastalıklar ve kanser alt 6 rapor edilmiştir. Yaygınlığı ve benzersiz yapısı sayesinde, bu küçük moleküllerin biyolojik, tedavi ajanları ve / veya hedeflerin nesil olması muhtemeldir. MiRNA ifade araştırmak için kullanılan yöntemler SYBR yeşil Ben boya dayalı olarak Taqman-prob tabanlı qPCR içerir. MiRNA&#39;lar etkili bir klinik uygulamada kullanılacak olursa, taze ve / veya muhafaza klinik örneklerde bunların algılama, tekrar üretilebilir ve sp olması şarttırecific. qPCR yaygın tür mikroarray çalışmaları 7 gibi tüm genom analizlerinde miRNA&#39;lar ifade doğrulamak için kullanılmıştır. Bu protokolde kullanılan örnekleri klinik lokalize prostat kanseri için radikal prostatektomi yapılan hastaların oldukları gözlenirken, diğer doku ve hücre hatları ek dondurulmuş rezeksiyon sonrası sıvı azot vardı Prostat numuneler in ikame edilebilir. Klinik değişkenler ve her hasta için izlem bilgileri 8 sonraki analiz için toplandı. Prostat tümörü örneklerinde miRNA düzeylerinin belirlenmesi. Tümörlerin qPCR analizinde temel adımları şunlardır: miRNA-spesifik primerler kullanılarak total RNA ekstraksiyonu, cDNA sentezi ve qPCR ürünlerinin tespiti. MRNA, miRNA ve diğer küçük RNA içerir RNA, TRIzol reaktif kullanılarak örneklerden çıkarıldı. Qiagen Kullanıcı miScript Sistemi cDNA sentez ve qPCR (Şekil 1) gerçekleştirmek için kullanılmıştır. Endojen miRNA&#39;lar po değildirTers transkripsiyon işlemi sırasında bu nedenle, lyadenylated, bir poli (A) polimeraz miRNA polyadenylates. MiRNA oligo-dT ve Reverse Transcriptase kullanılarak cDNA sentezi için bir şablon olarak kullanılmıştır. Oligo-dT primerlerin 5 &#39;ucunda bir evrensel bir etiket dizisi PCR adımda cDNA&#39;sının çoğaltılmasında kolaylaştırır. PCR ürünü amplifikasyon SYBR Green, çift sarmallı DNA&#39;ya intercalates bir boya tarafından yayılan floresan düzeyi tespit edilir. Evrensel etiketi dizisi bağlanan bir Universal Astar ile birlikte Özgül miRNA astar, özel miRNA dizileri yükseltmek olacaktır. MiScript Primer Tahliller binin üzerinde insana özgü miRNA&#39;lar ve fare özgü miRNA&#39;lar yüzlerce mevcuttur. Nispi miktar yöntemi miRNA&#39;lar ifadesi ölçmek için kullanılmıştır. Farklı örnekleri arasında değişkenlik düzeltmek için, bir hedef miRNA ifade seviyelerini bir referans genin ekspresyon düzeyleri normalize edilir. Bir GE seçimine analiz Göreceli ölçüm yöntemi kritik hedeflerinin ifade normalleştirmek için olduğu açıktır. Genellikle bu kapasite kullanılan referans gen örnekleri bunlar stabil en örnekleri üzerinden ifade edilmesi olarak kabul edildiğinden, küçük RNA RNU6B, RNU44 ve RNU48 vardır. Bu protokolde, RNU6B referans gen olarak kullanılır.

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

Kantitatif Real Time PCR (qPCR) tümör örneklerinde çeşitli mikroRNA ifade seviyeleri (miRNA) molekülleri araştırmak için hızlı ve duyarlı bir yöntemdir. Farklı miRNA moleküller yüzlerce bu yöntem ifadesi kullanılarak, amplifiye miktarı, ve aynı cDNA şablon ile analiz edilebilir.

Kantitatif Gerçek-zamanlı PCR (qPCR) tarafından Prostat tümörlerinde mikroRNA Algılama

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

microrna-detection-prostate-tumors-quantitative-real-time-pcr

Research

JoVE Journal

Medicine

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

24.4K Görüntüleme.  University of Toronto. Kantitatif Real Time PCR (qPCR) tümör örneklerinde çeşitli mikroRNA ifade seviyeleri (miRNA) molekülleri araştırmak için hızlı ve duyarlı bir yöntemdir. Farklı miRNA moleküller yüzlerce bu yöntem ifadesi kullanılarak, amplifiye miktarı, ve aynı cDNA şablon ile analiz edilebilir.

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.

Jinekolojik Onkoloji Sentinel lenf nodu intraoperatif Algılama için Multispektral Gerçek zamanlı Floresans Görüntüleme

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 ...

Tıp

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.

Kanser Hücreleri Endotel Hücre Tek tabakalı Invasion Tedbir Gerçek zamanlı Elektrik Empedans Tabanlı Tekniği

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 Görüntüleme Gerçek Zamanlı tümörler Nanopartikül Alımı Değerlendirme

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.

Taqman By Çocuk Tabureler gelen Noroviruses tespiti ve Genogrouping Tek aşamalı 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.

Kantitatif İmmünofloresan kullanarak Tümörler Protein İfade heterojenite Haritalama

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

Tanımlanması<em&gt; Sleeping Beauty</emLinker-aracılı PCR kullanarak Solid Tümörlerde&gt; Transposon Eklemeler

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.

Hareket Performans Gerçek zamanlı geri bildirim ile yeniden eğitimi

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.

Rekabetçi bir mürin Kemik İliği Transplantasyon Modelinde Donör Hücre Aşı belirleme Kantitatif Gerçek-zamanlı PCR kullanarak

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.

Kantitatif Real Time PCR kullanarak Beyin Omurilik Sıvısı MicroRNA Profil

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.

İnsan Tüm Kan gerçek zamanlı sitotoksisite deneyleri

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