Biz onun Real Time PCR makine kullanımı için Dr Charles Greenberg ederim. Bu çalışma MUSC Hollings Kanser Merkezi Başlangıç ​​Fonu, Hollings Kanser Merkezi ACS IRG, ASCO Kanseri Vakfı Kariyer Geliştirme Ödülü Conquer, NIH 1K08HL 103780-01A1 ve NIH 3P30CA138313-01S3 tarafından desteklenmektedir. Içeriği sadece yazarların sorumluluğundadır ve mutlaka Sağlık veya diğer finansman ajanlar Ulusal Enstitüleri resmi görüşlerini temsil etmemektedir.

1. Kemik İliği Hücre İzolasyonu <ol><li> Erkek donör FVB / NJ fareler ve servikal dislokasyon ile takip CO2 yöntemi kullanılarak dişi FVB / NJ fareler Euthanize. Dişi FVB / NJ kemik iliği hücreleri rekabetçi hücreler olarak kullanılır. </li><li> Farelerin femur ve tibiaes üzerinden incelemek ve% 5 ısı inaktive FBS ile 6 ml buz-soğuk RPMI1640 içeren 60 mm doku kültür kaplarına koyun, küçük bir makas ve forseps kullanın. Kas ve diğer dokuları kaldırmak için Kimwipe doku kullanın. Her bir çanak içinde kemik şaftın her iki ucuna kes. </li><li> 3 cc şırınga 23G iğne ile kemik ucunu, yemeğin içine% 5 ısı inaktive FBS ile RPMI1640 ile kemik iliği dışarı floş. Aynı iğne kullanılarak tekrarlanmalıdır özlemleri tarafından kemik iliği doku Ayrık. 15 ml&#39;lik bir santrifüj tüpüne hücre süspansiyonu aktarın. </li><li> 400 x g&#39;de 5 dakika için hücreler aşağı Spin, süpernatanın kaldırmak (155 mM p oda sıcaklığında kırmızı kan hücresi lizis tamponu, 1 ml hücre tekrar süspansiyonotassium bikarbonat, 10 mM amonyum klorid, EDTA, pH = 7.4, 0.1 mM) ve 5 dakika boyunca oda sıcaklığında inkübe edilir ve sonra% 5 ısı ile aktifliği giderilmiş FBS içeren RPMI 1640 5-10 ml eklenir. </li><li> Bir hücre süzgecinden hücreleri geçirir. Yeni bir tüp aracılığıyla akış toplayın. 400 x g&#39;de 5 dakika boyunca aşağı spin. Süpernatantı; hücre pelletini herhangi bir kırmızı renk içermemelidir. Kırmızı renk yokluğu, kırmızı kan hücrelerinin bir tam olarak çıkarılması gösterir. % 5 ısı ile aktifliği giderilmiş FBS RPMI1640 10 ml hücre pelet yeniden süspanse edin. Emin olmak için yavaşça girdap hücre süspansiyonu tamamen üniforma olduğunu. Bir kısım alın ve bir hemasitometre hücreleri saymak. Hesapla ne kadar kemik iliği transplantasyonu ve kısım yeterli hücre için gerekli hücrelerin hacim ve 05:02 oranında rakip dişi hücreleri erkek donör hücreleri karıştırın. 400 x g&#39;de 5 dakika boyunca aşağı Spin, 2, 5 x 10 6 / ml, ve rakip hücrelerini verici hücrelerin nihai konsantrasyon ile PBS içinde, PBS ile tekrar süspansiyon yıkama x 10 6 </sup&gt; / ml. </li></ol> 2. Rekabetçi Kemik İliği Transplantasyonu <ol><li> Kemik iliği nakli öncesi 11Gy 4-6 saat tek bir dozda 137Cs gama ışınları radyatör ile saçmak dişi alıcı fareler (yaş 8-12 hafta). </li><li> Bir fare süzgeç olarak ışınlanmış dişi fareler yerleştirin. Her fare 5 × 10 5 donör hücreleri ve 2 × 10 5 yarışmacı kemik iliği hücreleri aldığı gibi toplam hacminin 0,1 ml kuyruk veni yoluyla karışık donör ve rakip hücreleri enjekte edilir. </li></ol> 3. Örnek Toplama Çift hafta kemik iliği transplantasyonu sonrası, anestezi koşulu altında retroorbital kanama kadın alıcı fare periferal kan örnekleri (~ 50 ul) toplamak. Örnekleri EDTA kaplı tüpler içerisine toplanır. BM örnekleri de açıklandığı önceki aynı prosedürleri (Adım 1) ile, genellikle en az 4 ay transplantasyon sonrası, deney sonunda tahsil edilebilir; genellikle% 20-401 tibiada BM hücrelerinin DNA yeterli miktarda yapmak için yeterli. Kan veya BM örneklerinde yaş eşleştirilmiş normal dişi ve erkek farelerde de standart eğrisi hazırlamak için toplanır. 4. Genomik DNA İzolasyonu <ol><li> Her kan örneği oda sıcaklığında RBC lizis tamponu 4 × hacmi (~ 200 ul) ekleyin. İyice karıştırın ve 5 dakika oda sıcaklığında inkübe edin. PBS 1 ml ekleyin, daha sonra parçalanan RBC çoğunu kaldırmak için aşağı doğru döndürün. </li><li> Bir kan DNA ekstraksiyon kiti (QIAmp DNA Kan ekstraksiyon kiti) kullanılarak DNA izole edin. 37 Pre-sıcak elüsyon tampon (AE) ° C Elüsyonlanan DNA verimi artırmak için. Standart eğri için Erkek ve kadın DNA&#39;ları benzer izole edilmiştir. </li><li> (İsteğe bağlı) Genomik DNA, daha ileri saflaştırıldı ve 3 M sodyum asetat (pH = 5.5) varlığında, EtOH çökeltme yöntemi kullanılarak konsantre edilebilir. DNA pelletleri hemen sonra analiz için 40-50 ul damıtık su içinde tekrar süspanse edilir veya ileride kullanılmak üzere -20 ° C&#39;de saklanır. </li><li> DNA concentr ölçünNanodrop ND-1000 spektrumları fotometre ile ation. OD 260 / 1.8-2.0 arası 280 numuneler daha fazla analiz için kullanılır. </li><li> 50 ul toplam hacim içinde 4ng/μl için DNAase serbest H2O ile DNA seyreltin. </li></ol> 5. Standart Eğri Hazırlama 4 ng / ml &#39;lik bir konsantrasyon ile DNA erkek ve dişi DNA seyreltilir, ve Table1 göre DNA karışım yapmak <table border="1"><tbody><tr><td> Kadın Arkaplan erkek DNA&#39;sının% </td><td> 4ng/μl erkek DNA&#39;sının hacmi (ul) </td><td> 4ng/μl kadın DNA hacmi (ul) </td><td> Toplam hacim (ul) </td></tr><tr><td> 0.2 </td><td> 1 </td><td> 499 </td><td> 500 </td></tr><tr><td> 0.5 </td><td> 1 </td><td> 199 </td><td> 200 </td></tr><tr><td> 2.5 </td><td> 5 </td><td> 195 </td><td> 200 </td></tr><tr><td> 12.5 </td><td> 25 </td><td> 175 </td><td> 200 </td></tr><tr><td> 50 </td><td> 100 </td><td> 100 </td><td> 200 </td></tr><tr><td> 87.5 </td><td> 175 </td><td> 25 </td><td> 200 </td></tr><tr><td> 100 </td><td> 200 </td><td> 0 </td><td> 200 </td></tr></tbody></table> Standart eğri için Tablo 1. Numune hazırlama. 8-12wks çağındaki Normal erkek ve dişi FVB / NJ fareler sakrifiye edildi. Kan ve BM hücrelerinin toplanmıştır. Erkek ve dişi hücreler hücrelerden DNA 4ng/μl &#39;lik bir konsantrasyonda izole edildi ve yeniden süspansiyon haline getirildi. Erkek ve dişi DNA&#39;lar DNA standardının örneği karışımları üretmek için çeşitli oranlarda karıştırılmıştır. 6. Real-time PCR <ol><li> Astar ve genomik DNA ile SYBR Green Supermix reaktifi karıştırılarak PCR reaksiyonu plakasını yukarı ayarlayın. Reaksiyon hacmi (20 ul), her reaksiyon bölgesindeki her bir primer ve kan hücresi genomik DNA (4ng/μl x5 ul = 20 ng) 5 ul 400 nM içerir. DNA samples ve standartları üç tekrarlı olarak kurulmuştur. Primerlerin dizileri Tablo 2 &#39;de gösterilmiştir. </li></ol><table border="1"><tbody><tr><td> Gene adı </td><td> Ileri </td><td> Ters </td></tr><tr><td> Bcl2 </td><td> 5&#39;-AAGCTGTCACAGAGGGGCTA </td><td> 5&#39;-CAGGCTGGAAGGAGAAGATG </td></tr><tr><td> Zfy1 </td><td> 5-TGGAGAGCCACAAGCTAACCA </td><td> 5&#39;-CCCAGCATGAGAAAGATTCTTC </td></tr></tbody></table> Mürin Bcl2 ve Zfy1 için Tablo 2. RT-PCR primer sekans. <ol start="2"><li> Aşağıdaki koşullara BioRad iQ5 PCR makinesi kullanılarak PCR reaksiyonu gerçekleştirin: 95 ° C&#39;de 3 dakika, 95 42 amplifikasyon döngüleri ° 10 sn için C, 58 ° 15 sn 25 sn ve 72 ° C&#39;de, bir erime eğrisi izledi adım. </li><li> Bio-Rad iQ5 2.1 Standard Edition Optik Sistem tarafından Cycle threshold (Ct) değerleri elde edilir. Hesapla δCt (Ct Zfy1 &lt;/) Değeri&gt;-Ct Bcl2 sup ve Zfy1 ekspresyon seviyesi 2-δ Ct değeri olarak hesaplanır. </li><li> Bilinen kadın / erkek standart karışımlarından 2-δCt okuma kullanarak her reaksiyon serisi için standart eğrileri oluşturmak. Standart eğriler doğrusal regresyon uydurma ile karışım içinde% bilinen erkek DNA, kopyalardaki 2-δCt değer ortalama çizim tarafından oluşturulur. </li></ol>

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Biz hiçbir mali çıkarlarının ifşa rekabet var.

Mevcut çalışmanın amacı, izleyicilere rekabetçi bir kemirgen kemik iliği nakli modelinde donör hücre uyumunu ölçmek için PCR temelli bir yöntem sağlamaktır. Çeşitli çalışmalarda, transplantasyonu modelleri 9-10 donör hücrelerini saptamak için RT-PCR kullanılarak bildirilmiştir. Dr Schwarzenberger grubunun ilk y kromozomu özgü 8 yükseltmek için real-time PCR kullanarak bir kemirgen kemik iliği nakli modeli geliştirilmiştir. Bu yöntem, HSC etkinlik 11 Gli-1...

Mürin kemik iliği (Kİ) nakli model ilk 1960 1 geliştirilmiştir. Bu model yaygın donör hematopoetik kök hücre (HSC) bir ana alıcı fare biyoloji çalışma için kullanılır olmuştur. Mürin kemik iliği nakli modeli HSC fonksiyonları ve düzenlenmesi ile ilgili değerli bilgi ile bize sağladı ve HSC araştırma vazgeçilmezdir. C56Bl/6J CD45.2-B6.SJL CD45.1 gibi C57BL/6J H2b-Balb / C H2d veya congenic nakli modeli gibi Allojenik kemik iliği nakli modelinin etkisi, HSC aktivitesi 2 geninin işlevini incelemek için birçok laboratuarlarda kullanılmaktadır graft-versus-host hastalığı (GVHH) 4 olarak HSC fonksiyonu 3 veya transplantasyon ilişkili hastalıklar üzerinde ilaç tedavisi. Bu tür MHC haplotip veya CD45.1 gibi hücre yüzey belirteçleri yaygın olarak alıcı kökenli hücrelerden ayırt verici türetilmiş hücreler için kullanılmaktadır. C57BL/6J H2b, CD45.2 </sudonör hücre katkısı kolayca CD45.1 vs CD45.2 veya H2b vs ölçüm akım sitometri ile tespit edilebilir, çünkü p&gt;, Balb / C H2d ve B6.SJL CD45.1 kemik iliği nakli en yaygın olarak kullanılan fare suşları vardır H2d. Bununla birlikte, bu tür FVB / NJ 5 ve C3H gibi birçok diğer bakteriler aynı zamanda sık sık genetik mühendisliği transjenik fareler veya nakavt üretmek için kullanılır. Bu fareler, doğuştan bir hat için backcrossed ve bir karışık MHC / genetik arka plan içinde muhafaza edilebilir. Donör spesifik hücre yüzey belirteçleri mevcut olmayabilir bu durumda, donör hücre uyumunu ve HSC fonksiyonu belirlenmesi zor olabilir. Sex-uyumsuz kemik iliği nakli güney blot donör erkek hücrelerini saptamak için Y-kromozomu-spesifik DNA prob kullanarak ilk Dr Miwa grubu 6 tarafından geliştirilmiştir. Daha sonra, cinsiyet belirleyici bölge Y için bir gerçek-zamanlı PCR ma miktarının belirlenmesi için bir doğru ve büyük ölçüde belirli bir yöntem olduğu bulundumaternal kan sistemi 7 le fetal hücreler. Bu kavram, donör hücre uyumunu 8 belirlemek için bir kemirgen kemik iliği nakli modelinde, bir gerçek-zamanlı PCR tekniğinin geliştirilmesi için Dr Schwarzenberger grubu tarafından adapte edilmiştir. Biz daha FVB / NJ fare kemik iliği nakli modelinde donör hücre engraftman ölçümü için bu yöntemi değiştirilmiş. Bu yöntem, şu anda geniş olarak HSC biyoloji Pim1 kinaz rolünü incelemek için, grup halinde kullanılmaktadır.

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td></tr><tr><td> RPMI 1640 </td><td> Hyclone </td><td> SH30255.01 </td><td></td></tr><tr><td> FBS </td><td> Invitrogen </td><td> 16140-017 </td><td> Inaktive Isı </td></tr><tr><td> Amonyum klorid </td><td> MP Biomedicaals </td><td> 194806 </td><td></td></tr><tr><td> Potasyum Bikarbonat </td><td> Balıkçı </td><td> P184-500 </td><td></td></tr><tr><td> QIAamp DNA Kan MINIKIT </td><td> Qiagen </td><td> 51106 </td><td></td></tr><tr><td> Hücre süzgeç </td><td> Biyobilim BD </td><td></td><td></td></tr><tr><td> iQ SYBR Green Supermix </td><td> BioRad </td><td> 170-8882 </td><td></td></tr><tr><td> iQ5 gerçek zamanlı PCR makinesi </td><td> BioRad </td><td></td><td></td></tr><tr><td> Spectra fotometre </td><td> ND-1000 Nanodrop </td><td> ND-1000 </td><td></td></tr></tbody></table>

using quantitative real-time pcr to determine donor cell engraftment in a competitive murine bone marrow transplantation model

Mürin kemik iliği nakli modelleri hematopoetik kök hücre (HSC) fonksiyonları ve belirleyici gen / HKH&#39;lerin düzenleyen molekülleri ölçen önemli bir araç sağlar. Bu transplant modeli sistemlerinde HKH&#39;lerin fonksiyonu sokmak ve sulandırmak öldürücü ışınlanmış alıcı fareler için bu hücrelerinin yeteneği belirlenir. Genellikle, verici katkı hücre / melezleşmesi akış sitometrisi kullanılarak özel bir verici-hücre yüzey proteinleri, antikorlar tarafından ölçülür. Ancak, bu yöntem ağır özgüllük ve tüm fare suşları için geçerli olmayabilir alıcının kaynaklı hücreler, gelen donör kaynaklı hücreler ayırt etmek için hücre yüzey belirteci yeteneğine bağlıdır. Piyasada genetiği değiştirilmiş fare suşlarının çeşitli geçmişlere dikkate alındığında, bu hücre yüzey / akım sitometri tabanlı yöntem özellikle congenic alıcı ce ayrı donör hücreleri iyi tanımlanmış yüzey belirteçleri yoksun fare suşları önemli sınırlamaları vardırlls. Burada, transplant alıcıları farelerde donör hücre engraftman / katkısını belirlemek için PCR-temelli teknik bildirdi. Biz lethally ışınlanmış congenic dişi farelere erkek donörün kemik iliği HKH&#39;lerin nakledilen. Periferal kan numuneleri farklı zaman noktalarında, nakil sonrası toplandı. Kemik iliği örnekleri deneyler sonunda elde edildi. Genomik DNA izole edildi ve Y kromozom özgü gen, Zfy1, kantitatif Gerçek zamanlı PCR ile amplifiye edildi. Dişi alıcı farelerde erkek donör kaynaklı hücrelerinin aşılanması erkek vs kadın DNA&#39;ların bilinen yüzdesi, standart bir eğriye karşı hesaplanır edildi. Bcl2 toplam DNA miktarının normale döndürmek için bir gen referans olarak kullanılmıştır. Bizim verilerimiz bu yaklaşım güvenilir donör hücre uyumunu belirler ve fare kemik iliği nakli modelleri hematopoetik hücre sulandırma ölçmede yararlı, henüz basit bir yöntem sağlar önerdi. Bizim yöntemi rutin en laboratuarlarda yapılabilir çünkü hiçbirgibi flow sitometri gibi pahalı ekipman gereklidir.

Şekil 1 ve 2 standart eğri örnekleri erkek DNA yüzdeleri karşı 2-δCt ortalama değerleri ile çizilen gösterdi. Şekil 1A, sırasıyla 78.5 ° C&#39;de ve 88.5 ° C&#39;de lokalize Bcl2 ve Zfy1 amplikonlar için spesifik erime sıcaklığı göstermiştir. Bcl2 her bir PCR reaksiyonu içinde yüklenmiş DNA&#39;nın toplam miktarı normalleştirmek için bir referans gen olarak kullanılır. Her standart numune için Bcl2 amplifikasyon eğrileri (Log görünümü) yü...

Watch this Scientific Journal Video about Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model at JoVE.com

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

Donör hücre engraftman belirleme iyi tanımlanmış fenotipik belirteçlerin yoksun fare kemik iliği nakli modelleri bir meydan okuma sunuyor. Biz kadın transplant alıcı fareler erkek donör hücre uyumunu ölçmek için bir metodoloji tanımlamıştır. Bu yöntem, HSC fonksiyonların çalışması için tüm fare suşu olarak da kullanılabilir.

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 ...

using-quantitative-real-time-pcr-to-determine-donor-cell-engraftment

Research

JoVE Journal

Medicine

15.3K Görüntüleme.  Medical University of South Carolina. Donör hücre engraftman belirleme iyi tanımlanmış fenotipik belirteçlerin yoksun fare kemik iliği nakli modelleri bir meydan okuma sunuyor. Biz kadın transplant alıcı fareler erkek donör hücre uyumunu ölçmek için bir metodoloji tanımlamıştır. Bu yöntem, HSC fonksiyonların çalışması için tüm fare suşu olarak da kullanılabilir.

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

A Mouse Model of in Utero Transplantation

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus. For example, in utero transplantation (IUT) of hematopoietic stem cells has been used to successfully treat patients with severe combined immunodeficiency.1,2 In several other conditions, however, IUT has been attempted without success.3 Given these mixed results, the availability of an efficient non-human model to study the biological sequelae of stem cell transplantation and gene therapy is critical to advance this field. We and others have used the mouse model of IUT to study factors affecting successful engraftment of in utero transplanted hematopoietic stem cells in both wild-type mice4-7 and those with genetic diseases.8,9 The fetal environment also offers considerable advantages for the success of in utero gene therapy. For example, the delivery of adenoviral10, adeno-associated viral10, retroviral11, and lentiviral vectors12,13 into the fetus has resulted in the transduction of multiple organs distant from the site of injection with long-term gene expression. in utero gene therapy may therefore be considered as a possible treatment strategy for single gene disorders such as muscular dystrophy or cystic fibrosis. Another potential advantage of IUT is the ability to induce immune tolerance to a specific antigen. As seen in mice with hemophilia, the introduction of Factor IX early in development results in tolerance to this protein.14 
In addition to its use in investigating potential human therapies, the mouse model of IUT can be a powerful tool to study basic questions in developmental and stem cell biology. For example, one can deliver various small molecules to induce or inhibit specific gene expression at defined gestational stages and manipulate developmental pathways. The impact of these alterations can be assessed at various timepoints after the initial transplantation. Furthermore, one can transplant pluripotent or lineage specific progenitor cells into the fetal environment to study stem cell differentiation in a non-irradiated and unperturbed host environment.
The mouse model of IUT has already provided numerous insights within the fields of immunology, and developmental and stem cell biology. In this video-based protocol, we describe a step-by-step approach to performing IUT in mouse fetuses and outline the critical steps and potential pitfalls of this technique.

A Mouse Model of in Utero Transplantation

The mouse model of in utero transplantation is a versatile tool that can be used to study the potential clinical applications of stem cell transplantation and gene therapy in the fetus. In this protocol, we present a general approach to performing this technique

Bir Fare Modeli Utero Organ Nakli

The transplantation of stem cells and viruses in utero has tremendous potential for treating congenital disorders in the human fetus.  For example, in ...

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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.

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

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

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 ...

Pre-clinical Evaluation of Tyrosine Kinase Inhibitors for Treatment of Acute Leukemia

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are attractive druggable therapeutic targets. Here we describe an efficient four-step strategy for pre-clinical evaluation of tyrosine kinase inhibitors (TKIs) in the treatment of acute leukemia. Initially, western blot analysis is used to confirm target inhibition in cultured leukemia cells. Functional activity is then evaluated using clonogenic assays in methylcellulose or soft agar cultures. Experimental compounds that demonstrate activity in cell culture assays are evaluated in vivo using NOD-SCID-gamma (NSG) mice transplanted orthotopically with human leukemia cell lines. Initial in vivo pharmacodynamic studies evaluate target inhibition in leukemic blasts isolated from the bone marrow. This approach is used to determine the dose and schedule of administration required for effective target inhibition. Subsequent studies evaluate the efficacy of the TKIs in vivo using luciferase expressing leukemia cells, thereby allowing for non-invasive bioluminescent monitoring of leukemia burden and assessment of therapeutic response using an in vivo bioluminescence imaging system. This strategy has been effective for evaluation of TKIs in vitro and in vivo and can be applied for identification of molecularly-targeted agents with therapeutic potential or for direct comparison and prioritization of multiple compounds.

Receptor tyrosine kinases are ectopically expressed in many cancers and have been identified as therapeutic targets in acute leukemia. This manuscript describes an efficient strategy for pre-clinical evaluation of tyrosine kinase inhibitors for the treatment of acute leukemia.

Akut lösemi tedavisi için Tirosin Kinaz İnhibitörleri klinik öncesi değerlendirilmesi

Receptor tyrosine kinases have been implicated in the development and progression of many cancers, including both leukemia and solid tumors, and are ...

Murine Cervical Heart Transplantation Model Using a Modified Cuff Technique

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are available to perform mechanistic in vivo studies. While heart transplantation models using a suture technique were first successfully developed in rats, the translation into an equally widespread used murine equivalent was never achieved due the technical complexity of the microsurgical procedure. In contrast, non-suture cuff techniques, also developed initially in rats, were successfully adapted for use in mice1-3. This technique for revascularization involves two major steps I) everting the recipient vessel over a polyethylene cuff; II) pulling the donor vessel over the formerly everted recipient vessel and holding it in place with a circumferential tie. This ensures a continuity of the endothelial layer, short operating time and very high patency rates4.
Using this technique for vascular anastomosis we performed more than 1,000 cervical heart transplants with an overall success rate of 95%. For arterial inflow the common carotid artery and the proximal aortic arch were anastomosed resulting in a retrograde perfusion of the transplanted heart. For venous drainage the pulmonary artery of the graft was anastomosed with the external jugular vein of the recipient5.
Herein, we provide additional details of this technique to supplement the video.

The murine cervical heart transplantation model is well suited for immunological as well as ischemia reperfusion injury studies. We modified the procedure using a non-suture cuff technique and performed more than 1,000 successful transplants with this approach.
Herein, we provide additional details of this technique to supplement the video.

Modifiye Manşet Tekniği Sıçangiller Servikal Kalp Nakli Modeli

Mouse models are of special interest in research since a wide variety of monoclonal antibodies and commercially defined inbred and knockout strains are ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

İlköğretim İnsan Kemik İliği ve Maligniteleri Eğitim Bir Üç boyutlu Doku Kültürü Modeli

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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 ...

Murine Corneal Transplantation: A Model to Study the Most Common Form of Solid Organ Transplantation

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each year. While several animal models exist for this procedure and mice are the species that is most commonly used. The reasons for using mice are the relative cost of using this species, the existence of many genetically defined strains that allow for the study of immune responses, and the existence of an extensive array of reagents that can be used to further define responses in this species. This model has been used to define factors in the cornea that are responsible for the relative immune privilege status of this tissue that enables corneal allografts to survive acute rejection in the absence of immunosuppressive therapy. It has also been used to define those factors that are most important in rejection of such allografts. Consequently, much of what we know concerning mechanisms of both corneal allograft acceptance and rejection are due to studies using a murine model of corneal transplantation. In addition to describing a model for acute corneal allograft rejection, we also present for the first time a model of late-term corneal allograft rejection.

Mice have been used as a model for studying many forms of transplantation, including corneal transplantation. We describe in this report a murine model for both acute and late-term corneal transplantation.

Sıçangiller Kornea Nakli: Katı Organ Nakli En Yaygın Formu Eğitim için Bir Model

Corneal transplantation is the most common form of organ transplantation in the United States with between 45,000 and 55,000 procedures performed each ...

Evaluation of Lung Metastasis in Mouse Mammary Tumor Models by Quantitative Real-time PCR

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated death 1. Common sites of metastatic spread include lung, lymph node, brain, and bone 2. Mechanisms that drive metastasis are intense areas of cancer research. Consequently, effective assays to measure metastatic burden in distant sites of metastasis are instrumental for cancer research. Evaluation of lung metastases in mammary tumor models is generally performed by gross qualitative observation of lung tissue following dissection. Quantitative methods of evaluating metastasis are currently limited to ex vivo and in vivo imaging based techniques that require user defined parameters. Many of these techniques are at the whole organism level rather than the cellular level 3-6. Although newer imaging methods utilizing multi-photon microscopy are able to evaluate metastasis at the cellular level 7, these highly elegant procedures are more suited to evaluating mechanisms of dissemination rather than quantitative assessment of metastatic burden. Here, a simple in vitro method to quantitatively assess metastasis is presented. Using quantitative Real-time PCR (QRT-PCR), tumor cell specific mRNA can be detected within the mouse lung tissue.

This protocol describes how to use quantitative Real-time PCR (QRT-PCR) to detect tumor cell specific mRNA representing metastasis within the mouse lung tissue.

Kantitatif gerçek zamanlı PCR ile fare meme tümör modellerinde akciğer metastazı Değerlendirilmesi

Metastatic disease is the spread of malignant tumor cells from the primary cancer site to a distant organ and is the primary cause of cancer associated ...

Murine Cervical Aortic Transplantation Model using a Modified Non-Suture Cuff Technique

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. However, only minor improvement in the long-term results of transplanted solid organs could be observed over the past decades. In this context, chronic allograft vasculopathy (CAV) still represents the leading cause of late organ failure in cardiac, renal and pulmonary transplantation.
Thus far, the underlying pathogenesis of CAV development remains unclear, explaining why effective treatment strategies are presently missing and emphasizing a need for relevant experimental models in order to study the underlying pathophysiology leading to CAV formation. The following protocol describes a murine heterotopic cervical aortic transplantation model using a modified non-suture cuff technique. In this technique, a segment of the thoracic aorta is interpositioned in the right common carotid artery. With the use of the non-suture cuff technique, an easy to learn and reproducible model can be established, minimizing the possible heterogeneity of sutured vascular micro anastomoses.

Here, we present a protocol of heterotopic aortic transplantation in mice using the non-suture cuff technique in a cervical murine model. This model can be used to study the underlying pathology of chronic allograft vasculopathy (CAV) and can help evaluate new therapeutic agents in order to prevent its formation.

Modifiye Dikişsiz Manşet Tekniği Kullanılarak Murine Servikal Aort Transplantasyon Modeli

With the introduction of powerful immunosuppressive protocols, distinct advances are possible in the prevention and therapy of acute rejection episodes. ...

Flow Cytometry Analysis of Immune Cell Subsets within the Murine Spleen, Bone Marrow, Lymph Nodes and Synovial Tissue in an Osteoarthritis Model

Osteoarthritis (OA) is one of the most prevalent musculoskeletal diseases, affecting patients suffering from pain and physical limitations. Recent evidence indicates a potential inflammatory component of the disease, with both T-cells and monocytes/macrophages potentially associated with the pathogenesis of OA. Further studies postulated an important role for subsets of both inflammatory cell lineages, such as Th1, Th2, Th17, and T-regulatory lymphocytes, and M1, M2, and synovium-tissue-resident macrophages. However, the interaction between the local synovial and systemic inflammatory cellular response and the structural changes in the joint is unknown. To fully understand how T-cells and monocytes/macrophages contribute towards OA, it is important to be able to quantitively identify these cells and their subsets simultaneously in synovial tissue, secondary lymphatic organs and systemically (the spleen and bone marrow). Nowadays, the different inflammatory cell subsets can be identified by a combination of cell-surface markers making multi-color flow cytometry a powerful technique in investigating these cellular processes. In this protocol, we describe detailed steps regarding the harvest of synovial tissue and secondary lymphatic organs as well as generation of single cell suspensions. Furthermore, we present both an extracellular staining assay to identify monocytes/macrophages and their subsets as well as an extra- and intra-cellular staining assay to identify T-cells and their subsets within the murine spleen, bone marrow, lymph nodes and synovial tissue. Each step of this protocol was optimized and tested, resulting in a highly reproducible assay that can be utilized for other surgical and non-surgical OA mouse models.

Here, we describe a detailed and reproducible flow cytometry protocol to identify monocyte/macrophage and T-cell subsets using both extra- and intracellular staining assays within the murine spleen, bone marrow, lymph nodes and synovial tissue, utilizing an established surgical model of murine osteoarthritis.