The authors thank all previous and present members of the department of Reproduction and Development of the Erasmus MC for helpful and stimulating discussions during the past years. We also want to thank the three anonymous reviewers for providing helpful feedback. Work in the Gribnau lab is supported by funding from the Dutch research council (NWO-VICI) and an ERC starting grant.

1.. Kadın Embriyonik Kök Hücreleri Farklılaşma endüklenmesi X kromozomu inaktivasyonu tetikleyebilecek NOT: Bayan fare embriyonik kök hücreleri (istek üzerine) fare embriyonik fibroblastlar (MEF&#39;ler) ile kaplanmış Jelatinize kültür yemekleri standart ES hücre koşullarında yetiştirilir. Biz burada okuyucu standart hücre kültürü teknikleri 39-41 aşina olduğunu varsayalım. Farklılaşmasını teşvik etmek için, T25 tabaklarında büyütülmüş ES hücreler MEF&#39;ler ayrılabilir, ve farklılaşma ortam içinde kaplı olacaktır. <ol><li> ES hücre kültüründen ES orta çıkarın ve hücre kültürü iki kez PBS ile yıkayın. </li><li> 1.5 ml tripsin-EDTA (37 ° C&#39;ye önceden ısıtılmış) ekleyin ve 7 dakika boyunca 37 ° C&#39;de hücreler inkübe edilir. 3.5 dakika sonra, koloniler kırmak için hafifçe çanak sallayın. </li><li> Hücrelere farklılaştırma ortamının 3.5 ml eklenerek tripsin-EDTA etkisiz hale getirirler. Pipet hücreler yukarı ve aşağı tek hücreli çözüm elde ve toplamakbir 15 ml tüp içinde. 200 x g&#39;de 5 dakika boyunca Spin ve farklılaştırma ortamının 10 ml hücreleri toplamak. </li><li> Olmayan bir jelatinleştirilmiş kültür tabağına hücre süspansiyonu ekleyin ve bir hücre kültürü kuluçka makinesi içinde 1 saat boyunca inkübe edilir. NOT: ES hücreleri süspansiyon içinde kalacaktır MEF&#39;ler, non-jelatinleştirilmiş kültür tabağına yapışma mümkün olacaktır. </li><li> Bu arada, 6-çukurlu plakalara steril cam lamelleri (24 x 24 mm) ekleyin% 0.2 jelatin ekleyin ve oda sıcaklığında en az 5 dakika boyunca inkübe edin. </li><li> 1 saat sonra, esas olarak non-yapışan ES hücre oluşacak, ön kaplama ES hücre kültürü süpernatan toplamak. Lamelleri içeren 6 oyuklu plakalara plaka ES hücreleri. NOT: ⅙ 6 oyuklu plakanın 6 kuyu için bir birleşik T25 çanak th. Bu da farklılaşma 3 gün sonra, bir birleşik farklılaşma sağlayacaktır. Günlük olarak orta değiştirin. Uzun farklılaşma zamanlı dersler, bakteriyel yemekleri farklılaşma ortamda plaka ön kaplama ES hücreleri ve inkubasyon içinHücre kültürü inkübatörü içine entegre edilmiş bir hücre çalkalayıcıda yedi. Bu hücreler, 1-2 gün önce tespit etmek için lamelleri kaplama olabilir embriyoit organları oluşturmak için izin verir. </li></ol> Daha sonraki DNA-RNA FISH Analizi için Ayrılmış ES Hücre 2. Fixation <ol><li> Farklılaşma kültürlerden farklılaşma orta çıkarın ve hücre kültürü iki kez PBS ile yıkayın. NOT: hücreler yıkanır önlemek için dikkatli bir şekilde PBS ekleyin. </li><li> Hücre kültürü PBS çıkarın ve% 4 paraformaldehit (PFA) / PBS eklenerek hücreleri düzeltmek ve oda sıcaklığında 10 dakika boyunca inkübe edilir. DİKKAT: PFA toksik ve inhale veya zaman Gözlere veya cilde temas edilirken önemli sağlık riskine neden olabilir. Ayrıca, PFA kanserojen. </li><li> % 4 PFA / PBS çıkarın ve% 70 etanol ile lamelleri 3x yıkayın. NOT: PFA herhangi bir iz sonraki BALIK adımlarla engel olacak şekilde Doğru yıkama, önemlidir. Lameller önce F birkaç ay boyunca -20 ° C &#39;de% 70 etanol içinde saklanabilirISH analizi. </li></ol> FISH Deneyler için DNA Proses 3. Etiketleme Not: ilgi konusu genin (minimum uzunluğu&gt; RNA balık ya da&gt; DNA FISH için 20 kb 2 kb) ya da ilgi konusu geni içeren bir BAC bir DNA fragmanını elde edilir. Büyük ihtimalle transkripsiyon eş zamanlı olarak tespit edilebilir çok transkript ile sonuçlanacaktır Başlangıcı RNA saptanması için, daha küçük bir sonda, DNA tespiti ile karşılaştırıldığında yeterli olabilir. Tek bir kopya DNA şeridi üzerinde güçlü bir sinyal için, daha uzun bir sonda dahil haptenlerin yeterli sayıda tespit edebilmek için gereklidir. Tercihen, bir non-transkribe genomik bölge için DNA probunun tasarlanması için seçilir. RNA tespit etmek için daha küçük bir sonda kullanılarak Buna ek olarak, bu tamamen göz ardı edilemez olsa da RNA transkripsiyonu edildiği genomik lokusları, birleşik DNA-RNA FISH yaklaşımla tespit ediliyor önlenecektir. Fare Xist algılamak için, bir 5.5 kb cDNA probu kullanılmıştır. Det içinX kromozomu ection çeşitli BAC problar kullanılabilir. İyi sonuçlar Xist lokusuna yakın bulunan BAC RP23-100E1 ve BAC CT7-474E4, bir karışımı elde edilmiştir. Ilgi konusu gen veya kromozom bağlı olarak, çeşitli problar en iyi sonuçları elde etmek için gerekli olabilir. RNA-FISH için kullanılacak malzemeler ile çalışan temiz bir ortamda steril filtre ipuçları, RNAse serbest H 2 O ve iş kullanımı zaman. <ol><li> DNA 1 ug alın ve 16 ul bir toplam hacimde, H2O içinde seyreltin. , 4 digoksijenin ul veya biotin etiketleme çözeltisi (reaktif bakınız) ekle vorteks ile karıştırın ve 90 dakika boyunca 16 ° C&#39;de inkübe edin. Not: biotin ya da digoksigenin-etiketli nükleotidler hemen nik-çevirisi ile dahil edilecektir. </li><li> Bu arada, sigara entegre nükleotidler kaldırmak için G50 sütunları hazırlamak. , 2 ml şırınga alın stamper kaldırmak ve şırınga içine sterilize pamuk ekleyin. Çözüm t G50 topları Ekleo, 1 dakika boyunca 642 x g, 15 ml bir tüp ve santrifüj içinde şırınga, şırınga doldurun. NOT: şırınga% 80 civarında şimdi G50 ile dolu olmalıdır. Az G50 mevcut olduğunda tekrarlayın. </li><li> 90 dakika sonra, çentik-translasyon reaksiyon karışımına, H2O içinde 40 ul ekleyin ve G50 kolonuna reaksiyonu ekleyin. Sütunun altında boş bir tüp ekleyin ve 2 dakika boyunca 642 x g &#39;de santrifüj. Bir reaksiyon tüpü yoluyla akışını toplamak. </li><li> Bir pipet ile üzerinden akışı hacmini ölçün ve 200 ul toplam hacme ulaşmak için, H 2 O ekleyin. 13.3 ul somon sperm DNA&#39;sı (10 mg / ml), 13.3 ul maya tRNA (10 mg / ml), 26.7 ul fare Cot-1 DNA (1 mg / ml) ve 28 ul 2 M NaAc, pH ekleyerek yoluyla akışını Çökelti 5.6. Vorteks ile karıştırın ve 533 ul buz soğukluğunda% 100 etanol ilave edin. 4 ° C&#39;de 30 dakika boyunca 16,200 xg&#39;de tüp ve santrifüj sallayın </li><li> Süpernatant kaldırmak ve% 70 etanol ile iki kez pelet yıkayın. 5 dk boyunca 16,200 x g&#39;de santrifüj4 ° C ve hava pelet kurutun. 50 + melezleştirme çözeltisi 50 ul ve çözülmesini kolaylaştırmak için 30 dakika boyunca 37 ° C&#39;de inkübe edin. Not: Etiketli prob -20 ° C&#39;de birkaç yıl boyunca depolanabilir DİKKAT: 50 + melezleme çözüm, toksik deri, gözleri ve solunum sistemini tahriş edebilir ve aynı zamanda bir teratojen formamid içerir. </li></ol> 4.. Permeabilization, Kombine DNA-RNA FISH için Sabit Hücreler tedavi öncesi ve Hibritleşme <ol><li> % 70 etanol ve 6-delikli plakalara hücre kültürü PBS ilave kaldırarak, lamelleri ile sabitleştirilmiş hücreler rehidrate. 5 dakika boyunca inkübe edin. Toplam üç kez tekrarlayın. </li><li> Hücre kültürü PBS çıkarın ve hücreleri permeabilize, 6-çukurlu plakalara% 0.2 pepsin ekleyin. 4 dakika boyunca 37 ° C&#39;de bir su banyosu içinde 6 oyuklu inkübe edin. Pepsin kaldırmak ve RNAse serbest H 2 O. ile inaktive </li><li> Oda sıcaklığında 5 dakika boyunca% 4 PFA / PBS ile inkübe edilmesi ile post-fix hücreleri. Fixati sonraon, 5 dakika boyunca hücre kültürü iki kez PBS ile yıkayın. </li><li> 3 dakika sonra 3 dakika için 3 dakika ve% 100 etanol için% 90 etanol,% 70 etanol ile inkübe hücreleri kurutmak. Gelirleri 6-çukurlu dışarı lamelleri, ve birkaç dakika boyunca hava kuru lamelleri. </li><li> Bir ısı plaka üzerinde, 1 saat boyunca 65 ° C&#39;de inkübe edilerek lamelleri Yaş hücreleri. </li><li> Analiz edilecek her bir lamel için bir araya eklenerek, fare Cot-1 DNA ile FISH prob önceden hibridize, 50 + hibridizasyon çözeltisi, 0.5 ul fare Cot-1 DNA, biyotin-etiketli prob Xist 1 ul ve digoksijenin 1 ul 25 ul X kromozomu tespit etiketli BAC prob. Vorteks ile karıştırılmakta ve 5 dakika boyunca 99 ° C&#39;de denatüre. Katlanır-1 DNA probları mevcut dizilerini tekrar hibridize izin vermek için, 45 dakika boyunca 37 ° C &#39;de hemen inkübe edin. </li><li> Bekletildikten sonra cam slayt üzerinde (SSC/10 mM fosfat tampon maddesi formamide/2x% 70 arasında oluşan) denatürasyon tampon maddesi 50 ul nokta ve hücreler towar bakacak şekilde üst üste koymak lamelleridenatürasyon tampon ds. 2 dakika boyunca 65 ° C&#39;de inkübe edin. </li><li> 5 dakika boyunca buz gibi soğuk% 70 etanol içinde inkübe edilerek geri 6-çukurlu plakalara lamelleri ve post-fix hücreleri ekleyin. </li><li> 3 dakika sonra 3 dakika için 3 dakika ve% 100 etanol için% 90 etanol,% 70 etanol içinde daha sonraki kuluçka hücreleri kurutmak. 6-delikli plakadan lamelleri çıkarın ve birkaç dakika kurumasını sağlar. </li><li> Bir cam slayt üzerinde ön melezleşmiştir sonda nokta, ve üstüne lamel kuluçkaya yatmaktadır. Yer SSC formamide/2x 50 ml% 50 ile dolu nemli bir oda içinde slaytlar, 37 ° C de bir gece inkübe </li></ol> 5. Olarak hibridizasyon sonrası yıkamaların ve Probe Antikor Aracılı Algılama <ol><li> 2x SSC ile 6 oyuklu plakalar doldurun ve önceden 42 ° C sıcak kadar Gece boyunca inkübasyondan sonra lamelleri ekleyin ve 5 dakika boyunca 42 ° C&#39;de inkübe edin. </li><li> 2x SSC çıkarın ve 42 ° C&#39;de 10 dakika boyunca% 50 formamide/2x SSC ile lamelleri inkübe Toplam 3 kez (tamamen yıkama 30 dakika) tekrarlayın. </li&gt; <li> SSC formamide/2x% 50 kaldırmak ve oda sıcaklığında 2 x 5 dakika boyunca Tris-salin-Tween (TST) ile slaytlar yıkayın. </li><li> Nokta 50 ul yeni cam slaytlar üzerinde lamel başına Tris-salin-BSA (TSBSA) ve TST-nemlendirilmiş karanlık bir oda içinde oda sıcaklığında 30 dakika boyunca bloke edilmesi için ters lamelleri inkübe edin. </li><li> Aktarım arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. </li><li> Nokta 50 yeni cam slaytlar üzerinde lamel başına koyun-anti-dig antikoru (TSBSA olarak 1:500) ve ul ve TST-nemlendirilmiş karanlık bir oda içinde oda sıcaklığında 30 dakika boyunca inkübasyon birinci adım için lamelleri inkübe ters. </li><li> Aktarım arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. </li><li> Nokta 50 yeni cam slaytlar üzerinde lamel başına FITC (TSBSA olarak 1:250) ile konjüge edilmiş tavşan anti-koyun antikoru ul ve TST-nemlendirilmiş karanlık bir oda içinde oda sıcaklığında 30 dakika boyunca ikinci bir inkübasyon aşaması için ters lamelleri inkübe . </li><li> Transfer arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. </li><li> Nokta 50 yeni cam slaytlar üzerinde lamel başına FITC (TSBSA olarak 1:250) ile konjuge edilmiş keçi anti-tavşan antikoru ul ve TST-nemlendirilmiş karanlık bir oda içinde oda sıcaklığında 30 dakika boyunca inkübasyon üçüncü aşama için baş aşağı inkübe lamelleri . </li><li> Aktarım arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. </li><li> Nokta 50 yeni cam slaytlar üzerinde lamel başına fare-anti-biotin antikor ul (TSBSA olarak 1:200) ve TST-nemlendirilmiş karanlık bir oda içinde oda sıcaklığında 30 dakika boyunca inkübasyon dördüncü aşama için ters lamelleri inkübe edin. </li><li> Aktarım arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. </li><li> Nokta 50 yeni cam slaytlar üzerinde lamel başına Rodamin (TSBSA olarak 1:250) ile konjuge edilmiş eşek anti-fare antikorunun ul ve ters bir TST-nemlendirilmiş karanlık odacıklı içerisinde oda sıcaklığında 30 dakika boyunca inkübasyon beşinci aşama için lamelleri inkübeer. </li><li> Aktarım arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. </li><li> Nokta 50 yeni cam slaytlar üzerinde lamel başına Rodamin (TSBSA olarak 1:250) ile konjuge edilmiş keçi anti-at antikor ul ve TST-nemlendirilmiş karanlık bir oda içinde oda sıcaklığında 30 dakika boyunca inkübasyon altıncı aşama için baş aşağı inkübe lamelleri . NOT: keçi anti-at antikor eşek anti-fare antikoru tanıyacaktır; zaman araştırmacı mevcut, bir keçi-anti-eşek antikoru de işe yarayacaktır. </li><li> Aktarım arka 6-çukurlu plakalara lamelleri ve TST ile 2 x 5 dakika yıkayın. Tris-salin (TS) ilave 5 dakika yıkanır. </li><li> 3 dakika sonra 3 dakika için 3 dakika ve% 100 etanol için% 90 etanol,% 70 etanol içinde daha sonraki kuluçka hücreleri kurutmak. 6-delikli plakadan lamelleri çıkarın ve birkaç dakika kurumasını sağlar. </li><li> Bir cam slayt üzerinde DAPI ile montaj orta bir damla (reaktiflerimize bakın) Nokta ve baş aşağı üstüne lamel ekleyin. Tırnak cilası ile slaytlar mühür vefloresan mikroskopisi ile FISH sonuçlarının değerlendirilmesi (Şekil 1). </li></ol>

<ol>
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Yazarlar ifşa hiçbir şey yok.

DNA FISH için uygun koşullar daha az istikrarlı RNA molekülleri için çok sert olabilir gibi kombine DNA-RNA FISH, teknik olarak zor olabilir. Çeşitli yaklaşımlar hem DNA hem RNA&#39;yı tespit probları ile aynı anda inkübasyon için ihtiyaç engellemeyi, aynı hücre içerisinde DNA ve RNA hem de çalışma uygulanmıştır. Örneğin, bir bindirilmesi yaklaşımda, birinci RNA FISH gerçekleştirilir ve hücre görüntülenmiş ve koordinatlar alınır. Daha sonra, aynı slaytlar RNA FISH sinyalinin kaybol...

In situ hibridizasyon (FISH) floresan vasıtasıyla hücre ve dokuları inceleyerek 1970&#39;lerin 1 yılından bu yana, araştırmacılar subselüler seviyesinde genler, kromatin organizasyonu ve gen ifadesini incelemek için izin vardır. DNA FISH sık sitogenetik, karyotipleme 2, kanser teşhis 3 ve pre-implantasyon genetik tarama 4&#39;te kullanılan ve onların doğal ortamında nükleik asitlerin saptanmasını sağlar çünkü moleküler araştırma 5-6 önemli bir role sahiptir. RNA FISH ile tek hücre ekspresyon analizleri yerli primer transkript algılayabilir ve kodlamayan RNA&#39;lar kromozom transkripsiyonu, ve aralarında örneğin bir nüfus düzeyinde gen ifadesini değerlendirmek diğer tekniklerden avantaj sunuyor kantitatif RT-PCR, genom ekspresyon analizi veya Kuzey lekeleme . Geldikleri sitelerinden doğrudan kaynaklanan RNA transkript görselleştirerek, örneğin olmuşturgen ekspresyon stokastik 7, ve bazen 8 alel spesifik olabilir fark ettim. Tekniğin gelişmeler bile hücreler 9-11 olan bir mRNA moleküllerinin algılama ve ölçümü izin vermektedir. FISH temel ilkesi, son derece spesifik, Watson ve Crick&#39;in baz eşleştirme aracılığıyla bir nükleik asit sondasına hücre içinde nükleik asitlerin hibridizasyonu oluşur. Sonda, ya doğrudan veya dolaylı olarak tespit edildi, mikroskobik olarak görselleştirilebilen bir sinyal elde olabilir. Başlangıç ​​girişimleri güvenlik sorunları sınırlı uzamsal çözünürlük ve bir süre 12-14 yalnızca bir hedef tespit etme yeteneğine göre sakıncaları vardı radyoaktif olarak etiketlenmiş problar, oluşuyordu. Fluorochromes, haptenlere ve enzimler içeren radyoaktif olmayan etiketler, sonraki gelişme, rutin moleküler biyoloji tekniği olarak FISH yaygın kullanımına izin vermiş. FISH prob ya doğrudan fluoroch ile etiketlenmiş olabilirnükleik asit floresan moleküller ile kimyasal bağlanması romes 15 ve floresanla işaretlenmiş nükleotidlerin 16-19, ya da dolaylı olarak prob (biyotin ve digoksijenin dahil) haptenlerin entegrasyonu ve konjüge edilmiş hapten özgü antikorlarla haptenlerin immünolojik tespiti sonra görselleştirilebilir entegrasyonu dizilerinin flüoresan raportör moleküllerin 20-21. İkinci yaklaşım orijinal sinyal geliştirmek için kullanılan floresan işaretli antikorlar çeşitli katmanları kullanarak sinyal büyütülmesine olanak tanır, ve düşük seviyelerde ifade edilmiştir RNA türünün saptanmasını sağlar. Doğrudan ve dolaylı etiketleme teknikleri ve çeşitli haptenleri birleştirerek, bazı hedefler aynı anda aynı hücre içinde görselleştirilebilir. FISH protokolleri en önemli adımlardan biri hedefine probun hibridizasyonu. Teorik olarak, bir prob özel uygulamada, hedefine bağlanan tek olsa da, bu özgünlüğüprobları homolog bölgelere bağlanabilir her zaman olduğu gibi, elde edilememesi ve hibridizasyon koşulları daima belirli bir DNA bölgesi ya da RNA türleri için ideal olmayabilir olacaktır. Bunlar FISH prosedürün sıkılığının artırabilir ve arka plan gürültüsü yüksek düzeyde olmasına neden olur FISH prob, spesifik olmayan bağlanmasını önlemek gibi hibridizasyon sonrası yıkamalar, bu nedenle özel bir önem taşımaktadır. RNA molekülleri tek telli gibi kolayca bir BALIK hibritlenmiştir olabilir. Buna karşılık, iki iplikçikli DNA molekülü, birinci bir sonda melezleşebilir sonra denatürasyon adımı gerekmektedir. Bu genellikle DNA&#39;nın denatürasyonu ile sonuçlanan numunenin ısıtılması ile elde edilir. Ancak bu sert koşullar altında, kırılgan, tek sarmallı RNA molekülleri kaybolmuş olabilir. Bu nedenle, birleşik DNA-RNA FISH koşullarının önemli optimizasyon gerektirir ve sadece RNA veya DNA&#39;nın ayrı bir algılama göre daha teknik açıdan zor. Burada presenta detaylı bizi dişi fare embriyonik kök hücreleri 22-24 ayırt X kromozomu inaktivasyonu (XCI) okuttu kombine, eşzamanlı DNA-RNA FISH protokolü. XCI önemli bir epigenetik dişi embriyonik gelişme için bir mekanizma 25 ve heterochromatinization ile sonuçlanır ve bu nedenle dişi bireyler 26-27 iki X kromozomu birinin susturma. Bu işlem için gerekli 22-23 RNF12 ve REX1 24 proteinler tarafından düzenlenir kodlayıcı olmayan RNA Xist 28-30 vardır. Xist sentezleme embriyonik gelişimi sırasında ya da in vitro ES hücre farklılaşması üzerine gelecek etkin X kromozom (Xi) üzerinde yukarı regüle olur ve X kromozomu boyunca yayılır ve böylece X kromozomu 31 transkripsiyonel kapanmasına neden kromatin yeniden enzimleri de olabilir. Bu Xist RNA yayılan X kromozomal bir kaplama olarak RNA FISH ile görselleştirilebilirbazıları, aynı zamanda bir bulut Xist olarak ifade edilmektedir. Kadın ES hücreleri genomik istikrarsızlık nedeniyle X kromozomu birini kaybedebilir yana, biz ve diğerleri emin sadece karyotipik istikrarlı hücreleri, bu önemli sürecin 22,32 analizinde değerlendirilir emin olmak için, XCI çalışma kombine DNA-RNA FISH istihdam var -34. Her moleküler biyoloji tekniğinde olduğu gibi, çok sayıda farklı mükemmel protokoller 35-38 yayınlanmıştır. Burada izin vermek için, fare ES hücreleri, hücrelerin tespit Nick-çeviri, sabit hücrelerin ön-muamelesi ile FISH prob etiketleme farklılaşmasının indüksiyonu başlayarak eden yöntem mevcut permeabilizasyon ve daha sonra prob alımı, için sondanın hibridizasyon Hedef ve son olarak floresan etiketli antikorlar ile prob tespiti. Burada sunulan protokol Xist RNA sadık algılama ve iki günlük bir süre içinde X kromozomu sağlar ve bu tekniğin temelleri muhtemel ot adapte edilebilirOnun sistemleri ve araştırma alanları.

<table border="0" cellpadding="0" cellspacing="0" width="874">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">female mouse embryonic stem cells</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">will be made available upon request</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse embryonic fibroblasts</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>can be derived from E13.5 embryos, or via commercial sources e.g Millipore</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell culture medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>DMEM, 15% foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 0.1 mM &#223;-mercaptoethanol, and 1000 U ml-1 LIF. Filter sterilize and store at 4&#186;C for maximal 2 weeks.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">LIF</td>
			<td style="width:124px;">Chemicon</td>
			<td style="width:119px;">ESG1107</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">DMEM</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">11960085</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Fetal Calf Serum&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td align="right" style="width:119px;">10099141</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Penicillin&#8211;streptomycin (100&#215;)</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Non-essential aminoacids&#160;</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">11140-050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">&#223;-mercaptoethanol&#160;</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">ES cell differentiation medium</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>IMDM-Glutamax, 15% heat inactivated foetal calf serum, 100 U ml-1 penicillin, 100 mg ml-1 streptomycin, non-essential amino acids, 37.8 &#956;l/l monothioglycerol and 50 &#956;g/ml ascorbic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">IMDM-Glutamax</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">31980-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Ascorbic acid (50 mg/ml)</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>A4403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">monothioglycerol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>M6145</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">T25 cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">690160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">6-well cell culture dishes</td>
			<td style="width:124px;">Greiner Bio-one</td>
			<td align="right" style="width:119px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">cell culture grade PBS</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Trypsin-EDTA 0.25% (v/v) trypsin/0.2% (w/v) EDTA in PBS&#160;</td>
			<td style="width:124px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">falcon tubes</td>
			<td style="width:124px;">Falcon</td>
			<td align="right" style="width:119px;">352196</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">24x24 mm coverslips</td>
			<td style="width:124px;">Menzle</td>
			<td align="right" style="width:119px;">780882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">gelatin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G1890-100G</td>
			<td>prepare a 0.2% gelatin/cell culture PBS solution, and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">paraformaldehyde</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6148</td>
			<td>prepare 4% PFA/PBS solutions, by dissolving PFA in PBS. Stir at 70 &#7484;C until completely dissolved and cool down to room temperature. Aliquots can be stored at -20&#7484;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">absolute 100%&#160; ethanol</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">32221-2.5L</td>
			<td>use absolute 100% ethanol and RNAse free water to make 70% and 90% ethanol solutions</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">RNAse free water</td>
			<td style="width:124px;">Baxter</td>
			<td>TKF7114</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">sterile filter tips</td>
			<td style="width:124px;">Rainin Bio Clean</td>
			<td style="width:119px;">GP-L10F, GP-L200F, RT-1000F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">digoxigenin-labeling kit (DIG-Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745816910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">biotin-labeling kit (Biotin Nick translation)</td>
			<td style="width:124px;">Roche</td>
			<td align="right" style="width:119px;">11745824910</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Sephadex G50</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">G5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2 ml syringe</td>
			<td style="width:124px;">BD Plastipak</td>
			<td align="right" style="width:119px;">300013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">sterilized cotton</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf tubes</td>
			<td style="width:124px;">Eppendorf</td>
			<td align="right" style="width:119px;">30120086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">yeast tRNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15401-029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Salmon Sperm DNA 10 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">15632-011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">mouse cot-1 DNA&#160; 1 mg/ml</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">18440-016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">eppendorf bench top microcentrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5417R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Eppendorf Refrigerated Centrifuge</td>
			<td style="width:124px;">Eppendorf</td>
			<td style="width:119px;">Model 5810R&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M NaAc, pH 5.6</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td style="width:119px;">S7670</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">50+ hybridization solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>50% formamide, 2x SSC, 50 mM phosphate buffer pH 7, 10% dextran sulfate pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">formamide</td>
			<td style="width:124px;">Acros organics</td>
			<td align="right" style="width:119px;">3272350000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">dextran sulfate</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">31403</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">pepsin</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P6887</td>
			<td>prepare a 0.2% solution in RNAse free water, and add 84 &#181;l 37% HCL per 100 ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">HCL, 37% solution&#160;</td>
			<td style="width:124px;">Fluka</td>
			<td align="right" style="width:119px;">84422</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">object glasses</td>
			<td style="width:124px;">Starfrost</td>
			<td style="width:119px;">8037/1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">20x SSC</td>
			<td style="width:124px;">Invitrogen</td>
			<td style="width:119px;">AM9763</td>
			<td>dilute to 2x SSC in RNAse free water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">10 mM Phosphate buffer, pH 7</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>add 57.7 ml of 1M Na2HPO4 and 42.3 ml 1M NaH2PO4; use DEPC treated water and autoclave</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">denaturation buffer</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>70% Formamide/2xSSC/10mM phosphate buffer pH 7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tween-20</td>
			<td style="width:124px;">Sigma-Aldrich</td>
			<td>P2287</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">2M Tris pH 7.5 solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">Tris-Saline-Tween washing solution (TST)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>100 ml 10x Saline, 50 ml 2M Tris, 500 &#181;l Tween-20, add RNAse free water till 1 liter&#160;</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">10x Saline solution</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>dissolve 85 g NaCl in 1 liter RNAse free H2O, and autoclave</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline-BSA (TSBSA)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>500 &#956;l 10x Saline, 250 &#956;l 2 M Tris, 1 ml 100x BSA, 3,25 ml RNAse free H2O</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:464px;">BSA, purified, 100x&#160;</td>
			<td style="width:124px;">New England Biolabs</td>
			<td style="width:119px;">B9001S</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">sheep-anti-dig antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:500 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">rabbit-anti-sheep antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-rabbit antibody conjugated with FITC</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">mouse-anti-biotin antibody</td>
			<td style="width:124px;">Roche diagnostics</td>
			<td style="width:119px;"></td>
			<td>use 1:200 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">donkey-anti-mouse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">goat-anti-horse antibody conjugated with Rhodamine</td>
			<td style="width:124px;">Jackson labs</td>
			<td style="width:119px;"></td>
			<td>use 1:250 diluted in TSBSA</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:464px;">Tris-Saline washing solution (TS)</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td>10 ml 10x Saline, 5 ml 2 M Tris, 85 ml RNAse free H2O</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vectashield mounting medium with DAPI</td>
			<td style="width:124px;">Vectorlabs</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nail-varnish</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:464px;">fluorescent miscroscope</td>
			<td style="width:124px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

combined dna-rna fluorescent in situ hybridization (fish) to study x chromosome inactivation in differentiated female mouse embryonic stem cells

In situ hibridizasyon (FISH) floresan hücrelerdeki nükleik asitlerin saptanmasını sağlayan bir molekül tekniktir. DNA FISH genellikle sitogenetiği ve kanser teşhisinde kullanılan ve çoğu zaman önemli klinik etkileri vardır genomun, sapmaları tespit edebilir. RNA FISH hücrelerde RNA molekülleri tespit etmek için kullanılabilir ve gen ekspresyonunun düzenlenmesinde önemli bilgiler sağlamıştır. DNA FISH için uygun koşullar kırılgan, tek sarmallı RNA molekülleri için çok sert olabilir gibi aynı hücre içinde DNA ve RNA BALIK birleştiren, teknik olarak zordur. Biz burada X kromozomu tarafından kodlanan Xist RNA ve DNA kombine eşzamanlı algılamayı sağlayan kolay uygulanabilir bir protokol mevcut. Bu kombine DNA-RNA FISH protokol muhtemelen RNA ve DNA hem de tespit edilmesi gereken diğer sistemlere de uygulanabilir.

Kombine DNA-RNA FISH için yukarıda belirtilen protokolü kullanarak, dişi embriyonik kök hücreleri farklılaştırarak X kromozomu inaktivasyonu görselleştirmek mümkün olmuştur. 1. biz Xist (ki her ikisi de tespit edilen DNA-RNA FISH deney, temsili bir örneğini göstermektedir inaktif X kromozomu üzerindeki bir sözde Xist RNA bulut ve aktif X kromozomu üzerinde bir bazal transkripsiyon iğne ucu) ve bir iğne ucu sinyali olarak görülebilir X kromozomu,...

Watch this Scientific Journal Video about Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells at JoVE.com

Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

In situ hibridizasyon (FISH) floresan hücre içinde kendi doğal ortamında nükleik asitlerin saptanmasını sağlar. Burada fare embriyonik kök hücreleri X kromozomu inaktivasyon incelemek için kullanılabilir FISH vasıtasıyla RNA ve DNA birleşik, eş zamanlı olarak saptanması için bir protokol açıklar.

Kombine DNA-RNA Floresan<em&gt; In situ</em&gt; Hibridizasyon (FISH) Farklılaştırılmış Kadın Fare Embriyonik Kök Hücreleri X kromozomu inaktivasyonu Eğitim için

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in ...

combined-dna-rna-fluorescent-situ-hybridization-fish-to-study-x

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Combined DNA-RNA Fluorescent In situ Hybridization (FISH) to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Unit 2

Studying DNA and RNA

SCIENTISTS IN ACTION

27.5K Görüntüleme.  Erasmus MC - University Medical Center.  In situ hibridizasyon (FISH) floresan hücre içinde kendi doğal ortamında nükleik asitlerin saptanmasını sağlar. Burada fare embriyonik kök hücreleri X kromozomu inaktivasyon incelemek için kullanılabilir FISH vasıtasıyla RNA ve DNA birleşik, eş zamanlı olarak saptanması için bir protokol açıklar. 

Video: Combined DNA-RNA Fluorescent In situ Hybridization FISH to Study X Chromosome Inactivation in Differentiated Female Mouse Embryonic Stem Cells

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

Biyoloji

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Floresan In situ İnsan Sperm hibridizasyon (FISH) Protokolü

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

Fare Embriyonik Germinal Hücreleri İzolasyon ve türetilmesi

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures are lengthy and technically demanding with multiple important steps that collectively contribute to the quality of the final result. This protocol describes in detail several key quality control steps for optimizing probe labeling and performance. Overall, our protocol provides a detailed description of the critical steps necessary to reproducibly obtain high quality results. First, we describe the generation of digoxygenin (DIG) labeled RNA probes via in vitro transcription of DNA templates generated by PCR. We describe three critical quality control assays to determine the amount, integrity and specific activity of the DIG-labeled probes. These steps are important for generating a probe of sufficient sensitivity to detect endogenous mRNAs in a whole mouse embryo. In addition, we describe methods for the fixation and storage of E8.5-E11.5 day old mouse embryos for in situ hybridization. Then, we describe detailed methods for limited proteinase K digestion of the rehydrated embryos followed by the details of the hybridization conditions, post-hybridization washes and RNase treatment to remove non-specific probe hybridization. An AP-conjugated antibody is used to visualize the labeled probe and reveal the expression pattern of the endogenous transcript. Representative results are shown from successful experiments and typical suboptimal experiments.

Whole Mount in Situ Hybridization of E8.5 to E11.5 Mouse Embryos

This whole mount in situ hybridization protocol discusses critical steps that ensure reproducible high quality results for gene expression studies in E8.5-E11.5 day old mouse embryos.

Tüm Dağı<em&gt; In situ</emE11.5 fare embriyolarına E8.5 ve&gt; Hibridizasyon

Whole mount in situ hybridization is a very informative approach for defining gene expression patterns in embryos. The in situ hybridization procedures ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Floresans tarafından viral RNA tespit edilmesi<em&gt; In situ</em&gt; Hibridizasyon (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 protein variants encoded by the var genes.
The haploid P. falciparum genome harbors approximately 60 different var genes of which only one has been believed to be transcribed per cell at a time during the blood stage of the infection. How such mutually exclusive regulation of var gene transcription is achieved is unclear, as is the identification of individual var genes or sub-groups of var genes associated with different receptors and the consequence of differential binding on the clinical outcome of P. falciparum infections. Recently, the mutually exclusive transcription paradigm has been called into doubt by transcription assays based on individual P. falciparum transcript identification in single infected erythrocytic cells using RNA fluorescent in situ hybridization (FISH) analysis of var gene transcription by the parasite in individual nuclei of P. falciparum IE1.
Here, we present a detailed protocol for carrying out the RNA-FISH methodology for analysis of var gene transcription in single-nuclei of P. falciparum infected human erythrocytes. The method is based on the use of digoxigenin- and biotin- labeled antisense RNA probes using the TSA Plus Fluorescence Palette System2 (Perkin Elmer), microscopic analyses and freshly selected P. falciparum IE. The in situ hybridization method can be used to monitor transcription and regulation of a variety of genes expressed during the different stages of the P. falciparum life cycle and is adaptable to other malaria parasite species and other organisms and cell types.

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescent in situ hybridization (FISH) to identify mRNA transcripts in individual cells allows analysis of polygenic activity such as the simultaneous transcription of more than one member of the var multigene family in Plasmodium falciparum infected erythrocytes 1. The technique is adaptable and can be used on different types of genes, cells and organisms.

RNA Floresan tarafından Tek hücreli Gen Transkripsiyon Analizi<em&gt; In Situ</em&gt; Hibridizasyon (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Floresan ile birlikte Kromozom çoğaltılıyor Zamanlama<em&gt; Yerinde</em&gt; Hibridizasyon

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Tüm Dağı RNA Floresan<em&gt; In situ</emVe&gt; Hibridizasyon<em&gt; Drosophila</em&gt; Embriyolar

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories 1-5. Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

3D Nükleer teşkilatında değişiklikler Eğitim için 3D-korunmuş Tane Çekirdekle ilgili Kombine İmmünofloresan ve DNA FISH

Fluorescent in situ hybridization using DNA probes  on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA  FISH) represents the ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Floresan kullanarak mRNA Moleküllerin Görselleştirme ve Analiz<em&gt; Yerinde</emIn&gt; Hibridizasyon<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...