Bu eser Singapur NRF bursu (NRF-2011NRF-NRFF001-025) yanı sıra CBRG (NMRC / CBRG / 0070/2014) tarafından finanse edildi.

NOT: Bu çalışma kapsamında ilgi RNA AR 3&#39;UTR&#39;sinden bir parçasıdır. Biotin-etiketli RNA hazırlanması 1. <ol><li> RNA elde edilmesi için, bir ticari kit kullanılarak ve imalatçının talimatları 11, aşağıdaki in vitro transkripsiyon ve ardından, PCR ile, T7-ar-oligo, yükseltmek. Not: PCR primerleri, Tablo 4&#39;te listelenmiştir: T7-AR-F ve T7-AR-R. </li><li> Spesifik olmayan bağlanma kontrol için, in vitro transkripsiyon ve ardından psiCHECK-2 plasmidinden PCR ile ateşböceği lusiferaz (FL) DNA, bir kısmi sekansını yükseltmek. Not: PCR primerleri, Tablo 4&#39;te listelenmiştir: hluc (+) - T7-prob F hluc (+) - sonda-R, Tablo 4 PCR amplifikasyonu için şablon ve primer dizileri... </li><li> 50 | il PCR reaksiyonu için 50 ng DNA (oligo veya plazmit), 4 ul dNTP (her bir dNTP 2.5 mM), 5 ul 10x reaksiyon tamponu, 1 ul 2.5 U / ve karışım #181 L DNA polimeraz, 2 ul 10 uM ileri primer, 2 ul 10 uM ters primeri ve nükleaz içermeyen su. <ol><li> Aşağıdaki programı kullanılarak, bir termal döngü PCR amplifikasyonu çalıştırın. Adım 1: 2 dakika süreyle 95 ° C&#39;lik bir döngü; Adım 2: 30 saniye 95 ° C 35 döngü, 30 saniye için 55 ° C ve 30 sn (AR) ya da 60 saniye (FL) 72 ° C; Adım 3: 10 dakika boyunca 72 ° C&#39;lik bir döngü. </li></ol></li><li> Imalatçının talimatlarına 11 uygulanarak, bir in vitro kopyalama kiti kullanılarak biyotinile edilmiş RNA&#39;ları sentez. Bu deneyde, RNA sentezi için şablon olarak PCR ürünleri kullanır. 20 ul in vitro transkripsiyon reaksiyonu için, 200 ng PCR ile çoğaltılan DNA, her bir NTP 2 ul, 1 ul 10 mM biyotin-CTP 2 ul 10x reaksiyon tamponu ve 2 ul T7 RNA polimeraz karışımı. 4 saat 37 ° C&#39;de inkübe karışımı. </li><li> 37 &amp; # de in vitro olarak transkripsiyonu, 1 ul 2 U / ml DNaz ile muamele reaksiyon karışımının aşağıdakı176 C 15 dk şablon DNA aşağılamak için. Son olarak, daha fazla saflaştınlmadan 60 ul bir toplam hacme biyo-küçültülmüş RNA seyreltin. </li><li> Imalatçının talimatlarına 12 Aşağıdaki saflaştırma sütunu kullanarak tuzlarını ve nükleotidleri gidermek için biyotinile edilmiş RNA&#39;ları saflaştınlır. daha sonra açılır deneyleri için -80 ° C&#39;de RNA konsantrasyonu ve mağaza ölçün. </li><li> 2 dakika, 2 dakika boyunca buzda 90 ° C&#39;de RNA-protein etkileşimi, ısı 50 ul biyotinile edilmiş RNA (50 pM) daki RNA için uygun sekonder yapısını sağlamak için, daha sonra 50 ul 2x RNA yapısı tamponu (Tablo sahip kaynağı 3), ve 30 dakika 13,14 oda sıcaklığında katlama RNA için izin verir. </li></ol> RNA eşlenik boncuklar 2. hazırlanması Not: manyetik boncuklar ve süpernatan ayrılması için manyetik bir stand kullanın. <ol><li> Kısaca vorte orijinal şişede streptavidin kaplı manyetik boncuklar süspanseXING üreticisinin yönergeleri izleyerek. 1.5 ml tüp boncuk yeniden süspansiyon haline getirildi, 150 ul aktarın. 1 dakika boyunca mıknatıs tüp yerleştirin. Süpernatantı atın. boncuk pelet tutun. Not: RNA açılan deneylerinde orijinal şişeden yeniden süspansiyon haline boncuk aliquoting olduğunda, MS, ardından bir deneyde Western blotting ile ve ardından bir deneyde 50 ul boncuk ve 200 ul boncuk kullanır. </li><li> Yeniden süspansiyon üreticinin tavsiye 1x W ve B için 1 ml boncuk pelet (bağlanma ve yıkama) tampon maddesi (Tablo 3) bir kez boncuk yıkayın, bir rotatör içine oda sıcaklığında 5 dakika boyunca tüpün döndürülmesi takip etmektedir. Süpernatantı atın. boncuk pelet tutun. </li><li> 400 ul tampon A Tampon A ile topuk kısmı pelet yeniden süspansiyon haline getirilerek, her boncuk ilgili RNAaz ari inaktive iki boncuk yıkamak için 0.1 M NaOH ve 0.05 M NaCl (Tablo 3) ihtiva eden bir alkali bir çözümdür. rotator oda sıcaklığında 2 dakika boyunca bir tüp döndürün. Süpernatantı atın. boncuk pelet tutun. </li> &lt;li&gt; rotator oda sıcaklığında 2 dakika için tüpün döndürülmesi ve ardından yeniden süspansiyon 400 ul tampon B ile boncuk pelet (Tablo 3) ile iki kez, boncuk yıkama boncuklardan her NaOH kaldırın. Süpernatantı atın. boncuk pelet tutun. </li><li> 300 ul 2x W &amp; B tamponu ile süspanse boncuk pelet, split boncuk eşit ve üç 1.5 ml tüpler (tüp başına 100 ul boncuklar) içine aktarın. 100 ul RNaz ücretsiz su ile boncuk içeren tüpler kaynağı 100 ul FL RNA ve sırasıyla AR 3&#39;UTR RNA biyotinile 100 ul biyotinile. </li><li> dönüşü ile, oda sıcaklığında 1 saat boyunca, her boncuk karışımı inkübe edin. 1 dakika boyunca mıknatıs üzerinde tüpler yerleştirin. tüm süpernatant atın. boncuk pelet tutun. </li><li> 400 ul 1 x W &amp; B tamponu ile her kordon pelet yeniden süspansiyon haline getirilerek, iki kez her seferinde boncuk yıkayın, bir rotatör içine oda sıcaklığında 5 dakika boyunca tüpler döner eklenmiştir. </li><li> 1 dakika boyunca mıknatıs üzerinde tüpler yerleştirin. tüm süpernatant atın. her kordon Pelle yeniden süspanse50 ul çekirdek yalıtım tamponu (Tablo 3) T. </li></ol> 3. Preadibosit İzolasyon ve adipogenesis İndüksiyon <ol><li> Önceki yayınlarda 5&#39;te tarif edildiği gibi, interskapular kahverengi yağ pedi Preadipositler (C57BL6 vahşi tip fare, 3 haftalık) keser. </li><li> Büyümek ve in vitro preadipositlerden 5 ayırt. Hücreler 4-5 gün farklılaşma başlandıktan sonra aşağı uygulama için hazır. </li></ol> Birincil adipositlerden Hücresel lizat 4. hazırlanması NOT: Hücre lizatı hazırlık tüm işlemleri emin olun buz üzerinde yapılır ve tüm tamponlar buz üzerinde soğutulmuş edilir. buz üzerinde önceden soğuk Dounce cam homojenizatörler. <ol><li> Büyümek ve 15 cm yemekleri 5 (bakınız bölüm 3.2) &#39;de preadipositlerden ayırt. Her yemeğin bir RNA açılan tahlil için yeterli hücreleri sağlar. günde 4 veya farklılaşması, ıskarta hücre kültür ortamının 5 gün ve yıkama CEbir zamanlar 1x PBS ile LLS. </li><li> Oda sıcaklığında 5 dakika boyunca 200 x g&#39;de santrifüj ile 50 ml&#39;lik bir tüp içinde hücreleri ve pelet hücreleri toplamak için kazıma, hücreler hasat hücrelere 10-15 mi 1x PBS ekleyin. 1 ml pipet kullanarak süpernatant atın. </li><li> Tekrar süspansiyon hücre 2 ml pelet hipotonik tampon maddesi (Tablo 3), ve 15 dakika süreyle buz üzerinde hücrelerin inkübe. Transferi 15 ml Dounce cam homojenizatör hücreleri ve mekanik buz üzerinde 20 vuruş ile kesme hücreleri. </li><li> 4 ° C&#39;de 15 dakika boyunca 3300 x g&#39;de santrifüjleme ile pellet hücre çekirdekleri. ileride kullanılmak üzere buz üzerinde çekirdekleri pelet tutmak (bölüm 4.6). 1.5 ml tüpler süpernatantı aktarın, 4 ° C&#39;de 15 dakika boyunca 20,000 x g&#39;de bir son 150 mM konsantrasyonuna ve spin elde etmek üzere süpernatana 3M KCI ekleyin. </li><li> santrifüj sonrasında, dikkatli 1 ml pipet kullanarak üst lipidlayeron kaldırmak ve sitoplazmik hücresel lizat olarak supernatant toplamak. </li><li> çekirdek pelletini 1 ml nükleer izolasyon tamponunda (bakınız bölüm 4.4). Transfer 1 ml pipet kullanarak 15 ml Dounce cam homojenizatör çekirdekler ve mekanik 100 vuruş ile buz üzerinde kayma çekirdekler. 4 ° C&#39;de 15 dakika boyunca 20,000 x g&#39;de santrifüj ile nükleer yıkıntıları pelet haline ve nükleer hücre lizatı olarak supernatant toplamak. </li><li> Ya nükleer ve sitoplazmik hücresel lizatları birleştirmek, ya da aşağı açılan uygulama için, ayrı ayrı fraksiyonu kullanın. Bu tanıtım deneyde, hücre lizatı, bu iki fraksiyon 1 bir hacim oranında birleştirildi: 1 arasındadır. </li><li> 0.5 U / ul son konsantrasyon elde etmek için, bu bölünmemiş hücre lizatı RNaz önleyici. Batı lekeleme için 15 giriş kontrol olarak hücre lizatının bir kenara 100 ul ayarlayın. </li></ol> 5. Bağlayıcı ve RBP elüsyonu <ol><li> Bölünmüş hücre, üç eşit kısma lisatı (1.5 ml bir tüp içinde 1 ml hücre lisatı) ve boş bir kuluçkalanması, FL RNA konjüge ve AR 3&#39;UTR RNA eşlenik boncuklar 4 ° &#39;de 3 saat boyunca, sırası ile, (bakınız bölüm 2.8) Cdönüşü ile. </li><li> 1 dakika boyunca mıknatıs üzerinde tüpler yerleştirin. RNA bütünselliği onaylamak için süpernatanından RNA izolasyonu, ardından 1 ml pipet kullanılarak süpernatan toplamak. Üreticinin talimatlarına (Tablo 2), aşağıdaki bir asit guanidinyum tiosianat-fenol solüsyonu kullanılarak RNA izole edin. ribozomal RNA 28S ve 18S alt birimleri analiz ederek RNA Bozulmamışlık değerlendirin. buz üzerinde boncuk pelet tutun. </li><li> rotator oda sıcaklığında 2 dakika için tüpler döner ve ardından 40 U RNaz inhibitör ve% 0.25 IGEPAL içeren 1 mi, nükleer yalıtım tamponu her kordon topağın tekrar askıda bırakılmasıyla manyetik boncuk altı kez, her seferinde yıkayın. manyetik boncuklar ve süpernatan ayrılması için manyetik bir stand kullanın. tüm süpernatant atın. buz üzerinde boncuk pelet tutun. </li><li> Western blotting için boncuklardan RNA-protein kompleksleri elüte etmek için aşağıdaki adımları kullanın. İlk olarak, 75 ul nükleer izolasyon tamponunda her boncuk pelet; Daha sonra, 25 ul 4x western blot yükleme buf eklemekfer (SDS içeren) 100 ul toplam hacim elde edilmiştir. Son olarak, 5 dakika boyunca 95 ° C &#39;de, bu 100 ul çözelti içinde boncuk kaynatın. NOT: adımları MS için boncuk RNA-protein kompleksleri Zehir için aşağıdaki. Adım 1: 100 ul elüsyon tamponu (Tablo 3), her topuk kısmı pelet; Adım 2: rotasyon ile oda sıcaklığında 2-3 saat süreyle boncuk örnekleri inkübe; Adım 3: santrifüjden sonra protein ihtiva eden süpernatan toplamak; Adım 4: MS için sunulmadan önce 20-30 ul protein çözümleri konsantre. </li><li> Santrifüj, her 100, 4 ° C&#39;de 30 saniye boyunca 12,000 x g&#39;de boncuk örnek ul ve supernatant toplanır. western blot analizi (Bölüm 6.1 ve 6.2) için Kısım 15 ul süpernatant. gece boyunca, (1000 sulandırma 1) ile seyreltildi Hur fare monoklonal antikoru ile elde edilen membran kontrol edin. </li></ol> RBP doğrulanması için 6. Western Blot <ol><li> standart teknikler kullanılarak, SDS-PAGE ile ayrı RBPS. </li><li> biz Davranışkıç ilgi RNA ile ilişkili RBPS için sondalama tarafından standart teknikler kullanılarak kurutma. NOT: Bu demo deneyde, Hür fare monoklonal antikoru Hur proteininin İmmuno için kullanılmıştır. </li><li> seyreltilmiş Hür antikor ile sonuçlanan membran inkübe: a / h yağsız süt, 1x TBS% 5 (1 1,000 seyreltme), % 0.1 Tween - 20 4 at Gecede, nazik sallayarak ° C. 1x TBST ile membran üç kez yıkayın. 1 saat süre ile oda sıcaklığında (anti-fare IgG-HRP işaretli keçi) ikincil antikor ile membran inkübe edin. Yıkama membran. Geliştirmek ve kayıt sinyali. </li></ol>

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Çıkar çatışması ilan etti.

lncRNAs ve RBPS ancak bu moleküllerin moleküler mekanizmalar tam olarak anlaşılmamıştır, hastalık ve sağlıkta önemli roller üstlenir. lncRNA molekülleri ile etkileşime proteinlerin tanımlanması düzenleyici mekanizmaların tanıtılması yolunda önemli bir adımdır. RNA açılan deney sistemi ile RBPS zenginleştirilmesi, bir hücre lizatından proteinler seçici streptavidin manyetik taneleri bağlanmış biyo-küçültülmüş RNA yemler kullanılarak elde edilebilir, böylece kompleks etkileşim iç...

A correction to the author list was made to: <a href="http://www.jove.com/video/54207/detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte">Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture</a>.
The author list has been updated from:
Qianfan Bai1*, Zhiqiang Bai1*, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
*These authors contributed equally
to:
Qianfan Bai1*, Zhiqiang Bai1*, Shaohai Xu3, Lei Sun1,2
1Cardiovascular and Metabolic Disorders Program, Duke-NUS Medical School
2Institute of Molecular and Cell Biology, Singapore
3Division of Bioengineering, School of Chemical &#38; Biomedical Engineering, Nanyang Technological University
*These authors contributed equally

A correction to the author list was made to: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture.

Erratum: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RBPS hücrelerde, tek veya çift sarmallı RNA bağlanan ve RNA-protein komplekslerinin oluşturulması katılmak proteinlerdir. RBPS mRNA ve uzun kodlamayan RNA&#39;lar (lncRNAs) de dahil olmak üzere RNA türlerinin, çeşitli bağlamak, ve post-transkripsiyonel seviyede nüfuzlarını olabilir. Hem RBPS ve lncRNAs adipoz geliştirme gibi yeni regülatörleri ortaya çıkan ve 1,2,3 işlev görmektedir. özel bir RNA molekülü ve bir ya da daha RBPS arasındaki etkileşimi saptamak için çoğu zaman gerekli olan, RBP- mekanizmasını ve hücresel yolların lncRNA-aracılı düzenlenmesinin anlamak için, ve bazen de bir RNA protein ortakları tam spektrum tespit etmek transkript. Ancak, deneme nedeniyle adipositlerdeki lipidlerin içeriği yüksek zor olabilir. Burada tarif edilen RNA açılan protokolü primer kültürlerinde 4,5 arasında adiposit lisatlanndan özel bir RNA protein ortakları almak için de kullanılabilir. Bu proto Gerekçesişöyle col özetlenmiştir. Bir RNA yem, bir DNA şablonu in vivo transkribe olan, biyotin-etiketli streptavidin kaplı manyetik boncuklar 4 ve konjuge. RNA, yem, daha sonra, bir manyetik stand aşağı çekilir RNA-protein kompleksleri oluşumuna olanak sağlamak için hücresel lizatları ile inkübe edilir. Daha spesifik olarak, RNA açılan protokolü aşağıda açıklanan HÜR proteini, birincil kültür theadipocytelysate gelen mRNA 6,7 3&#39;UTR&#39;sinden bağlı bir evrensel ifade RBP almak için yem olarak AR 3&#39;UTR&#39;sinden bir RNA parçası kullanın. Bu deneyde bağlanma spesifitesini test etmek için, uygun olmayan bir RNAas da boş streptavidin boncuk kontrol olarak dahil edilir. Bu protokol belirli RBP yakalama teyit etmek veya çekilen RBPS sırasıyla 8 tam repertuarını tespit etmek western blotting ya da kütle spektrofotometresi (MS) ile uyumludur. Çeşitli teknikler RNA-protein etkileşimi incelemek için kullanılabilirs. Belirli bir RBP, RIP (RNA immünopresipitasyon) ve CLIP (UV çapraz ve immünopresipitasyon) bağlı RNA&#39;lar ortaya çıkarmak için uygulanabilir. Buna karşılık, belirli bir RNA protein ortakları belirlemek için, RNA açılır, cıvıltı (RNA saflaştırma ile kromatin izolasyon), ABC (RNA hedeflerinin yakalanması hibridizasyon analizi) ve RAP (RNA antisens arıtma) uygulanabilir. Daha sonra olanlar ile karşılaştırıldığında, RNA açılan teknik kurmak için daha az çaba gerektirir. In vitro ve in vivo proteinler yakalamak için kullanılabilir. Onun in vitro meslektaşı daha teknik olarak daha zor RNA pull, in vivo yaklaşım hücrelerinde çapraz bağlama RNA-protein etkileşimleri korur, hücrelerin ilgi aptamer-etiketli RNA&#39;lar yakalar ve daha sonra bağlı RBPS algılar. RNA açılan düşük bol RBPS ettirecek ve izole edilmesi ve hücresel düzenleme 9,10 kontrol farklı fonksiyonel rolleri var RNA-protein kompleksleri tespit etmek için kullanılabilir </s&gt; Yukarı.

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			<td height="21" style="height:21px;">NucAway spin columns&#160;</td>
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			<td height="21" style="height:21px;">Goat anti-mouse IgG-HRP(sc-2005)&#160;</td>
			<td>Santa Cruz Biotechnology</td>
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			<td height="21" style="height:21px;">Hypure Molecular Biology Grade Water (nuclease-free)&#160;</td>
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			<td height="21" style="height:21px;">Pfu Turbo DNA polymerase&#160;</td>
			<td>Agilent Technologies</td>
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			<td height="21" style="height:21px;">TRIsure</td>
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			<td height="21" style="height:21px;">Major equipment used in this study.</td>
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			<td height="21" style="height:21px;">Sorvall Legend Micro 21R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
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			<td height="21" style="height:21px;">Sorvall Legend X1R centrifuge&#160;</td>
			<td>Thermo Scientific</td>
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			<td height="21" style="height:21px;">Bio-Rad T100 thermal cycler&#160;</td>
			<td>Bio-Rad</td>
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			<td height="21" style="height:21px;">Nanodrop 2000&#160;</td>
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			<td height="21" style="height:21px;">BOECO rotator Multi Bio RS-24&#160;</td>
			<td>BOECO,Germany</td>
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			<td height="21" style="height:21px;">Protein gel electrophoresis and blotting apparatus&#160;</td>
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detection of rna-binding proteins by in vitro rna pull-down in adipocyte culture

RNA bağlayıcı proteinler (RBPS) adipoz gelişiminde ve fonksiyonunda bir düzenleyici bir tabaka olarak ortaya çıkmaktadır. RBPS, hedef mRNA stabilitesi ve dönüşüm etkinliğini etkileyerek transkripsiyon sonrası seviyelerde gen ifadesinin düzenlenmesinde önemli bir rol oynamaktadır. RNA açılan tekniği yaygın mekanizmasının RBPS &#39;hem de uzun kodlayıcı olmayan RNA&#39;lar&#39; (lncRNAs) işlevini yatan aydınlatmak için gerekli olan RNA-protein etkileşimlerini incelemek için kullanılmıştır. Ancak, adipositlerde yüksek lipid bolluk bu deneyi yapan bir teknik sorun teşkil etmektedir. İşte detaylı RNA açılan protokol birincil adiposit kültürü için optimize edilmiştir. androjen reseptörün (AR) 3 &#39;çevrilmemiş bölge adiposit lystate gelen, onun RBP ortağı Hür proteini almak nasıl göstermek için bir örnek olarak kullanılan bir adenilat-üridilat zengin elementwas içeren (3&#39;UTR) bir RNA fragmanı. Burada tarif edilen yöntem ile etkileşimini tespit etmek için uygulanabilirRBPS ve kodlayıcı olmayan RNA&#39;lar, hem de arasındaki RBPS ve kodlama RNA.

Bu tanıtım deneyde, AR RNA fragmanı kendi bağlanma proteini Hur yakalamak için yem olarak kullanılmıştır. Hem Hur proteinine ilgili olmayan bir FL RNA yem ve konjüge edilmemiş boş streptavidin boncuk bir alikosu negatif kontrol olarak görev yapmıştır. RNA-protein etkileşimleri çekirdek veya sitoplazmada ya da oluşabilir ve bu açılan protokolü total veya parçalanmış (nükleer ve sitoplazmik) hücre lizatlarında, ya da uygulanabilir. Western blotting ile proteinle...

Watch this Scientific Journal Video about Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture at JoVE.com

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

Bir RNA açılan protokolü RNA bağlayıcı proteinlere (RBPS) ve kodlama yapmayan ve kodlama RNA arasındaki etkileşimin saptanması için optimize edilmiştir. androjen reseptörü (AR) için bir RNA fragmanı primer kahverengi adipositlerin lystate onun RBP almak için nasıl kullanılacağını göstermek için bir örnek olarak kullanılmıştır.

RNA bağlama proteinlerinin tespiti ile<em&gt; İn Vitro</emAdiposit Kültür&gt; RNA pull-down

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

detection-rna-binding-proteins-vitro-rna-pull-down-adipocyte

Research

JoVE Journal

Biology

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

23.5K Görüntüleme.  Duke-NUS Medical School.  Bir RNA açılan protokolü RNA bağlayıcı proteinlere (RBPS) ve kodlama yapmayan ve kodlama RNA arasındaki etkileşimin saptanması için optimize edilmiştir. androjen reseptörü (AR) için bir RNA fragmanı primer kahverengi adipositlerin lystate onun RBP almak için nasıl kullanılacağını göstermek için bir örnek olarak kullanılmıştır. 

Video: Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

PAR-Klip - RNA Cilt Proteinler Transcriptome geniş Cilt Siteleri Belirlemek Amacıyla Bir Yöntem

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Biyoloji

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Sitokrom Sitoplazmik Mikroenjeksiyon Apoptoz aktivasyonu C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

Pull-down of Calmodulin-binding Proteins

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca2+ (Ca2+-CaM) as opposed to its Ca2+-free state (ApoCaM)3.
While most target proteins bind Ca2+-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin4, neurogranin (Ng)5, and certain myosins6. These proteins have been shown to play important roles in presynaptic function7, postsynaptic function8, and muscle contraction9, respectively. Their ability to bind and release CaM in the absence or presence of Ca2+ is pivotal in their function. In contrast, many proteins only bind Ca2+-CaM and require this binding for their activation. Examples include myosin light chain kinase10, Ca2+/CaM-dependent kinases (CaMKs)11 and phosphatases (e.g. calcineurin)12, and spectrin kinase13, which have a variety of direct and downstream effects14.
The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca2+-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca2+-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.
Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca2+, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca2+ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca2+/CaM signaling and proteins that interact with CaM.

Calmodulin (CaM) pull-down assay is an effective way to investigate the interaction of CaM with various proteins. This method uses CaM-sepharose beads for efficient and specific analysis of CaM-binding proteins. This provides an important tool to explore CaM signaling in cellular function.

Kalmodulin bağlayıcı Proteinler, Pull-down

Calcium (Ca2+) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca2+ signaling is mediated through the ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa Hücre Transfeksiyon Ardından Gaussia Lusiferaz mRNA in vitro transkripsiyon ve Kapatma yılında

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA Saflaştırma tarafından Kromatin İzolasyon (chirp)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Yetişkin Fare Kalvaryal Kemik İliği içinde Hematopoietik kök hücrelerin Vivo 4-Boyutlu Takip

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular biology has been greatly fueled by the discovery of noncoding RNAs as major players in post-transcriptional regulation. Such a revolutionary perspective has been accompanied and triggered by the development of powerful technologies for profiling short RNAs expression, both at the high-throughput level (genome-wide identification) or as single-candidate analysis (steady state accumulation of specific species). Although several state-of-art strategies are currently available for dosing or visualizing such fleeing molecules, Northern Blot assay remains the eligible approach in molecular biology for immediate and accurate evaluation of RNA expression. It represents a first step toward the application of more sophisticated, costly technologies and, in many cases, remains a preferential method to easily gain insights into RNA biology. Here we overview an efficient protocol (Enhanced Northern Blot) for detecting weakly expressed microRNAs (or other small regulatory RNA species) from Drosophila melanogaster whole embryos, manually dissected larval/adult tissues or in vitro cultured cells. A very limited amount of RNA is required and the use of material from flow cytometry-isolated cells can be also envisaged.

Enhanced Northern Blot Detection of Small RNA Species in Drosophila Melanogaster

The aim of this publication is to visualize and discuss the operative steps of an Enhanced Northern Blot protocol on RNA extracted from Drosophila melanogaster embryos, cells, and tissues. This protocol is particularly useful for the efficient detection of small RNA species.

Küçük RNA Türlerin Northern Blot Algılama geliştirilmiş<em&gt; Drosophila melanogaster</em

The last decades have witnessed the explosion of scientific interest around gene expression control mechanisms at the RNA level. This branch of molecular ...

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures (MAAC)

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments for T2D, more physiologically relevant in vitro adipocyte models are required. This study describes a new technique to isolate and culture mature human adipocytes. This method is entitled MAAC (membrane mature adipocyte aggregate culture), and compared to other adipocyte in vitro models, MAAC possesses an adipogenic gene signature that is the closest to freshly isolated mature adipocytes. Using MAAC, adipocytes can be cultured from lean and obese patients, different adipose depots, co-cultured with different cell types, and importantly, can be kept in culture for 2 weeks. Functional experiments can also be performed on MAAC including glucose uptake, lipogenesis, and lipolysis. Moreover, MAAC responds robustly to diverse pharmacological agonism and can be used to study adipocyte phenotypic changes, including the transdifferentiation of white adipocytes into brown-like fat cells.

Isolation and Culture of Human Mature Adipocytes Using Membrane Mature Adipocyte Aggregate Cultures MAAC

Membrane mature adipocyte aggregate culture (MAAC) is a new method to culture mature human adipocytes. Here we detail how to isolate adipocytes from human adipose and how to set up MAAC.

Membran Olgun Adiposit Agrega Kültürleri (MAAC) Kullanarak İnsan Olgun Adipositlerin İzolasyon ve Kültürü

White adipose tissue (WAT) dysregulation plays a central role in development of insulin resistance and type 2 diabetes (T2D). To develop new treatments ...