Yazarlar mükemmel teknik yardım için parazit ve Christina Holm genotipleme için Michael Alifrangis ve Ulla Abildtrup teşekkür etmek istiyorum. Bu çalışma Howard Hughes Tıp Enstitüsü (hibe 55005511), Lundbeck Vakfı (hibe R9-A840) tarafından ve Niels Bohr Vakfı tarafından finanse edildi.

1. Taze Seçilen Enfekte Eritrositler Nesil PfEMP1 proteininin yüzeyde ekspresyonu için seçilen taze kültür kullanılarak, bu test için, en iyi sonuçlar elde edilir. Bu özel deney 3D7 P. falciparum soyu önce 1 açıklandığı gibi spesifik antikorlar kullanılarak seçildi. 1. Gün <ol><li> Bir S. dan paketlenmiş kan hücrelerinin hasat 200 ul falciparum kültürü oda sıcaklığında 8 dakika boyunca 800 x g&#39;de santrifüj ile% 2-5 geç safha IE içeren. </li><li> 37. 2 ml kan pelet yeniden askıya ° C sıcak% 0.75 jelatin bir steril 14 ml tüp içinde çözelti ve tortu ile enfekte olmamış ve halka-aşamalı IE izin vermek için 37 ° C sıcaklıkta 15-20 dk için bırakın. </li><li> Son aşama IE hasat etmek, yeni bir 14 ml tüp içine, üst faz, yani, 37 ° C sıcak RBC-yıkama ortamı 10 mL ile iki kez yıkanır. </li><li> Belirli bir anti-PfEMP 50 ul seyreltin37 2 ml ° C sıcak RBC yıkama medya ve steril filtre 1 antikorları. </li><li> 37, 30 dakika boyunca 14 ml steril tüp ile inkübe steril seyreltilmiş antiserumlar ° hafif ajitasyon ile C ile yıkanmış son aşama IE karıştırın. </li><li> Oda sıcaklığında 8 dakika boyunca 800 xg&#39;de aşağı Spin, ve 37 ° C&#39;de sıcak bir RBC-yıkama ortamı 10 mL ile iki kez yıkanır. </li><li> Bir DynaMaq-15 mıknatıs kullanarak RBC-yıkama medya ile bir süspansiyon kez Dynabead Protein 50 ul yıkayın. </li><li> 300 ul RBC yıkama medya Dynabeads süspanse ve yıkanmış geç evre IE ekleyebilirsiniz. Nazik çalkalama ile 37 ° C &#39;de 30 dakika süre ile inkübe edin. </li><li> DynaMaq-15 mıknatıs tüp aktarın. 1-2 dakika bırakın ve tüm sıvı kaldırmak. </li><li> RBC-yıkama ortamı 4-5 ml Dynabeads yeniden süspanse edin. Mıknatıs üzerine tüpü geri aktarın ve tüm sıvı çıkarmadan önce 1-2 dakika bekletin. Kez yıkama adımı yineleyin. </li><li> 25 cm 2 steril kültür f yıkanmış boncuklar aktarın200 ul taze paketlenmiş kırmızı kan hücreleri içeren LASK. Kültürün içine Albumax medyanın 5-6 ml ekleyin. 37% 5 CO2 de bir gece ° C&#39;de saklayın </li></ol> 2. Gün <ol start="12"><li> Seçilen IE bir steril 14 ml tüp için tüm kültür aktararak taze kırmızı kan hücreleri reinvaded olan kültürden Dynabeads çıkarın. DynaMaq-15 mıknatıs üzerine tüpü koyun ve 1-2 dakika bekletin. Daha sonra, bir kültür şişesine tüm sıvı geri dökülür. </li><li> Olduğunca az% 5-10 parazitemi kadar mümkün olduğunca döngüleri ve parazitler için Kültür ringstage altındadır. </li><li> IE doğru yüzey fenotipini ya PFD1235w and/orPF11_0008 yani protein ekspresyonu 1 elde edilmiştir emin olmak için FACS tarafından analiz edilmelidir. </li></ol> 2. Sondalar hazırlanması Antisense RNA probları PFD1235w ve PF11_0008 var olan en değişken bölgelerin elde edildi &lt;P. den / em&gt; genler falciparum 3D7 genomik DNA. DNA, üreticinin tanım (Roche) &#39;ye göre, sırasıyla, PCR ile amplifiye edilir ve transkripsiyon için pSPT18 vektör ya da 19 içine klonlandı. Sondalar (580 baz çifti (bp) ve uzunluğu 590 bp) sırasıyla DIG RNA etiketleme Kiti veya Biyotin RNA etiketleme Mix kullanarak digoxigenin (DIG) veya Biyotin ile işaretlendi. İkimiz de Northern blot analizleri ile ve tek etiketli FISH analizi 1 ile probların özgüllüğü doğruladı. 3. İn Situ Hibridizasyon İnce Smear ve öncesinde Parazitler Fiksasyon 1. Gün Tüm adımlar RNase içermeyen bir ortamda gerçekleştirilir ve tüm reaktifler RNase içermeyen ya da dietil pyrocarbonate (DEPC) ya da RNaz Zap (Invitrogen) ile önceden tedavi edilen vardır. Slaytlar ve kapak camları artık üretim gres kaldırmak için alkol ile temizlenmelidir. Bu adımlar arasında herhangi bir duraklama olmadan protokolü gerçekleştirmek için önemlidirnükleaz kontaminasyon riskini en aza indirmek için. <ol><li> Standart yayma yöntemi 3 ile ince bir kan film yapın. Mikroskop 100x yağı objektif lens kullanarak IE saymak ve kültür IE yüzdesi hesaplanır. Parazit aşamalarını halka ve IE döngüsünün erken trofozoit aşamalarında, yaklaşık senkronize olduğunu kontrol edin. Bu ön-çoğaltma, tek çekirdekli aşamaları, ifade var gen mRNA ve bu tip tek bir hücre FISH transkripsiyon testleri için de uygundur. </li><li> 1.5 ml RNaz ücretsiz Eppendorf tüp,% 5-10 parazitemi de, parazitin kültür 50 ul aktarın. Oda sıcaklığı altında 2.000 rpm hızında 1 dakika için santrifüjleyin. </li><li> Ortamı çıkarın ve DEPC-işlenmiş su 50 ul PBS pH 7.2 ekleyin. Tüpü yavaşça çalkalayın. Medya ve hücre artıkları ücretsiz hücreleri yıkamak için iki santrifüj sedimantasyon adımı yineleyin. </li><li> Dört kaliteli ince smear olun. Her smear için, üzerine bir karalama yapmakiyi bir RNaz ücretsiz kaputu bir 4 sıra slayt, toplam yani dört cam slaytlar, (önemli bir adım) biri 11 mm çapında. Dalga kuru havaya kısaca kayar. Belirli bir prob, RNaz tedavi ve ilgilenilen genin ifade IE içeren üçüncü bir slayt için başka bir slayt kullanarak başka alakasız gen için tek prob boyama için kontroller-bir negatif slayt için üç ekstra slaytlar olun. 10-20 dakika tezgah üzerinde kurumasına slaytlar bırakın. </li><li> Kuyu içine% 4 paraformaldehid ve% 5 asetik asit içeren, tespit solüsyonu 60 ul ilave edilerek ince filmlerin düzeltildi. Oda sıcaklığında, tezgah üzerinde 10 dakika bekletin. </li><li> , Oda sıcaklığında nazik çalkalama ile 5 dakika için 2 x SSPE tampon içinde slaytlar yıkayın. Kısaca bir kağıt mendille hafifçe hücreleri içeren kuyular dokunarak aşırı sıvı kaldırmak. </li><li> 37 ° C&#39;de (çok önemli bir adım 2 dakika süreyle, 0.01 M HCl içinde% 0.01 pepsin ile slaytlar Kapak). Oda sıcaklığında 5 dakika için 2 x SSPE, slaytları yıkayın. </li><li> 10 ug / ml &#39;lik nihai bir konsantrasyona kadar, RNaz çözeltisi ile seyreltilir. RNaz solüsyonu 60 ul ile negatif kontrol yayma kaplayın. 37 ° C de 30 dakika inkübe ve daha sonra da oda sıcaklığında 5 dakika için 2 x SSPE, slaytları yıkayın. </li><li> Hibridizasyon adımı hemen geçin. </li></ol> 4. Sabit Slaytlara RNA problar Hibritleşmesi 1. Gün <ol><li> Böyle bir Thermostar 100 HC4 veya RNaz Zap ile püskürtme ve bir kağıt mendille silerek temizleyin, temiz bir fırın koymak boş bir pipet ucu kutusu olarak hibridizasyon odasının, hazırlayın. Hibridizasyon odasının veya slaytlar hibridizasyon sırasında kurumasına olmayacak emin olmak için DEPC-işlenmiş su ile kutunun alt kanalları doldurun. 48 odası ve ön ısıtma kapatın ° C. </li><li> 12 ng / ul nihai konsantrasyon için hibridizasyon probları çözelti içine seyreltilir. Bir ısıtma bloğu içinde 5 dakika için 65 ° C sonda çözelti denatüre. Hemen buz üzerinde soğutun. </li><li> Kısaca hava 2x SSPE yıkamadan sonra slaytları kurulayın. Dikkatlice bir kağıt mendille kuyu çevresinde aşırı sıvı kaldırmak. </li><li> Hibridizasyon odasının açın ve odasında slayt yerleştirin. Bir ya da kuyu başına 60 ul toplam hacim içinde, her iki sensör uygulanır. Dikkatlice steril forseps kullanarak bir RNaz içermeyen kapak kayma ile sıvı damlası kapsamaktadır. </li><li> Oda kapatın ve gece boyunca en az 16 saat süreyle 48 ° C &#39;de slaytlar melezleşir. </li></ol> 5. Antikor Konjugasyon ve Floresans Amplifikasyon 2. Gün Aşağıdaki adımları Perkin Elmer dan TSA Artı Flüoresans Palet sistemi dayanmaktadır. Tüm adımlar oda sıcaklığında yapılır. <ol><li> Nazik ajitasyon ile 5 dakika boyunca TNT tampon slaytlar 3 kere yıkayın. </li><li> 30 dakika için TNB tampon 150 ul kuyu Blok. </li><li> 1:500 oranında, TNB tamponu içinde 1:100 &#39;lük bir oran ile HRP-eşlenikli α-biotin antikor de TNB tampon içinde HRP-eşlenikli α-DIG antikoru seyreltilir. Bir kağıt mendille slaytlar emdiği TNB tampon çıkarın. Aynı anda iki transkript tespit edildiğinde her iki antikor seferde gidebilirsiniz. Kuyu başına antikor-TNB çözeltisinden 100 ul toplam miktar uygulanır ve dikkatli bir şekilde bir kapak slipi ile kaplayın. 2 saat için slaytlar inkübe edin. </li><li> Dikkatle kapak camları çıkarın. 5 dakika boyunca 3 kez TNT tampon slaytlar yıkayınız. </li><li> 1:500 oranında tyramide amplifikasyon reaktif içinde FITC-fluorofor seyreltilir ve her oyuğa 100 ul çözelti uygulanır. Kapağı makbuzları ile kuyu örtün ve 12 dakika inkübe edilir. </li><li> Dikkatlice kapağı fişleri çıkarın ve yavaşça sallanarak TNT tamponu içinde 5 dakika boyunca slaytlar 3 kere yıkayın. </li><li> 1:500 oranında tyramide amplifikasyon reaktif olarak Cyan3-fluorofor seyreltilir. Başına 100 ul ekleyinBu çözüm de. Kapağı makbuzları ile kuyu örtün ve 12 dakika inkübe edilir. </li><li> Kapağını çıkarın dikkatli kayıyor ve sonra TNT tampon batırılarak hızla slaytlar durulayın. </li><li> 5 dk için TNT tampon ile slaytlar 3 kere yıkayın. </li><li> DEPC-işlenmiş su hızlı bir şekilde slaytlar daldırın. </li><li> Kurumaya slaytlar bırakın. DAPI içeren anti-solmaya reaktifi ile slaytlar monte edin. Oje ile kapak camları köşelerinde mühürleyin. </li><li> Slaytlar artık mikroskopi için hazırız. Ol 4&#39;te alüminyum folyo ile kaplanmış ve mağaza slaytlar ° C deney ertesi gün tamamlanması için ise. Slaytlar iki tam bir sızdırmazlık solmaya karşı reaktif uygulamadan sonra 24 saat üzerinden gerçekleştirilebilir. Bu protokol tamamlandıktan sonra, slaytlar bir hafta için yansıma sağlar. </li></ol> 6. Melezleşmiştir Sondalar Görselleştirme <ol><li> Mikroskop açın. Standart bir immünofloresan mikroskop suffic olduğunuEğer bir konfokal mikroskop erişiminiz varsa deney bu tür ient, ama aynı zamanda 3D görüntüleri için kullanabilirsiniz ya da boyanmış hücrelerin videolar elde etmek. Mikroskop 15-30 dakika ısınmasını bekleyin. Bilgisayar ve yazılım açın. </li><li> Immünofloresan mikroskop boyama kalitesini kontrol edin. Birkaç parazitler içeren bir nokta seçin. </li><li> Elle her lazerin kazanç ayarlayın. Piksel 4-6 m -1 s -1 ve 512 x 512 piksel adımları yaşamak ayarlayın. </li><li> Çerçeve Lambda kullanarak bir test resim çekin. Lazer kazanç yeniden ayarlayın. </li><li> Floresan tek hücre 20 iyi resimler en az atın. 5-20 parazitler içeren bir alanda en az 2-5 fotoğraf pozitif parazitlerin yüzdesi adil bir bakış elde etmek için gereklidir. </li></ol>

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Çıkar çatışması ilan etti.

RNA FISH analizi, bu gibi Northern blot ve RT-PCR gibi yöntemlerin tersine, tek bir hücre düzeyinde spesifik mRNA transkriptlerinin ayrımı izin verir. Bu, P., bu örnekte, bu mümkün transkripsiyonel olarak aktif ve aktif olmayan hücreler arasında ayrım yapan falciparum parazitik insan kırmızı kan hücreleri içinde protozoa. Böyle tam hücreli gözlemler genelde gereklidir ve önemlidir ve roman transkripsiyonel desenler 1 çözülmeye olabilir. Di?...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> Asetik asit </td><td> Sigma / Aldrich </td><td> 338826-100ml </td><td></td></tr><tr><td> Albumax medya: RPMI 1640 Glutamine çözümü </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml RPMI 1640 5 ml çözelti glutamin </td></tr><tr><td> Albumax medya: Gentamisin sülfat Albumax-çözüm </td><td> Lonza </td><td> BE02-012E </td><td> 2.5 ml gentamisin sülfat 50 ml Albumax çözelti </td></tr><tr><td> Albumax çözelti: Hipoksantin </td><td> Sigma-Aldrich </td><td> H9377 </td><td> 0,8 g Hipoksantin </td></tr><tr><td> Albumax çözelti: AlbuMAX II </td><td> Bennvitrogen </td><td> 11021-037 </td><td> 200 g Albumax </td></tr><tr><td> Albumax çözelti: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 4 litre RPMI 1640 Max mıknatıslı çözülür. 50 ° C. Filtre sterilize ve alikotları -20 ° C&#39;de saklayın. </td></tr><tr><td> Amberlit </td><td> Sigma </td><td> A5710-110G&#39;nin </td><td> Formamid Deiyonize için Reçine. </td></tr><tr><td> Anti-biotin keçi pAb Peroksidaz Konjugat </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> Anti-Dig antikoru </td><td> Novus Biyolojik </td><td> NB100-41330 </td><td></td></tr><tr><td> DAPI ile Anti-solmaya reaktifi </td><td> Invitrogen </td><td> P36931 </td><td> Montaj medya tamamen slayt sızdırmazlık önce 24 saat boyunca tedavi vardır. </td></tr><tr><td> Biotin RNA Etiketleme Mix </td><td&gt; Roche </td><td> 11685597910 </td><td></td></tr><tr><td> Kamera dijital </td><td> Nikon dijital görme DC-F11 </td><td></td><td></td></tr><tr><td> Lameller </td><td> Menzel-Glaser </td><td> 631-1570 </td><td></td></tr><tr><td> kültürü şişesi 25 cm 2 Nunclon yüzey </td><td> Nunc </td><td> 156340 </td><td></td></tr><tr><td> DEPC </td><td> Fluka / Sigma </td><td> 32490-100ml </td><td> 1000 ml su içinde deinonized 1 mL DEPC. Bir karıştırma çubuğu ekleyin ve 12 saat için karıştırın. 30 dakika süreyle otoklav. </td></tr><tr><td> Dijital fotoğraf makinesi </td><td> Nikon dijital görme DC-F11 </td><td></td><td></td></tr><tr><td> Dynabeads Protein A </td><td> Invitrogen </td><td> 10002D </td><td></td></tr><tr><td> DynaMaq-15 Mıknatıs </td><td> Invitrogen </td><td> 123-01D </td><td></td></tr><tr><td> Formamid bioultra 99% </td><td> Fluka/ Sigma </td><td> 47671-1l-F </td><td> Deiyonize formamid: formamid 100 ml iyon değiştirme reçinesi 5 g. 30 dakika karıştırın. Whatman kağıt süzülür. </td></tr><tr><td> . Jelatin 0 75% çözüm: Jelatin </td><td> Sigma-Aldrich </td><td> G2500 </td><td> 500 ml RPMI 1640 içerisinde 3.75 g jelatin. 56 Isı ° C eritin. Zaman 56 ° C sterilize Filtre Alikots içinde -20 ° C&#39;de saklayın. </td></tr><tr><td> . Jelatin 0 75% çözüm: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml RPMI 1640 içerisinde 3.75 g jelatin. 56 Isı ° C eritin. Zaman 56 ° C sterilize Filtre Alikots içinde -20 ° C&#39;de saklayın. </td></tr><tr><td> Glutamin çözüm: L-glutamin </td><td> Sigma-Aldrich </td><td> G3126 </td><td> 500 ml% 0,9 NaCl içinde 14,6 g L glutamin. Dağılma, filtre sterilize ve alikotları -20 ° C&#39;de saklayın. </td></tr><tr><td> Glutamin çözüm: HCl </td><td> Sigma </td><td> H1758 </td><td> 500 ml% 0,9 NaCl içinde 14,6 g L glutamin. Dağılma, sterilize filtre ve alikotları -20 ° C&#39;de saklayın. </td></tr><tr><td> Hibritleşme çözüm: Formamid Bio ultra% 99 SSC 20x </td><td> Fluka / Sigma </td><td> 47671-1l-F </td><td> Toplam 20 ml -20 donmuş tutmak ° C alikotları 10 ml deiyonize formamid </td></tr><tr><td> Hibritleşme çözüm: Denhardt kullanıcısının 50x konsantresi </td><td> Sigma </td><td> D2532 </td><td> 5ml 20xSSC </td></tr><tr><td> Hibritleşme çözüm: Maya tRNARoche engelleme reaktif </td><td> Sigma </td><td> R-6750 </td><td> 2 ml 50x Denhardt sitesi Ml maya tRNA başına 250 ul 20 mg </td></tr><tr><td><sSomon Sperm DNA:&gt; Hibridizasyon çözümü Trong </td><td> Fluka / Sigma </td><td> 31149-106GF </td><td> 0,4 g Roche reaktif engelleme Ml somon spermi DNA başına 10 mg (: denatüre somon spermDNA 96 5 dakika için ° C hibridizasyon çözeltisi ilave edilmeden önce Kritik) 1 mL </td></tr><tr><td> Hybridizer Thermostar 100 HC4 </td><td> Quantifoil Instruments GmbH </td><td> 1004-0011 </td><td> Hibridizasyon fırın ve DEPC su ile doldurulur RNaz ücretsiz hibridizasyon odaları tarafından değiştirilebilir. </td></tr><tr><td> Daldırma yağı UV şeffaf floresans ücretsiz </td><td> Sigma </td><td> 10976-1EA </td><td></td></tr><tr><td> İmmünofloresan veya konfokal mikroskop </td><td> Nikon D-Eclipse TE2000C </td><td></td><td></td></tr><tr><td> Nailpolish </td><td> Herhangi bir eczane mevcuttur </td><td></td><td></td></tr><tr><td> PBS 20x: NaCl KCl Na 2 HPO 4 x 2H 2 O KH 2 PO 4 DEPC deiyonize H 2 O </td><td></td><td></td><td> PH 7.4 &#39;e Ajust 160 g 4 g 23 g 4 g 1 l </td></tr><tr><td> Paraformaldehyde 4% </td><td> Fluka / Chemika </td><td> 76240 </td><td> 80 ml PBS / DEPC içinde 4 g PFA. PFA eriyene ° C kadar 65 Isı. 20 ml PBS ekleyin, çözüm soğumasını bekleyin. PH 7.4 &#39;e ayarlayın. Filtre. -20 ° C&#39;de alikotları Mağaza </td></tr><tr><td> Paraformaldehyde% 4 / Asit asetik 5% </td><td></td><td></td><td> 950 ul paraformaldhyde + 50 ul asetik asit </td></tr><tr><td> Pepsin </td><td> Sigma / Aldrich </td><td> P7000 </td><td></td></tr><tr><td> Peroksidaz-konjuge anti-biotin </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> RBC yıkama ortamı: RPMI 1640 Glutamine çözümü </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml RPMI 1640 çözeltisi 5 ml glutamin </td></tr><tr><td> RBC yıkama ortamı: Gentamisin sülfat </td><td> Lonza </td><td> BE02-012E </td><td> 2.5 ml gentamisin sülfat </td></tr><tr><td> RNaz </td><td> Sigma / Aldrich </td><td></td><td> Bir 10 mg / ml stok çözeltisi edin. </td></tr><tr><td> RNaz ücretsiz 1.5 ml tüp </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> RNaz Zap </td><td> Ambion </td><td> AM9780 </td><td></td></tr><tr><td> 11mm 4 kuyu Slaytlar </td><td> Thermo Scientific </td><td> MENZXER306W </td><td></td></tr><tr><td> SSC 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> 3 M NaCl (175 g / l), 0,3 M Na sitrat, 3 x H2O (88 g / l), 1M HCI ile pH 7.0 &#39;a ayarlayın </td></tr><tr><td> SSC 20x: Sodyum sitrat dihidrat </td><td> Sigma / aldrich </td><td> W302600 </td><td> 3 M NaCl (175 g / l), 0,3 M Na sitrat, 3 x H2O (88 g / l), 1M HCI ile pH 7.0 &#39;a ayarlayın </td></tr><tr><td> SSPE 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> 175.3 g NaCl </td></tr><tr><td> SSPE 20x: NaH 2 PO 4 </td><td> Sigma / Aldrich </td><td> S0751 </td><td> 27.6 g NaH 2 PO 4. </td></tr><tr><td> SSPE 20x: 4 EDTA toz </td><td></td><td></td><td> 9.4 g EDTA tozu DEPC su ekleyin, pH 7.4 ayarlayın. Oto20 dakika için Clave </td></tr><tr><td> TNB tampon: TNT tampon </td><td></td><td></td><td> 10 ml tampon TNT </td></tr><tr><td> TNB tampon: Engelleme reaktifi </td><td></td><td></td><td> 0.05g Engelleme reaktifi Bloke edici reaktif çözmek için, bir taraftan karıştırılarak bir saat süreyle 60 ° C ila çözelti ısıtın. -20 ° C&#39;de saklayın (Perkin Elmer TSA-protokol türetilen.) </td></tr><tr><td> TNT tamponu: Tris / HCl </td><td> Sigma </td><td> T1503 </td><td> 1 M Tris / HCl, pH 8.0 </td></tr><tr><td> TNT tamponu: NaCl </td><td> Sigma Aldrich </td><td> S9625 </td><td> 100 ml </td></tr><tr><td> TNT tamponu: Tween20 </td><td> Sigma </td><td> 93773 </td><td> 5 M NaCl içeren 30 ml 1 ml DEPC dH 2 O869 Ml Oda sıcaklığında, pH 7.5 &#39;e ayarlayın. (Perkin Elmer TSA-protokol türetilen.) </td></tr><tr><td> TSA artı Cyanine3 / Floresein sistemi </td><td> Perkin Elmer </td><td> NEL753000IKT </td><td> Deney başlamadan önce dikkatlice TSA Artı Flüoresans Palet Sistemi protokolü okuyun. </td></tr><tr><td> Steril tüpler 14 ml </td><td> Almeco - CM LAB Aps </td><td> 91016 </td><td></td></tr></tbody></table>

analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)

Sıtma enfeksiyonları sırasında insan endotel reseptörlerine Plasmodium falciparum enfekte eritrositler (IE) Yapışma var genler tarafından kodlanan PfEMP1 protein varyantları ifade aracılık etmektedir. Haploid P. falciparum genom tek bir enfeksiyonun kan aşaması sırasında her defasında hücre başına transkribe olduğu düşünülmektedir edilmiş olan yaklaşık 60 farklı var genler barındırır. Nasıl var gen transkripsiyonu gibi birbirini dışlayan düzenleme elde edilir belirsizdir, bireysel var genleri veya P. klinik sonuçlar üzerinde farklı reseptörleri ve diferansiyel bağlama sonucu ile ilişkili var genlerin alt gruplarının tanımlanmasıdır falciparum enfeksiyonlar. Son zamanlarda, birbirini dışlayan transkripsiyon paradigması bireysel P. esas transkripsiyon analizleri ile şüphe içine adı olmuştur Tek inf içinde falciparum transkript tespitiP. bireysel çekirdeklerinde parazit var gen transkripsiyonu in situ hibridizasyon RNA floresan kullanarak ected eritrotik hücreleri (FISH) analizi falciparum IE 1. Burada, P. tek-çekirdekleri var in gen transkripsiyonu analizi için RNA FISH yöntemi gerçekleştirmek için bir protokol ayrıntılı sunmak falciparum insan eritrosit etkilemiştir. Yönteminin kullanımını esas digoxigenin-ve TSA Artı Flüoresans Palet Sistemi 2 (Perkin Elmer), mikroskopik analiz ile taze seçilmiş S. antisens RNA probları kullanılarak biyotin-etiketli falciparum IE. In situ hibridizasyon metodu ile de P. farklı aşamaları sırasında ifade edilen genlerin transkripsiyonu ve çeşitli düzenleme izlemek için kullanılabilecek falciparum yaşam döngüsü ve diğer sıtma parazit türlerinin ve diğer organizmalar ve hücre tipleri uyarlanabilir.

Şekil 1, büyük adımlar ve RNA FISH yöntemin zaman süresinin bir akış şeması gösterilmektedir. Tek P. kullanarak iyi ve kötü korunmuş lekeli mRNA BALIK deneyler temsili görüntüleri bir dizi falciparum IE Şekil 2 &#39;de gösterilmiştir. Bu intrasellüler protozoon DAPI ile mavi lekeli bir küçük (1-1.5 mikron çapında) çekirdeğe sahip. Yirmi dört saat P. eritrosit işgalinden sonra falciparum schizo...

Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

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

Floresan<em&gt; In situ</emBireysel hücrelerde mRNA transkriptleri tanımlamak için&gt; hibridizasyon (FISH) bu tür birden fazla üyesinin eşzamanlı olarak transkripsiyon aktivitesi polijenik analizinin yapılmasını mümkün kılar<em&gt; Var</em&gt; Multigen aile<em&gt; Plasmodium falciparum</em&gt; Enfekte eritrositler<sup&gt; 1</sup&gt;. Bu teknik uyarlanabilir ve genler, hücreler ve organizmalar farklı türlerde kullanılır.

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 ...

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JoVE Journal

Biology

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

15.3K Görüntüleme.  University of Copenhagen. Floresan In situ hibridizasyon (FISH) bu tür birden fazla üyesinin eşzamanlı olarak transkripsiyon aktivitesi polijenik analizinin yapılmasını mümkün kılar Var Multigen aile Plasmodium falciparum Enfekte eritrositler 1. Bu teknik uyarlanabilir ve genler, hücreler ve organizmalar farklı türlerde kullanılır.

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

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 ...

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 ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radyoaktif In situ</emDoku Çeşitli Gen Patterns Saptayan> Hibridizasyon

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

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 ...

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 ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; Yerinde</emTüm Dağı Embriyolar ve Hücre histolojisi&gt; Hibridizasyon Deniz Elasmobranchs İçin Uyarlanmış

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

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 ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Ününüz Aşağıdaki Tek Hücre Düzeyinde Küresel RNA Sentezi Analizi

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...