Yazarların eleştirel okuma ve el yazması düzenleme için Dr. Cliona M. McHale teşekkür etmek istiyoruz. Bu çalışma, Ulusal Çevre Sağlığı Bilimleri, L Zhang Sağlığı Hibe R01ES017452 bölgesinin Enstitüsü Ulusal Enstitüsü tarafından finanse edildi.

1. Örnek slayt hazırlama <ul><li> OctoChrome FISH,,,, de dahil olmak üzere,, insan hücreleri, of metafaz spreadler in chromosomal değişiklikleri incelemek etmek olarak tasarlanmış, ancak,, kültürlü periferik kan hücreleri,, stem / progenitor hücreleri, ve hücre hatları etmek sınırlıdır değil edilir. Örnekleri hazırlanan metafaz 3,11-13 hasat için standart bir prosedür takip edilmelidir: hücreleri şişmeye metafaz, hipotonik tedavi hücreleri tutuklamak için colcemid tedavi istihdam ederek ve yaklaşık 0.5-1 x 10 6 hücre süspansiyon hücreleri gidermek için Carnoy Kullanıcı sabitleştirici ml başına. </li><li> 2 dakika süreyle saf etanol içine daldırma: 8-kare slayt (PMP 803, Cytocell Ltd, Cambridge, İngiltere, Şekil 2 OctoChrome Takımı, Katalog # sağlanan) temizleyin. </li><li> ,, Numune slaydın yaptıktan etmeden önce,,, hücre yoğunluğu, ogrenmek etmek en az,,, bir slayt üzerine hücreleri of, bir küçük bir bir miktarını düşürmeyin. ,, Hücre yoğunluğu,,, yüksek çok (çok olarak pek çok üst üste binen hücreleri) konumundadır varsa,, taze fixative,, ile birlikte süspansiyon seyreltmeyin. Hücre dansiteli, çok (çok az sayıda hücre çok, düşük konumundadır ayarlanmıştır ederse,,Slaytta ler), hücreleri aşağı spin ve taze fixative daha küçük bir hacim yeniden askıya. ,,,: Şablon slaydın:,, - 2/8 - 3/5 - 4/6 küçük kareler 1/7 alternan of, bir sekansı in of 8 alanlarda of her biri üzerine hücre süspansiyon of Pipette 5-10 mikrolitre. ,, Olanlar önce iki adet küçük kareler air-kurutulmuş sahiptir Once,,,, İçerikler aynı bir tarzda in geriye kalan küçük kareler belirleyilme. Bu, işlem,,,, engellemesini ile birlikte her biri, diğer, from hücre spreadler önlemek edecektir. </li><li> Kurutulmuş bir slaydın,, göre daha daha büyük bir hybridization daha önce 5 yaşından için,,,, nitrojen gaz atmosferinde in -20 ° C at depolanan uygulanmaz edebilirsiniz. </li></ul> 2. Bir otomasyon sistemi kullanılarak metafaz lokalizasyonu <ul><li> ,,, The slide of her biri yarım of center etmek olarak, 4 of 20, mikrolitre &#39;,6-diokso-diamidino-2-fenilindol (0,2 ug / ml&#39; &#39;), Apply (Uygula) ve sonra da, bir coverslip ile birlikte kapsamaz. </li><li> Metafaz hücreleri Metafer yazılım, (MetaSystems, Altlussheim, Almanya) kullanılarak yerelleştirilmesine örnek slayt otomatik bir tarama gerçekleştirin. Bu, tüm metafaz ve gelecekteki tüm cep görüntülerin yeniden değerlendirme yerini kolaylaştırır. </li><li> Elle tarama sonuçları ve non-metafaz eserler silin. </li></ul> 3. Melezleme <ol><li> Hazırlanması, örnek slayt ve OctoChrome cihaz </li><ul><li> , DAPI kadar uzak: yıkamak etmek 30, min, için: numune slaydın:,, oda sıcaklığında at internetin türü 2x SSC buffer ile in daldırınız. </li><li> Etanol yıkar (2% 70 dakika, her biri% 85 ve% 100 etanol) bir dizi örnek slayt kurutmak, kuru ve 5 dakika 37 ° C ocak yerleştirmek için izin verir. </li><li> ,,,, Bir (37) ° C su bath: in Chromoprobe Multiprobe: Hibridizasyon kullanılan kullanılan Chamber (OctoChrome bir Kit in sağlanan.) Yerleştirin ve Bir olmak üzere 37 dengelenmeye etmek bu izin vermek.. ° C (, + / - 1 ° C). </li><li> 37 ° C pipetleme ve Tekrarlayan OctoChrome cihaz başına ön-sıcak 25 ul tablet bir hibridizasyon çözüm (OctoChrome Kit) karıştırın. </li><li> Ön sıcak her OctoChrome bir cihaz 37 ° C cihaz etiket tarafı aşağı bir ocak koyarak (OctoChrome Kit sağlanan bkz. Şekil 2). T dokunmayın VERMEYİNkabartma yüzeyler o OctoChrome cihazın probları içerdiği gibi. </li></ul><li> OctoChrome cihaz üzerinden örnek slayt Konumlandırma (Şekil 2) </li><ul><li> It, 37 adresindeki - retmekte iken,,,, pre-ılındırılmış bir bir OctoChrome aygıt on sekiz adet alanlarda of her biri etmek,,, pre-ılındırılmış bir hybridization bir çözüm of 2 mikrolitre ekle ° C.. </li><li> Şimdi baş aşağı 1 numara, OctoChrome cihazın sağ üst el alan üzerinde bulunan böyle OctoChrome cihaz üzerinden şablon slayt dikkatlice ters çevirin. Kare 1 bulmalarına yardımcı olmak için, cihazın konumunu Orange işaretlendi. </li><li> Şablon slayt dikkatlice OctoChrome cihaz eşleşen alanları ile uyumlu olduğundan emin olun. , Hibridizasyon çözüm damla slayt temas yapmak o kadar dikkatle OctoChrome cihaz üzerinden slayt indirin. Nazik bir Uygula&#39;yı,, hatta,,,, hybridization bir çözüm,,,,, OctoChrome bir aygıt on raised alanlarda of her biri of kenarlarını etmek yayıldı edilir olduğunu, yürütülmesini sağlamak etmek zorlamak. </li><li> Asansörslayt / OctoChrome, cam slayt dikkatlice buzlu ucunu tutarak ve slayt OctoChrome cihazın altında olduğunu bu nedenle ters. Bu, problar çapraz kontaminasyona neden olabilir cihaz şablon slaytta smear değil emin olun. </li><li> , Bir (37) on Yer. ° C (+ / - 1. ° C),, 10 dakikada bir için ocak gözünün. </li></ul><li> Denatürasyon </li><ul><li> Özellikle dikkat çekici bir seviyede tutmanıza ocak için örnek slayt / OctoChrome cihaza aktarın. Örnek slayt eşit ocak gözü rehber olun. </li><li> ,, 75 at ocak gözünün on denatüre. ° C (, + / - 1 ° C.),, 5 dakika öncesinden için. </li></ul><li> Melezleme </li><ul><li> Chromoprobe Multiprobe Hibridizasyon Odası Yeri örnek slayt / OctoChrome cihaz. </li><li> Kapağını değiştirin ve 37 odası float ° C (+ / - 1 ° C) su banyosunda (karıştırma) gece. Lütfen dikkat: hibridizasyon odasında kapak mühür YAPMAYIN; kapağı su banyosu mühür YAPMAYIN; bir melezleşme VERMEYİNinkübatör. Bu adımlar, nem kontrolü için kritik öneme sahiptir. </li></ul><li> Post-hibridizasyon yıkar </li><ul><li> , Wash çözümleri of Hazırlanışı Çözüm 1:, 0.4x SSC içeren bir Coplin / Hellendahl kavanoz hazırlayın. Ile 72 dengelenmeye. ° C (+ / - 1 ° C ile),, bir su bath in ve, 7.0 &#39;ye pH değeri ayarlamak görüntüle. Çözüm 2: 2x SSC ve% 0.05 Tween 20 içeren bir Coplin / Hellendahl kavanoz hazırlayın. Oda sıcaklığında bekletin. </li><li> , Slayt dikkatle OctoChrome bir cihaz çıkarın ve 2 dakika Çözüm 1 slayt yerleştirin. </li><li> Çözüm 2 30 saniye boyunca slayt yerleştirin. </li></ul></ol> 4. Montaj ve sonuçların görselleştirme <ul><li> ,, The slide of her biri yarım of center etmek,, 20, mikrolitre of DAPI, Apply (Uygula) ve, bir coverslip uyguladıktan tıklayın. </li><li> ,,, Floresans mikroskopi kullanıcısı tarafından inceleyen ziyaretçilerin etmeden önce,, (azından 10) room sıcaklık dakika için koyu renk in inkübe edin. </li></ul> 5. Temsilcisi Sonuçlar Figure 3A için,,,, Meydanı&#39;na 2 in, bir tarafindan: Normal bir bir bir metafaz hücre gösterir. (1) - (3):, Kromozomlar 8, 21 ve 12 kırmızı, yeşil ve mavi boyalı, ve sırasıyla Texas Kırmızı, FITC ve Deac filtre ile görüntülenir; (4): / DAPI / FITC ile Görselleştirme Texas Red üçlü filtre. Genellikle, mavi ile boyanmış kromozom üçlü filtre ile net değil ve özel Deac altında filtre izlenmesi gerekir. Şekil 3B, lösemi-spesifik kromozomal translokasyon ve anöploidi temsili anormal hücreler gösterir. (1): t (8; 21), akut myeloid lösemi sık görülen bir kromozom translokasyonu; (2): Trizomi 21, 21 kromozom, lösemi ortak bir anöploidi üç nüsha. <img alt="Şekil 1," src="/files/ftp_upload/3619/3619fig1.jpg" /> Şekil 1. kromozom kombinasyonları OctoChrome cihaz ve beklenen kromozomal boyama sonuçları Düzenlenmesi. <img alt="Şekil 2," src= &quot;/ Files/ftp_upload/3619/3619fig2.jpg&quot; /&gt; Şekil 2 OctoChrome cihaz üzerinden örnek slayt Konumlandırma. Figure 3.: Temsilcisi sonuçlarOctoChrome BALIK (Kare 2) elde edilmiştir. <img alt="Şekil 3A" src="/files/ftp_upload/3619/3619fig3A.jpg" /> Bir Figure 3A için bu alanı kullanabilirsiniz. Meydanı&#39;na 2 &#39;in boyalı 12,, 21 chromosomes 8, ile ile birlikte A tarafindan: Normal bir bir bir hücre. <img alt="Şekil 3B," src="/files/ftp_upload/3619/3619fig3B.jpg" /> Şekil 3B Meydanı 2 lösemi-spesifik kromozomal translokasyon ve anöploidi Temsilcisi anormal hücreler.

<ol>
	<li>Levsky, J. M., Singer, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence+in+situ+hybridization:+past,+present+and+future.">Fluorescence in situ hybridization: past, present and future.</a> J. Cell. Sci. 116, 2833-2838 (2003).</li><li>Zhang, L., Eastmond, D. A., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nature+of+chromosomal+aberrations+detected+in+humans+exposed+to+benzene.">The nature of chromosomal aberrations detected in humans exposed to benzene.</a> Crit. Rev. Toxicol. 32, 1-42 (2002).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrations+in+chromosomes+associated+with+lymphoma+and+therapy-related+leukemia+in+benzene-exposed+workers.">Aberrations in chromosomes associated with lymphoma and therapy-related leukemia in benzene-exposed workers.</a> Environ. Mol. Mutagen. 48, 467-474 (2007).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benzene+increases+aneuploidy+in+the+lymphocytes+of+exposed+workers:+a+comparison+of+data+obtained+by+fluorescence+in+situ+hybridization+in+interphase+and+metaphase.">Benzene increases aneuploidy in the lymphocytes of exposed workers: a comparison of data obtained by fluorescence in situ hybridization in interphase and metaphase.</a> Environ. Mol. Mutagen. 34, 260-268 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+aneusomy+and+long+arm+deletion+of+chromosomes+5+and+7+in+the+lymphocytes+of+Chinese+workers+exposed+to+benzene.">Increased aneusomy and long arm deletion of chromosomes 5 and 7 in the lymphocytes of Chinese workers exposed to benzene.</a> Carcinogenesis. 19, 1955-1961 (1998).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+translocations+and+aneusomy+in+chromosomes+8+and+21+among+workers+exposed+to+benzene.">Increased translocations and aneusomy in chromosomes 8 and 21 among workers exposed to benzene.</a> Cancer. Res. 58, 2176-2181 (1998).</li><li>Bayani, J., Squire, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+the+detection+of+chromosomal+aberrations+using+spectral+karyotyping.">Advances in the detection of chromosomal aberrations using spectral karyotyping.</a> Clin. Genet. 59, 65-73 (2001).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromosome-wide+aneuploidy+study+(CWAS)+in+workers+exposed+to+an+established+leukemogen,+benzene.">Chromosome-wide aneuploidy study (CWAS) in workers exposed to an established leukemogen, benzene.</a> Carcinogenesis. 32, 605-612 (2011).</li><li>Rowley, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+chromosome+translocations+in+leukemogenesis.">The role of chromosome translocations in leukemogenesis.</a> Semin. Hematol. 36, 59-72 (1999).</li><li>Zhang, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+OctoChrome+fluorescence+in+situ+hybridization+to+detect+specific+aneuploidy+among+all+24+chromosomes+in+benzene-exposed+workers.">Use of OctoChrome fluorescence in situ hybridization to detect specific aneuploidy among all 24 chromosomes in benzene-exposed workers.</a> Chem. Biol. Interact. , 153-154 (2005).</li><li>Barch, M. J., Lawce, H. J., Arsham, M. S., Barch, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> In The ACT cytogenetics laboratory. , 17-30 (1991).</li><li>Zhang, L., Yang, W., Hubbard, A. E., Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonrandom+aneuploidy+of+chromosomes+1,+5,+6,+7,+8,+9,+11,+12,+and+21+induced+by+the+benzene+metabolites+hydroquinone+and+benzenetriol.">Nonrandom aneuploidy of chromosomes 1, 5, 6, 7, 8, 9, 11, 12, and 21 induced by the benzene metabolites hydroquinone and benzenetriol.</a> Environ. Mol. Mutagen. 45, 388-396 (2005).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroquinone,+a+benzene+metabolite,+increases+the+level+of+aneusomy+of+chromosomes+7+and+8+in+human+CD34-positive+blood+progenitor+cells.">Hydroquinone, a benzene metabolite, increases the level of aneusomy of chromosomes 7 and 8 in human CD34-positive blood progenitor cells.</a> Carcinogenesis. 21, 1485-1490 (2000).</li><li>Smith, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+understanding+benzene+health+effects+and+susceptibility.">Advances in understanding benzene health effects and susceptibility.</a> Annu. Rev. Public. Health. 31, 133-148 (2010).</li></ol>

Biz ifşa şey var.

Bu roman OctoChrome-FISH testi, aynı anda tek bir slayt tek bir hibridizasyon Tüm 24 insan kromozomlarını incelemek için olanak sağlar. ,, 8 küçük kareler on chromosome kombinasyonları of aranjman,,,,,,, en çok,, ortak bir lösemilerin ve lenfomalarin in non bir-rasgele bir bir bir chromosome Çevrilmeler of tanımlama kolaylaştırmak etmek olarak tasarlanmış&gt; edilmiştir&gt; gelmiştir. Böylece, deney aynı zamanda sayısal (anöploidi) yanı sıra yapısal (translokasyonlar) kromozom değişiklikleri...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar (isteğe bağlı) </td><tr><tr><td> OctoChrome Seti </td><td> Cytocell Ltd, Cambridge, İngiltere </td><td> PMP 803 </td><td></td></tr><tr><td> fitohemaglutininlerin (PHA) </td><td> Invitrogen Corporation&#39;ın </td><td> 10576-015 </td><td></td></tr><tr><td> Colcemid </td><td> Invitrogen Corporation&#39;ın </td><td> 15212-012 </td><td></td></tr><tr><td> Carnoy Kullanıcı sabitleştirici </td><td></td><td></td><td> Methanol: glasiye asetik asit, = 3: 1 </td></tr><tr><td> Floresans microscope </td><td></td><td></td><td> Farklı renkler aynı anda görüntülemek için, DAPI, Texas Kırmızı, FITC, ve KUMARİN spektrumları bireysel ve DAPI / FITC / Texas Red üçlü filtre filtreler ile donatılmış </td></tr><tr><td> Metafer yazılım </td><td> :, MetaSystems, Altlussheim, Almanya </td><td></td><td> Tüm metafaz bulmak için kolaylaştırılması ve için anormallikler yeniden değerlendirmek </td></tr></tbody></table>

chromosomics: detection of numerical and structural alterations in all 24 human chromosomes simultaneously using a novel octochrome fish assay

Floresan in situ hibridizasyon (FISH), spesifik DNA dizilerinin hücre çekirdekleri 1 metafaz veya interfaz kromozomlar üzerinde tespit edilmesini sağlayan bir tekniktir. Tekniği, tüm kromozom ya da belirli kromozomal bölgeler melezler eşsiz dizileri ile DNA probları kullanır ve klasik sitogenetik güçlü bir yardımcı olarak hizmet vermektedir. Örneğin, çok daha önceki çalışmalarda, klasik sitogenetik analiz 2 kullanılarak, benzen maruz kalma, benzen zehirlenmesi hasta ve benzen maruz sağlıklı işçi ile ilgili lösemi hastalarında artmış kromozom anormallikleri sık algılama bildirdi. BALIK kullanarak, lösemi-spesifik kromozomal değişiklikler, benzen kaynaklı lökomogenezisin cytogentic değişikliklerin kritik rolleri belirten, benzen 3-6 maruz görünüşte sağlıklı işçiler yükselmiş olduğu gözlenmiştir. Genellikle olarak,,, bir tek bir bir FISH assay inceliyor,, yalnızca birini ya da, bir birkaç bir tamamını chromosomes ileveya slayt başına belirli lokusları, birden çok hibridizasyonları birden çok slayt insan kromozomlarının tümünü kapsayacak şekilde yürütülmesi gerekir. Spektral karyotip (SKY), aynı anda tüm genom görselleştirme, ancak özel bir yazılım ve donanım limitlerini uygulama 7 için gereksinimi sağlar. Burada, bir roman FISH testi, kromozom çapında anöploidi çalışma olarak insan çalışmaları, bir slayt tüm 24 insan kromozomlarının eşzamanlı analizi (CWAS) olarak tanımlayan Chromosomics için, uygulanabilir OctoChrome-BALIK, tarif 8. ,,,,, Chromoprobe Multiprobe System olarak Cytocell kullanıcısı tarafından pazarlanan yöntemini,, of temelinde,,,,,,,, üç farklı bir bir tamamını chromosome painting probları (Figure sık kullanılan 1) taşır her biri hangi 8 küçük kareler, içine bölünmüş edilir, bir OctoChrome bir aygıt konumundadır. Her üç probları doğrudan farklı bir renkli florofor yeşil (FITC), kırmızı (Texas Kırmızı) ve mavi (KUMARİN) ile etiketlenmiştir. Kromozom kombinasyonlarının OctoChrom düzenleme,,, t (15,; 17), t (8; 21 e-aygıt;,, örneğine t (22 9,) için,,,, en çok,, ortak bir lösemilerin ve lenfomalarin in bulundu non-rasgele bir bir bir,,, yapısal chromosome değişikler (translakosyon belirlenmiş) of olduğunda kimlik tespitinin kolaylaştırılması için olarak tasarlanmış olmuştur ), t (14; 18) 9 izlediği görülmüştür. Ayrıca, kromozomlardaki sayısal değişiklikleri (anöploidi) aynı anda tespit edilebilir. The karşılık gelen bir şablon slaydın,, aynı zamanda, 8 küçük kareler içine metafaz spreadler bağlıdırlar (Figure 2), ve,, OctoChrome bir aygıt üzerinden konumlandırılmış edilir hangi üzerine: bölünmüş edilir. Prob ve hedef DNA yüksek sıcaklık ve nemli bir odasında melezleşmiştir denatüre olup, daha sonra 24 insan kromozomlarının aynı anda görüntülenebilmekte. OctoChrome BALIK lösemi ve lenfoma klinik teşhis ve bütün kromozomlarda anöploidiler tespit için gelecek vaat eden bir tekniktir. Biz, benzen maruz kalan Çinli işçilerin 8,10 CWAS çalışmada bu yeni kromozomal yaklaşım uyguladık.

Watch this Scientific Journal Video about Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay at JoVE.com

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Bir roman floresans<em&gt; In-situ</em&gt; Hibridizasyon (FISH) yöntemi, aynı anda, özellikle sayısal ve yapısal kromozom değişiklikleri, lösemi ve lenfoma ile ilişkili bir hibridizasyon tek bir cihaz tüm 24 insan kromozomlarının belirli kromozomal translokasyonlar, inceler açıklanmıştır.

Chromosomics: Sayısal ve Yapısal Değişmeler Algılama Tüm 24 İnsan Kromozomlar Aynı zamanda Roman OctoChrome BALIK Test kullanma

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

chromosomics-detection-numerical-structural-alterations-all-24-human

Research

JoVE Journal

Biology

18.7K Görüntüleme.  University of California, Berkeley. Bir roman floresans In-situ Hibridizasyon (FISH) yöntemi, aynı anda, özellikle sayısal ve yapısal kromozom değişiklikleri, lösemi ve lenfoma ile ilişkili bir hibridizasyon tek bir cihaz tüm 24 insan kromozomlarının belirli kromozomal translokasyonlar, inceler açıklanmıştır.

Video: Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

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 ...

Biyoloji

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, form a complex with Smad4 and translocate to the nucleus where they function as transcription factors to initiate BMP specific downstream effects 1. Traditional immuno-fluorescence techniques with antibodies against phospho-Smad peptides exhibit low sensitivity, high background and offer gross quantification as they rely on intensity of the antibody signal particularly if this is photosensitive fluorescent. In addition, phospho-Smads may not all be in complex with Smad4 and engaged in active transcription.
In situ PLA is a technology capable of detecting protein interactions with high specificity and sensitivity 2-4. This new technology couples antibody recognition with the amplification of DNA surrogate of the protein. It generates a localized, discrete signal in a form of spots revealing the exact position of the recognition event. The number of signals can be counted and compared providing a measurement. We applied in situ PLA, using the Duolink kit, with a combination of antibodies that allows the detection of the BMP signaling effectors phospho-Smad1/5/8 and Smad4 only when these are in proximity i.e. in a complex, which occurs only with signaling activation. This allowed for the first time, the visualization and measurement of endogenous BMP signaling with high specificity and sensitivity in a time course experiment under BMP4 stimulation.

Detection of Signaling Effector-Complexes Downstream of BMP4 Using in situ PLA, a Proximity Ligation Assay

Here we show how to use Proximity Ligation Assay (PLA), with a combination of antibodies to visualize Bone Morphogenetic Protein (BMP) signaling in fixed cells. This technique allowed us to follow the nuclear accumulation of endogenous BMP activated effector-complexes and quantify their levels over time under BMP4 stimulation.

Efektör-Kompleksleri kullanarak BMP4 Mansabında Sinyal Algılama In situ PLA, Proximity ligasyon Testi

BMPs are responsible for a wide range of developmental and biological effects. BMP receptors activate (phosphorylate) the Smad1/5/8 effectors, which then, ...

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace 
the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties 
of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the 
phenolic polymer lignin, that is also difficult to digest 1. In order to improve the digestibility of plant materials we need to discover the main bottlenecks 
for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification 2. These tasks require 
a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has 
been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes 
for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.
This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant 
materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded 
amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for 
analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this 
station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90&deg;C. The pretreatment solution 
is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme
mixture for a variable length of time at 50&deg;C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH 
colorimetric method.

A simple, rapid method for determining the saccharification potential of large numbers of plant biomass samples is described. The automated platform for this analysis involves the preparation of the plant biomass for analysis in 96 well plates and the subsequent performance of pretreatment, hydrolysis and quantification of the sugars released.

Lignocellulosic Malzemeler için Yüksek verim Saccharification Testi

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

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 ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

Ekson yakalama ve Devasa Paralel Dizilimine tarafından Tümör Örneklerinin somatik genetik değişiklikler tespit

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Verimli Üretimi ve Saflaştırılması Rekombinant murin kindlin-3 Biophysical Çalışmaları Böcek hücrelerinden

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Assessing Species-specific Contributions To Craniofacial Development Using Quail-duck Chimeras

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to the histological and morphological development of vertebrate embryos. In particular, the use of chimeric embryos has established the importance of neural crest in directing the species-specific morphology of the craniofacial complex. The method described herein utilizes two avian species, duck and quail, with remarkably different craniofacial morphology. This method greatly facilitates the investigation of molecular and cellular regulation of species-specific pattern in the craniofacial complex.&#160;Experiments in quail and duck chimeric embryos have already revealed neural crest-mediated tissue interactions and cell-autonomous behaviors that regulate species-specific pattern in the craniofacial skeleton, musculature, and integument. The great diversity of neural crest derivatives suggests significant potential for future applications of the quail-duck chimeric system to understanding vertebrate development, disease, and evolution.

This article describes a method to generate chimeric embryos that are&#160;designed to test the species-specific contributions of neural crest and/or other tissues to craniofacial development.

Bıldırcın-ördek Chimeras kullanma Kraniyofasiyal Kalkınma için Türlere özgü Katılımlar değerlendirilmesi

The generation of chimeric embryos is a widespread and powerful approach to study cell fates, tissue interactions, and species-specific contributions to ...

Recombinant Protein Expression for Structural Biology in HEK 293F Suspension Cells: A Novel and Accessible Approach

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293F cells.

The expression of recombinant proteins by mammalian systems is becoming an attractive method for producing protein complexes for structural biology. Here we present a simple yet highly efficient expression system using suspension grown mammalian cells to purify protein complexes for structural studies.

Bir Roman ve Erişilebilir Yaklaşım: Yapısal HEK 293F Askı Hücreleri Biyoloji Rekombinant Protein

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two ...

Detection of Intracellular Gene Expression in Live Cells of Murine, Human and Porcine Origin Using Fluorescence-labeled Nanoparticles

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, rat, human, pig and others. The verification of iPS clones mainly relies on the detection of the endogenous expression of different pluripotency genes. These genes mostly represent transcription factors which are located in the cell nucleus. Traditionally, the proof of their endogenous expression is supplied by immunohistochemical staining after fixation of the cells. This approach requires replicate cultures of each clone at this early stage to preserve validated clones for further experiments. The present protocol describes an approach with gene-specific nanoparticles which allows the evaluation of intracellular gene expression directly in live cells by fluorescence. The nanoparticles consist of a central gold particle coupled to a capture strand carrying a sequence complementary to the target mRNA as well as a quenched reporter strand. These nanoparticles are actively endocytosed and the target mRNA displaces the reporter strand which then start to fluoresce. Therefore, specific target gene expression can be detected directly under the microscope. In addition, the emitted fluorescence allows the identification, isolation and enrichment of cells expressing a specific gene by flow cytometry. This method can be applied directly to live cells in culture without any manipulation of the target cells.

This manuscript describes a novel technique which allows the detection of intracellular gene expression after endocytosis of fluorescence-labeled nanoparticles directly in live cells. The method does not require manipulation of the cells and is not restricted with respect to the target gene or species.

Floresan etiketli Nanopartiküller kullanma Fare, İnsan ve domuz Menşe Canlı hücreler hücre içi Gen İfade Algılama

The reprogramming of somatic cells to induced pluripotent stem cells (iPS) has successfully been performed in different mammalian species including mouse, ...

Studying Ribonucleotide Incorporation: Strand-specific Detection of Ribonucleotides in the Yeast Genome and Measuring Ribonucleotide-induced Mutagenesis

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be assessed by alkaline hydrolysis and gel electrophoresis as RNA is highly susceptible to hydrolysis in alkaline conditions. This, in combination with Southern blot analysis can be used to determine the location and strand into which the ribonucleotides have been incorporated. However, this procedure is only semi-quantitative and may not be sensitive enough to detect small changes in ribonucleotide content, although strand-specific Southern blot probing improves the sensitivity. As a measure of one of the most striking biological consequences of ribonucleotides in DNA, spontaneous mutagenesis can be analyzed using a forward mutation assay. Using appropriate reporter genes, rare mutations that results in the loss of function can be selected and overall and specific mutation rates can be measured by combining data from fluctuation experiments with DNA sequencing of the reporter gene. The fluctuation assay is applicable to examine a wide variety of mutagenic processes in specific genetic background or growth conditions.

Ribonucleotides are among the most abundant non-canonical nucleotides incorporated into the genome during eukaryotic nuclear DNA replication. If not properly removed, ribonucleotides can cause DNA damage and mutagenesis. Here, we present two experimental approaches that are used to assess the abundance of ribonucleotide incorporation into DNA and its mutagenic effects.

Eğitim Ribonucleotide ortaklık: Strand-özel hafiye-in Ribonucleotides Maya genom ve Ribonucleotide kaynaklı Mutagenesis ölçme

The presence of ribonucleotides in nuclear DNA has been shown to be a source of genomic instability. The extent of ribonucleotide incorporation can be ...