We thank Hongjae Sunwoo for helpful advice for oligonucleotide design in RNA FISH. We also thank Serenity Curtis for editing the manuscript. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad of the Japan Society for the Promotion of Science (JSPS). This work was supported by the NIH (RO1-GM102184), the March of Dimes Research Foundation (#6-FY12-337), and the Developmental Fund and Trustee Grant at Cincinnati Children's Hospital Medical Center to Y.O.

1. Prob Hazırlık <ol><li> 5&#39;-amino modifikasyon ile birden fazla benzersiz Xist oligonükleotitlerin (20-30 nükleotid uzunlukta oligonükleotitlerin, 63-65 ° C, erime sıcaklığı, Tablo 1) elde edilir ve su içinde askıya. </li><li> 5&#39;-amino modifikasyon (toplam 4.5 ug) ve imalatçının talimatları izlenerek, amin-reaktif bir floresan boya ile etiket ile oligonükleotidler havuzu, eşit molar miktarlarda. <ol><li> Nükleaz içermeyen suyun nihai 5 ul toplanmış oligonükleotidler çözülür ve oligonükleotit solüsyonuna 1 M sodyum bikarbonat ve 3 ul ekleyin. </li><li> Kısaca, amin-reaktif boya ve girdap şişeye 2 ul DMSO ekleyin. </li><li> DMSO içinde reaktif boya ile oligonükleotid çözeltisi karıştırılır ve karanlıkta 1 saat boyunca oda sıcaklığında karışım inkübe edin. </li></ol></li><li> Etanol ile çökeltme, ardından G-25 kolonu ile floresan etiketli oligonükleotid probları arındırın. <ol><li&gt; 750 xg 1 dakika süreyle Santrifüj G-25 sütunu fazla depolama tampon kaldırmak için. </li><li> Adım 1.2.3 üretilen etiketleme karışımına 40 ul nükleaz ücretsiz su ekleyin ve G-25 kolonuna uygulanır. 2 dakika boyunca 750 x g&#39;de santrifüjleyin. </li><li> Aşama 1.3.2 arıtılmış proba 50 ul nükleaz içermeyen su, 10 ul 3 M sodyum asetat (pH 5.2) ve 250 ul etanol ekleyin. 4 ° C&#39;de 15 dakika boyunca 21,000 x g&#39;de 30 dakika santrifüj için -80 ° C&#39;de saklayın. </li><li> % 70 etanol, 1 ml ile bir kez çöktürülerek oligonükleotidler yıkayın. </li><li> 50 ul içinde yeniden askıya nükleaz içermeyen su veya TE (10 mM Tris-HCI, pH 7.5; 1 mM EDTA) içinde yaklaşık 10 uM bir nihai konsantrasyonu. </li></ol></li><li> Hibridizasyon tampon maddesi ile 250 nM arasında bir nihai konsantrasyona floresan etiketli oligonükleotit problar seyreltin (% 10 formamid, 2X SSC [300 mM NaCI, 30 mM sodyum sitrat] 2 mg / ml BSA;% 10 dekstran sülfat). NOT: Bu hızlı BALIK protokolü ile, oligonükleotid pXist elbiseler tasarlanmış ve hibridizasyon için kuluçka süresini kısaltmak için, oligonükleotit problar kullanılarak normal BALIK protokollerine göre daha yüksek bir konsantrasyonda kullanılmıştır. </li></ol> 2. Slayt Hazırlama NOT: Slaytlar fare embriyonik kök (ES) veya embriyoid gövdesini (EB) kullanarak bağışıklık FISH için hazırlanabilir ya poli-lizin-tedavi slayt üzerinde doğrudan büyüyen veya hücre süspansiyonu ile sitospin kullanarak hücreleri 20, 21 farklılaştırarak. Burada, Sitospin slayt hazırlama gösterilir. <ol><li> 6 gözlü tabak içinde kültür ortamı aspire. İyice PBS (oda sıcaklığı) ve aspirasyon ile yıkayın. </li><li> 37 ° C&#39;de 5-10 dakika süreyle inkübe, 6-çukurlu tabağa tripsin 0.5 ml ilave edilir. </li><li> Ortamın 5 ml ilave edilir ve bir kaç kez özenle aşağı yukarı pipetleme hücreleri dağıtılır. </li><li> Oda sıcaklığında, 200 x g&#39;de 5 dakika boyunca, bir 15 ml tüp santrifüj hücreleri aktarın. </li><li> Ortam, bir atınyavaşça 2 ml PBS içinde hücrelerin süspansiyonu d. </li><li> Hemasitometre kullanarak hücreleri sayın ve PBS ile 4 x 10 5 hücre / ml hücre süspansiyonu seyreltin. </li><li> Bir slayt, bir filtre kartı ve bir klip ile bir odayı birleştirin ve Sitospin rotor uygun yuvalara yerleştirin. Sitospin monte birimlerin her odasına adım 2.6 hazırlanan her numunenin 250 ul ekleyin. Oda sıcaklığında 10 dakika boyunca 1500 rpm&#39;de santrifüj. </li><li> 40 ml buz-soğuğu PBS CSK tamponu her biri buz üzerinde 3 coplin kavanoz doldurun (10mM PIPES pH 6.8; 100 mM NaCI, 300 mM sakaroz, 3 mM MgCl2) ve CSKT tamponu (% 0.5 Triton CSK tampon X-100) idi. </li><li> Sitospin tamamlandıktan sonra hızla buz gibi soğuk PBS içine slayt aktarmak ve 5 dakika süreyle inkübe edilir. </li><li> Buz soğukluğunda CSK tampon içine slayt aktarın ve 1 dakika boyunca inkübe edin. </li><li> Buz soğukluğunda CSKT arabelleğe slayt aktarın ve 5 dakika süreyle inkübe edilir. </li><li> Buz-soğuk CSK buf geri slayt aktarınfer ve 1 dakika boyunca inkübe edin. </li><li> PBS içinde% 4 paraformaldehid içinde slayt aktarın ve oda sıcaklığında 10 dakika inkübe edilir. </li></ol> 3. İmmünofloresan <ol><li> Birkaç saniye (% 0.1 Tween-20 ile 1x PBS), PBST ile coplin kavanoza slaytlar yerleştirin. </li><li> Slayt aşağı akmasına tampon kalan izin eğik slaytlar yerleştirin. Dikkatle slayt kapalı aşırı sıvı silin ve su ile boş bir pipet ucu kutusuna yerleştirin. Yumuşak bir lamel, bloke etme çözeltisi 40 ul (1 x PBS,% 1 BSA,% 0.1 Tween-20) ile slayt Yerleşimi, ve oda sıcaklığında 10 dakika inkübe edilir. </li><li> Lamel Bloke etme çözeltisi uzaklaştırılır kaldırmak ve 1 bir konsantrasyona seyreltilmiştir Histon H3K27me3 birincil antikor 40 ul: 500 çözüm engelleme. Geri slayt lamel yerleştirin ve oda sıcaklığında 30 dakika inkübe edilir. Not: bloke çözeltisi içinde bir RNaz inhibitör bu RNA FIS RNA&#39;yı korumak için yararlı olabilirPoliklonal antikorlar kullanılmıştır lH protokolüdür. </li><li> Yıkama hafifçe yıkama sırasında sallayarak, 5 dakika her biri için, PBST ile dolu coplin kavanozlarda 2 kez kaydırın. </li><li> Slayt aşağı akmasına tampon kalan izin eğik slaytlar yerleştirin. Dikkatle aşırı sıvı silin ve tekrar ucu kutuya slayt yerleştirin. Ikincil antikor (1: 500) 40 ul Kaplama çözeltisi engelleme seyreltilmiş, bir lamel ve karanlıkta 15 dakika boyunca inkübe edin. Aşağıdaki adımları için, her zaman karanlıkta slayt tutun. </li><li> Adım 3.4 gibi PBST ile kavanoz Coplin 2 kez yıkayın. </li></ol> Oligonükleotid Probları 4. RNA BALIK <ol><li> Adım 4.3 RNA FISH için 37 ° C&#39;de kurumasını önlemek için slaytlar altındaki su ile boş bir pipet ucu kutusu Ön-sıcak. </li><li> Slayt aşağı akmasına tampon kalan izin eğik slaytlar yerleştirin. Dikkatle adım 3,6 slayt kapalı aşırı sıvı silin ve wat ile pipet ucu kutusu içine slayt yerleştirinalt er. Slayt üzerinde FISH yıkama tamponu (2x SSC,% 10 formamid) 500 ul ilave edin ve oda sıcaklığında 5 dakika inkübe edilir. </li><li> FISH yıkama solüsyonu çıkarın slayt üzerine oligonükleotid prob 8 ul ekleyin, bir kapak kayma yerleştirin ve alt su ile önceden ısıtılmış pipet ucu kutusu slayt aktarmak; 1-2 saat boyunca 37 ° C&#39;de inkübe edin. </li><li> BALIK yıkama tamponu, DAPI FISH yıkama tamponu ve 2 x SSC ile 37 ° C&#39;de bir su banyosu içinde 3 önceden ısıtılmış coplin kavanoz yerleştirin. </li><li> RNA FISH için 1-2 saat inkübasyondan sonra, önceden ısıtılmış BALIK yıkama tamponu içine slayt koydu. </li><li> Lamel slayt düşer kadar birkaç dakika için slayt inkübe. </li><li> Geri yıkama tamponu içine slayt yerleştirin ve 5 dakika inkübasyon devam ediyor. </li><li> 0.5 ng / ml DAPI ile önceden ısıtılmış BALIK yıkama tamponu ile bir coplin kavanoza slayt aktarın ve 5 dakika boyunca inkübe edin. </li><li> Önceden ısıtılmış 2x SSC ile başka coplin kavanoza slayt aktarın ve 5 mil inküben. </li><li> Slayt aşağı akmasına tampon kalan izin eğik slaytlar yerleştirin. Dikkatle aşırı sıvı silin ve kuru bir ucu kutuya geri slayt yerleştirin. Slayt üzerine DAPI ile antifade 4 ul ekleyin ve bir lamel yerleştirin. </li><li> Oje ile lamel Seal ve bir mikroskop altında gözlemlemek. Lam, bir kaç ay boyunca karanlıkta 4 ° C de muhafaza edilebilir. </li></ol>

<ol>
	<li>Payer, B., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+chromosome+dosage+compensation:+how+mammals+keep+the+balance.">X chromosome dosage compensation: how mammals keep the balance.</a> Annu Rev Genet. 42, 733-772 (2008).</li><li>Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gracefully+ageing+at+50,+X-chromosome+inactivation+becomes+a+paradigm+for+RNA+and+chromatin+control.">Gracefully ageing at 50, X-chromosome inactivation becomes a paradigm for RNA and chromatin control.</a> Nat Rev Mol Cell Biol. 12, 815-826 (2011).</li><li>Gendrel, A. V., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fifty+years+of+X-inactivation+research.">Fifty years of X-inactivation research.</a> Development. 138, 5049-5055 (2011).</li><li>Froberg, J. E., Yang, L., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guided+by+RNAs:+X-inactivation+as+a+model+for+lncRNA+function.">Guided by RNAs: X-inactivation as a model for lncRNA function.</a> J Mol Biol. 425, 3698-3706 (2013).</li><li>Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S., Brockdorff, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Requirement+for+Xist+in+X+chromosome+inactivation.">Requirement for Xist in X chromosome inactivation.</a> Nature. 379, 131-137 (1996).</li><li>Marahrens, Y., Panning, B., Dausman, J., Strauss, W., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xist-deficient+mice+are+defective+in+dosage+compensation+but+not+spermatogenesis.">Xist-deficient mice are defective in dosage compensation but not spermatogenesis.</a> Genes Dev. 11, 156-166 (1997).</li><li>Clemson, C. M., McNeil, J. A., Willard, H. F., Lawrence, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=XIST+RNA+paints+the+inactive+X+chromosome+at+interphase:+evidence+for+a+novel+RNA+involved+in+nuclear/chromosome+structure.">XIST RNA paints the inactive X chromosome at interphase: evidence for a novel RNA involved in nuclear/chromosome structure.</a> J Cell Biol. 132, 259-275 (1996).</li><li>Sado, T., Brockdorff, N. <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+chromosome+silencing+by+the+long+non-coding+RNA.">Advances in understanding chromosome silencing by the long non-coding RNA.</a> Xist. Phil Trans R Soc B. 368, 20110325-20 (2013).</li><li>Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polycomb+proteins+targeted+by+a+short+repeat+RNA+to+the+mouse+X+chromosome.">Polycomb proteins targeted by a short repeat RNA to the mouse X chromosome.</a> Science. 322, 750-756 (2008).</li><li>Plath, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+histone+H3+lysine+27+methylation+in+X+inactivation.">Role of histone H3 lysine 27 methylation in X inactivation.</a> Science. 300, 131-135 (2003).</li><li>Silva, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+histone+h3+methylation+on+the+inactive+X+chromosome+requires+transient+recruitment+of+Eed-Enx1+polycomb+group+complexes.">Establishment of histone h3 methylation on the inactive X chromosome requires transient recruitment of Eed-Enx1 polycomb group complexes.</a> Dev Cell. 4, 481-495 (2003).</li><li>Gall, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+synthesis+of+the+genes+for+ribosomal+RNA+during+amphibian+oogenesis.">Differential synthesis of the genes for ribosomal RNA during amphibian oogenesis.</a> Proc Natl Acad Sci U S A. 60, 553-560 (1968).</li><li>Femino, A. M., Fay, F. S., Fogarty, K., 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=Visualization+of+single+RNA+transcripts+in+situ.">Visualization of single RNA transcripts in situ.</a> Science. 280, 585-590 (1998).</li><li>Raj, A., van den Bogaard, P., Rifkin, S. A., van Oudenaarden, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+individual+mRNA+molecules+using+multiple+singly+labeled+probes.">Imaging individual mRNA molecules using multiple singly labeled probes.</a> Nat Methods. 5, 877-879 (2008).</li><li>Chaumeil, J., Augui, S., Chow, J. C., Heard, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combined+immunofluorescence,+RNA+fluorescent+in+situ+hybridization,+and+DNA+fluorescent+in+situ+hybridization+to+study+chromatin+changes,+transcriptional+activity,+nuclear+organization,+and+X-chromosome+inactivation.">Combined immunofluorescence, RNA fluorescent in situ hybridization, and DNA fluorescent in situ hybridization to study chromatin changes, transcriptional activity, nuclear organization, and X-chromosome inactivation.</a> Methods Mol Biol. 463, 297-308 (2008).</li><li>Katayama, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+transcription+in+the+mammalian+transcriptome.">Antisense transcription in the mammalian transcriptome.</a> Science. 309, 1564-1566 (2005).</li><li>Ogawa, Y., Xite Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-inactivation+intergenic+transcription+elements+that+regulate+the+probability+of+choice.">X-inactivation intergenic transcription elements that regulate the probability of choice.</a> Mol Cell. 11, 731-743 (2003).</li><li>Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X+inactivation+in+mouse+ES+cells:+histone+modifications+and+FISH.">X inactivation in mouse ES cells: histone modifications and FISH.</a> Methods Enzymol. 376, 419-428 (2004).</li><li>Khalil, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+human+large+intergenic+noncoding+RNAs+associate+with+chromatin-modifying+complexes+and+affect+gene+expression.">Many human large intergenic noncoding RNAs associate with chromatin-modifying complexes and affect gene expression.</a> Proc Natl Acad Sci U S A. 106, 11667-11672 (2009).</li><li>Martin, G. R., Evans, 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=Differentiation+of+clonal+lines+of+teratocarcinoma+cells:+formation+of+embryoid+bodies+in+vitro.">Differentiation of clonal lines of teratocarcinoma cells: formation of embryoid bodies in vitro.</a> Proc Natl Acad Sci U S A. 72, 1441-1445 (1975).</li><li>Lee, J. T., Lu, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+mutagenesis+of+Tsix+leads+to+nonrandom+X+inactivation.">Targeted mutagenesis of Tsix leads to nonrandom X inactivation.</a> Cell. 99, 47-57 (1999).</li><li>Zhang, L. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+RNAs+mark+sex+chromosomes+in+stem+cells.">Telomeric RNAs mark sex chromosomes in stem cells.</a> Genetics. 182, 685-698 (2009).</li><li>Raj, A., Tyagi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+individual+endogenous+RNA+transcripts+in+situ+using+multiple+singly+labeled+probes.">Detection of individual endogenous RNA transcripts in situ using multiple singly labeled probes.</a> Methods Enzymol. 472, 365-386 (2010).</li><li>Kimura, H., Hayashi-Takanaka, Y., Goto, Y., Takizawa, N., Nozaki, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organization+of+histone+H3+modifications+as+revealed+by+a+panel+of+specific+monoclonal+antibodies.">The organization of histone H3 modifications as revealed by a panel of specific monoclonal antibodies.</a> Cell Struct Funct. 33, 61-73 (2008).</li><li>Ogawa, Y., Sun, B. K., Lee, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersection+of+the+RNA+interference+and+X-inactivation+pathways.">Intersection of the RNA interference and X-inactivation pathways.</a> Science. 320, 1336-1341 (2008).</li><li>Kohlmaier, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+chromosomal+memory+triggered+by+Xist+regulates+histone+methylation+in+X+inactivation.">A chromosomal memory triggered by Xist regulates histone methylation in X inactivation.</a> PLoS Biol. 2, 171 (2004).</li><li>Wang, K. C., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanisms+of+long+noncoding+RNAs.">Molecular mechanisms of long noncoding RNAs.</a> Mol Cell. 43, 904-914 (2011).</li></ol>

No conflicts of interest declared.

Bu yazıda, H3K27me3 için slayt hazırlama, Xist RNA FISH ve immünoflüoresans tamamlamak için en az 5 saat sürer hızlı bir bağışıklık BALIK protokolü sundu. Genellikle RNA FISH gecede inkübasyon ihtiyacı Xist RNA saptanması, genel bağışıklık FISH yaklaşımı ile karşılaştırıldığında, bu protokol önemli ölçüde harcanan zamanı azaltır, aynı zamanda oligonükleotid probları kullanılarak bağışıklık FISH duyarlılığını artırır sadece. Xist yüksek...

Memeli X-kromozomu inaktivasyonu (XCI) kadınlarda iki X-kromozomu biri transkripsiyonel 1 inaktive burada XX ve XY arasındaki X-bağlantılı gen doz dengesizliği telafi etmek için bir stratejidir. X-kromozomu inaktivasyonu uzun olmayan kodlama RNA (lncRNA) araştırma için harika bir model sistemidir. X-kromozomu inaktivasyonu birden lncRNAs tarafından düzenlenir ve yoğun lncRNAs, transkripsiyon, kromatin yapısı ve nükleer organizasyonu 2, 3 arasındaki karışma mekanizmaları ortaya çıkarmak için son birkaç yılda çalışılmıştır. X kromozomunda bulunan X inaktivasyon merkezi (XIC) kodlayıcı olmayan RNA&#39;lar 4 üreten genlerin bir dizi içeren bir kompleks genetik lokus olduğu. X-aktif spesifik transkript (Xist) lncRNA eutherian memelilerde X kromozomu inaktivasyonu 5,6 çok önemli bir rol oynar böyle bir lncRNA olup. Xist transkript fu yerini çevreleyenTure Xi X-kromozomu inaktivasyonu başlatmak ve RNA FISH kullanılarak görüntülendi zaman bir bulut gibi görünmesini; Bu oluşum &quot;Xist Cloud&quot; 7 olarak adlandırılır. Xist RNA, çeşitli kromatin değiştirici enzimler ile etkileşim için, sessiz bir kromatin ve baskı transkripsiyon için farklı epigenetik değişiklikler ile Xist bulutlar eş-lokalizasyonu X kromozomu inaktivasyonu 8 süresince gözlenir. Örneğin, Xist RNA H3K27me3 sorumludur ve baskıcı bir kromatin devlet 9 uyaran Polycomb baskıcı kompleksi 2 (PRC2) ile etkileşime girer. Xi ve yoğun H3K27me3 modifikasyonu ile birlikte yerelleştirme Xist bulut oluşumu Xi 10,11 bir fakültatif heterokromatinin manzara temsil eder. Böyle DNA / RNA FISH gibi sitogenetik teknikler, gelişmiş hassasiyet ile son ve gelişmiş teknikler, radyoaktif işaretli problar 12 kullanılarak geleneksel yöntemle uzun bir yol katettikBirden fazla oligonükleotid sondaları 13,14 kullanılarak flüoresan görüntüleme. Immünofloresan ile birleştiğinde DNA / RNA BALIK rutin Spatiotemporal nükleer organizasyonu, RNA lokalizasyonu, kromatin yapısını ve değişiklikleri anlamak için bir sitolojik araç olarak kullanılmıştır. RNA, FISH için pek çok standart prob hazırlama plazmid ya da bakteriyel yapay kromozom (BAC) klonu ve çentik translasyonu veya rastgele prime 15 ya da daha sonraki etiketi kullanılmasını içerir. Bununla birlikte, farelerde genlerin yaklaşık% 70 ve insanlarda genlerin% 40 sens ve antisens transkriptleri ayırmak için bu nedenle bir büküm özel FISH yöntem gerektiren, sens ve antisens transkriptleri 16 bir üst üste göstermektedir. İn vitro transkribe RNA sondaları (riboprob ) genellikle iplik-spesifik RNA FISH 17,18 için kullanılır; bununla birlikte, bu, T7, SP6 veya T3 promoteri ve sentezlenmesi riboprobes bir plazmid klonu ya da PCR ürün hazırlama içerir. Ayrıca, genomunun elde riboproblarıic DNA veya cDNA sık sık yüksek arka plan gürültüsü neden non-spesifik bölgeleri ve tekrarlayan unsurlar içerir. Diğer konu da, uzunluğu bir kaç yüz nükleotidlerdir ve birden florofor içeren riboprobları, etkili bir çekirdek içine girmek edemezler. Bu aşmak için, ucunda tek bir florofor ile işaretlenmiş çok sayıda daha kısa oligonükleotid probları iyi hassasiyet, düzgün sinyal kuvveti ve saflaştırma kolaylığı ve 14 taşıma olduğu geliştirilmiştir. Buna ek olarak, DNA oligonükleotidleri, genel olarak RNA daha stabildir. Bu X kromozomu etkisizleştirme işlemi sırasında Xist lncRNA neden Xi epigenetik dinamiklerini anlamak için immünofloresan ile Xist RNA, FISH 19 oligonükleotidleri kullanılarak benzer bir strateji uygulanır. Bu protokol, oligonükleotid prob ve hücrelerin uygun hazırlama oluşturma, hem de bağışıklık ve RNA FISH kullanımını tarif etmektedir. Birden oligonucle kullanarak Xist RNA BALIKXist RNA BALIK kişinin laboratuvarında rutin olarak ise, SEK, uzun vadede maliyet etkin bir yaklaşımdır. Bu teknik aynı zamanda epigenetik değişiklikler ya da faktörleri ile birlikte-lokalizasyonunu haritalama ise hücrelerinde lncRNAs tanımlamak için kullanılabilir. Protokolün bir büyük avantajı kolayca kişinin araştırma çıkarlarına uygun şekilde değiştirmek için yeteneğidir.

<table><tbody><tr><td>oligonucleotides with 5&amp;rsquo;-amino modification</td><td>IDT</td><td></td><td></td></tr><tr><td>Alexa Fluor 488 Reactive Dye</td><td>Life Technologies</td><td>A32750</td><td></td></tr><tr><td>MicroSpin G-25 columns</td><td>GE Healthcare</td><td>27-5325-01</td><td></td></tr><tr><td>Cytospin 2</td><td>Shandon</td><td>59900102</td><td></td></tr><tr><td>Bovine Serum Albumin, Molecular Biology Grade (for hybridization buffer)</td><td>Roche</td><td>10715859103</td><td></td></tr><tr><td>Histone H3K27me3 antibody</td><td>Active Motif</td><td>61017</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse, highly cross-adsorbed</td><td>Life Technologies</td><td>A21424</td><td></td></tr><tr><td>ProLong Gold antifade reagent</td><td>Life Technologies</td><td>P36931</td><td></td></tr></tbody></table>

quick fluorescent in situ hybridization protocol for xist rna combined with immunofluorescence of histone modification in x-chromosome inactivation

Immünfloresans (immün-FISH) ile in situ hibridizasyon (FISH) RNA floresan proteinleri birleştiren, epigenetik değişiklikler ve diğer detaylar lokalizasyonu içine eşzamanlı bir bakış açısı ile RNA lokalizasyonu mekansal dinamiklerini tespit etmek için tek hücre düzeyinde istihdam edilebilir bir teknik oluşturur hangi immünofloresan vurgulanmış olabilir. X-kromozomu inaktivasyonu uzun olmayan kodlama RNA (lncRNA) aracılı gen susturulması için bir paradigma olduğunu. Memeli kadınlarda iki X-kromozom birinde (bir Xist bulut denir) X-inaktif özel transkript (Xist) lncRNA birikimi X-kromozomu inaktivasyonu başlatmak için kritik bir adımdır. Xist RNA doğrudan veya dolaylı olarak çeşitli kromatin-modifiye enzimler ile etkileşim ve inaktif X-kromozomu (Xi) için ayrı epigenetik manzara tanıttı. Xi biri bilinen epigenetik damgasını Histone H3 trimetil-lizin 27 (H3K27me3) değişiklik. Burada, biz basit ve hızlı bir immün FISH tarifXist RNA lokalizasyonu ve ilişkili epigenetik değişiklikleri incelemek için H3K27me3 immünofloransı ile birleştiğinde birden oligonükleotid probları ile RNA FISH kullanarak Xist RNA tespiti için protokol. Oligonükleotid probları daha kısa bir kuluçkalama süresi sonuçları in vitro ötelenmiş RNA probları (riboprobları) göre Xist RNA daha hassas algılama kullanılması. Bu protokol lncRNAs dinamiklerini ve ilgili epigenetik değişiklik, kromatin yapısı, nükleer organizasyon ve transkripsiyon düzenleme anlamak için güçlü bir araç sağlar.

Pratik immüno-FISH temsili görüntüleri Şekil 1A&#39;da gösterilmiştir. Xi Xist RNA bulut ve H3K27me3 sinyalinin Eş-lokalizasyonu kadın hücrelerin ayırt saptandı. Farklılaşması üzerine 12. günde EB hücrelerinin% 90&#39;dan daha fazla bir Xist bulut (Şekil 1B) vardı. (; 150% 97, n = Şekil 1C) Kısa oligonükleotid probları etkin bir şekilde neredeyse tüm H3K27me3 sinyalleri Xist RNA ile birlikte lokalize bir görselleştirme yol, çekirdeklerin iç...

Watch this Scientific Journal Video about Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation at JoVE.co

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level.

Hızlı Floresan<em&gt; In Situ</emX-kromozomu İnaktivasyonu içinde histon Modifikasyon İmmünofloresan ile Xist RNA Kombine için&gt; Hibridizasyon Protokolü

Combining RNA fluorescent in situ hybridization (FISH) with immunofluorescence (immuno-FISH) creates a technique that can be employed at the single cell ...

quick-fluorescent-situ-hybridization-protocol-for-xist-rna-combined

Research

JoVE Journal

Biology

Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

13.9K Görüntüleme.  Cincinnati Children's Hospital Medical Center. We developed an easily customized strand-specific fluorescent in situ hybridization (FISH) protocol combined with immunofluorescence. This allows for a detailed examination of RNA dynamics with simultaneous insight into the chromatin structure, nuclear organization, and transcriptional regulation at the single cell level. 

Video: Quick Fluorescent In Situ Hybridization Protocol for Xist RNA Combined with Immunofluorescence of Histone Modification in X-chromosome Inactivation

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development. The Xenopuslaevis embryo is very useful for studying organogenesis because their large size makes them very suitable for identifying organs at the earliest steps in organogenesis. At this time, the primary method used for identifying a specific organ or primordium is whole mount in situ hybridization with labeled antisense RNA probes specific to a gene that is expressed in the organ of interest. In addition, it is relatively easy to manipulate genes or signaling pathways in Xenopus and in situ hybridization allows one to then assay for changes in the presence or morphology of a target organ. Whole mount in situ hybridization is a multi-day protocol with many steps involved. Here we provide a simplified protocol with reduced numbers of steps and reagents used that works well for routine assays. In situ hybridization robots have greatly facilitated the process and we detail how and when we utilize that technology in the process. Once an in situ hybridization is complete, capturing the best image of the result can be frustrating. We provide advice on how to optimize imaging of in situ hybridization results. Although the protocol describes assessing organogenesis in Xenopus laevis, the same basic protocol can almost certainly be adapted to Xenopus tropicalis and other model systems.

Understanding Early Organogenesis Using a Simplified In Situ Hybridization Protocol in Xenopus

The Xenopus laevis embryo continues to be exceptionally useful in the study of early development due to its large size and ease of manipulation. A simplified protocol for whole mount in situ hybridization protocol is provided that can be used in the identification of specific organs in this model system.

Anlamak Basitleştirilmiş kullanma Erken Organogenez<em&gt; In Situ</emIçinde&gt; Hibridizasyon Protokolü<em&gt; Ksenopus</em

Organogenesis is the study of how organs are specified and then acquire their specific shape and functions during development.  The Xenopuslaevis embryo ...

Gelişim Biyolojisi

Using Fluorescence In Situ Hybridization (FISH) to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk of conceiving an aneuploid fetus increases dramatically and this phenomenon is known as the maternal age effect. One requirement for accurate chromosome segregation during the meiotic divisions is maintenance of sister chromatid cohesion during the extended prophase period that oocytes experience. Cytological evidence in both humans and model organisms suggests that meiotic cohesion deteriorates during the aging process. In addition, segregation errors in human oocytes are most prevalent during meiosis I, consistent with premature loss of arm cohesion. The use of model organisms is critical for unraveling the mechanisms that underlie age-dependent loss of cohesion. Drosophila melanogaster offers several advantages for studying the regulation of meiotic cohesion in oocytes. However, until recently, only genetic tests were available to assay for loss of arm cohesion in oocytes of different genotypes or under different experimental conditions. Here, a detailed protocol is provided for using fluorescence in situ hybridization (FISH) to directly visualize defects in arm cohesion in prometaphase I and metaphase I arrested Drosophila oocytes. By generating a FISH probe that hybridizes to the distal arm of the X chromosome and collecting confocal Z stacks, a researcher can visualize the number of individual FISH signals in three dimensions and determine whether sister chromatid arms are separated. The procedure outlined makes it possible to quantify arm cohesion defects in hundreds of Drosophila oocytes. As such, this method provides an important tool for investigating the mechanisms that contribute to cohesion maintenance as well as the factors that lead to its demise during the aging process.

Using Fluorescence In Situ Hybridization FISH to Monitor the State of Arm Cohesion in Prometaphase and Metaphase I Drosophila Oocytes

This manuscript presents a detailed method for generating X-chromosome arm probes and performing fluorescence in situ hybridization (FISH) to examine the state of sister chromatid cohesion in prometaphase and metaphase I arrested Drosophila oocytes. This protocol is suitable for determining whether meiotic arm cohesion is intact or disrupted in different genotypes.

Kol uyum içinde prometafaz ve metafaz durumunu izlemek için Floresans In Situ hibridizasyon (balık) kullanarak ben Drosophila yumurta

In humans, chromosome segregation errors in oocytes are responsible for the majority of miscarriages and birth defects. Moreover, as women age, their risk ...

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

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including genetic disorders, of which chromosomal abnormalities are the most common. Fluorescence in situ hybridization (FISH) is a well-known and frequently used method to detect chromosomal aberrations in humans. FISH is mainly used for the analysis of chromosomal abnormalities in the spermatozoa of males with numerical or structural chromosomal aberrations. Furthermore, this technique is also frequently applied in females to detect X chromosomal aberrations that are known to cause ovarian dysgenesis. However, information on the X chromosomal content of ovarian cells from females with X chromosomal aberrations in lymphocytes and/or buccal cells is still lacking.
The aim of this study is to advance basic research regarding X chromosomal aberrations in females, by presenting two methods based on FISH to identify the X chromosomal content of ovarian cells. First, a method is described to determine the X chromosomal content of isolated ovarian cells (oocytes, granulosa cells, and stromal cells) in non-grafted ovarian cortex tissue from females with X chromosomal aberrations. The second method is directed at evaluating the effect of chromosomal aberrations on folliculogenesis by determining the X chromosomal content of ovarian cells of newly formed secondary and antral follicles in ovarian tissue, from females with X chromosomal aberrations after long-term grafting into immunocompromised mice. Both methods could be helpful in future research to gain insight into the reproductive potential of females with X chromosomal aberrations.

Exploring X Chromosomal Aberrations in Ovarian Cells by Using Fluorescence In Situ Hybridization

This article presents two methods based on fluorescence in situ hybridization to determine the X chromosomal content of ovarian cells in non-grafted and grafted ovarian cortex tissue from females with X chromosomal aberrations.

Floresan In Situ Hibridizasyon Kullanarak Yumurtalık Hücrelerinde X Kromozomal Anormalliklerinin Araştırılması

Millions of people worldwide deal with issues concerning fertility. Reduced fertility, or even infertility, may be due to many different causes, including ...

Genetik

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Xenopus laevis'te Nöronal Öncül Karakterizasyonu için Floresan Kalsiyum Görüntüleme ve Daha Sonra Yerinde Hibridizasyon

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

Çift Floresan birleştiren<em&gt; In Situ</em&gt; Fare Beyin Bölüm Üç Genlerin İfade tespiti için immünoişaretleme ile Hibridizasyon

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

Nörobilim

Quantitative Fluorescence In Situ Hybridization (FISH) and Immunofluorescence (IF) of Specific Gene Products in KSHV-Infected Cells

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout the cell at specific times can be captured with fluorescence in situ hybridization (FISH) and immunofluorescence (IF). During lytic herpesvirus infection, the virus hijacks the host cell to preferentially express viral genes, causing changes in cell morphology and behavior of biomolecules. Lytic activities are centered in nuclear factories, termed viral replication compartments, which are discernable only with FISH and IF. Here we describe an adaptable protocol of RNA FISH and IF techniques for Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells, both adherent and in suspension. The method includes steps for the development of specific anti-sense oligonucleotides, double RNA FISH, RNA FISH with IF, and quantitative calculations of fluorescence intensities. This protocol has been successfully applied to multiple cell types, uninfected cells, latent cells, lytic cells, time-courses, and cells treated with inhibitors to analyze the spatiotemporal activities of specific RNAs and proteins from both the human host and KSHV.

Quantitative Fluorescence In Situ Hybridization FISH and Immunofluorescence IF of Specific Gene Products in KSHV-Infected Cells

We describe a protocol utilizing fluorescence in situ hybridization (FISH) to visualize multiple herpesviral RNAs within lytically infected human cells, either in suspension or adherent. This protocol includes quantification of fluorescence producing a nucleocytoplasmic ratio and can be extended for simultaneous visualization of host and viral proteins with immunofluorescence (IF).

KSHV Enfekte Hücrelerde Spesifik Gen Ürünlerinin Situ Hibridizasyonunda (FISH) ve İmmünfloresansında (IF) Kantitatif Floresan

Mechanistic insight arrives from careful study and quantification of specific RNAs and proteins. The relative locations of these biomolecules throughout ...

İmmünoloji ve Enfeksiyon

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

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

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

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

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

Fluorescent in situ hybridization (FISH) is a molecular technique which enables the detection of nucleic acids in cells. DNA FISH is often used in cytogenetics and cancer diagnostics, and can detect aberrations of the genome, which often has important clinical implications. RNA FISH can be used to detect RNA molecules in cells and has provided important insights in regulation of gene expression. Combining DNA and RNA FISH within the same cell is technically challenging, as conditions suitable for DNA FISH might be too harsh for fragile, single stranded RNA molecules. We here present an easily applicable protocol which enables the combined, simultaneous detection of Xist RNA and DNA encoded by the X chromosomes. This combined DNA-RNA FISH protocol can likely be applied to other systems where both RNA and DNA need to be detected.

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

Fluorescent in situ hybridization (FISH) allows the detection of nucleic acids in their native environment within cells. We here describe a protocol for the combined, simultaneous detection of RNA and DNA by means of FISH, which can be used to study X chromosome inactivation in mouse embryonic stem cells.

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

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

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

DNA in situ hybridization (DNA ISH) is a commonly used method for mapping sequences to specific chromosome regions. This approach is particularly effective at mapping highly repetitive sequences to heterochromatic regions, where computational approaches face prohibitive challenges. Here we describe a streamlined protocol for DNA ISH that circumvents formamide washes that are standard steps in other DNA ISH protocols. Our protocol is optimized for hybridization with short single strand DNA probes that carry fluorescent dyes, which effectively mark repetitive DNA sequences within heterochromatic chromosomal regions across a number of different insect tissue types. However, applications may be extended to use with larger probes and visualization of single copy (non-repetitive) DNA sequences. We demonstrate this method by mapping several different repetitive sequences to squashed chromosomes from Drosophila melanogaster neural cells and Nasonia vitripennis spermatocytes. We show hybridization patterns for both small, commercially synthesized probes and for a larger probe for comparison. This procedure uses simple laboratory supplies and reagents, and is ideal for investigators who have little experience with performing DNA ISH.

Simple Method for Fluorescence DNA In Situ Hybridization to Squashed Chromosomes

Here, we present a simple method for performing fluorescence DNA in situ hybridization (DNA ISH) to visualize repetitive heterochromatic sequences on slide-mounted chromosomes. The method requires minimal reagents and it is versatile for use with short or long probes, different tissues, and detection with fluorescence or non-fluorescence-based signals.

Hızlı Floresan

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Anlamak Basitleştirilmiş kullanma Erken Organogenez

Kol uyum içinde prometafaz ve metafaz durumunu izlemek için Floresans In Situ hibridizasyon (balık) kullanarak ben Drosophila yumurta

Zebra balığı Tüm Dağı Yüksek Çözünürlüklü Çift Floresan Yerinde Hibridizasyon

Floresan In Situ Hibridizasyon Kullanarak Yumurtalık Hücrelerinde X Kromozomal Anormalliklerinin Araştırılması

Xenopus laevis'te Nöronal Öncül Karakterizasyonu için Floresan Kalsiyum Görüntüleme ve Daha Sonra Yerinde Hibridizasyon

Çift Floresan birleştiren

KSHV Enfekte Hücrelerde Spesifik Gen Ürünlerinin Situ Hibridizasyonunda (FISH) ve İmmünfloresansında (IF) Kantitatif Floresan

Tüm Dağı RNA Floresan

Kombine DNA-RNA Floresan

Floresan DNA için Basit Bir Yöntem