Name: transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform
Uploaded: 2017-01-30T13:00:00.000+00:00
Duration: 9 min 4 s
Description: Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Research supported by Alberta Livestock and Meat Agency, Alberta Innovates - BioSolutions, Alberta Milk, and Livestock Research Branch, Alberta Agriculture and Forestry.

Bu çalışmanın hayvan parçası Alberta Hayvan Bakımı ve Kullanımı Komitesi Üniversitesi tarafından tüm hayvan deney prosedürleri onaylı (Protokol # AUP00000131) ile, Alberta, Edmonton, Kanada Üniversitesi Metabolik Araştırma Birimi&#39;nde yürütülen ve hayvanlar göre bakım için Hayvan Bakımı yönergelere Kanada Konseyi (1993). 1. Embriyo Üretim, Toplam RNA ve DNaz Tedavi İzolasyonu <ol><li> Hayvan deneysel protokollerin ve embriyo toplama önceki yayınlarda 17, 18&#39;de başvurun. </li><li> Havuz ve sıvı azot içinde her ineğin benzer sahne embriyoların dondurularak ek ve daha sonra RNA ekstraksiyonu kadar -80 ° C tutun. </li><li> (Kit bilgisi Malzeme Listesinde mevcut) piko ölçekli örneklerinden RNA ayıklamak sağlayan bir sütun tabanlı RNA ekstraksiyon kiti sayesinde toplam RNA ayıklayın. NOT: Tavsiye edilen kit içerir: Klima Buffer, Ekstraksiyon tamponfer,% 70 Etanol, yıkama tampon çözeltisi 1, Yıkama Tamponu 2, elüsyon tamponu, toplama tüpleri ve mikrosantrifüj tüpleri RNA saflaştırma sütunlar. </li><li> Tüp içeren embriyo 10 ul ekstraksiyon tampon ekleyin ve 42 ° C&#39;de 30 dakika inkübe edilir. 2 dakika boyunca 800 xg&#39;de santrifüj tüpü mikrosantrifüj tüp içine hücre ekstresi toplamak için. </li><li> arıtma sütun filtresi zar üzerine 250 ul Klima Buffer Pipet. Oda sıcaklığında 5 dakika boyunca Tertibatı Tamponu ile RNA saflaştırma sütunu inkübe edin. 1 dakika boyunca 16,000 x g&#39;de toplama tüpü arıtma sütun santrifüjleyin. </li><li> Pipet RNA ekstraksiyon tampon maddesinin ve embriyo karışımına% 70 Etanol 10 uL. Yukarı ve aşağı pipetleme iyice karıştırın. şartlandırılarak arıtma sütuna Pipet karışımı. </li><li> 100 x g&#39;de 2 dakika süre ile RNA, santrifüj saflaştırma kolona bağlanacağı hemen akışı çıkarmak için 30 saniye süreyle 16,000 xg&#39;de santrifüj uygulayın. </li><li> pipet 10081; 8000 x g, 1 dakika için arıtma kolonu ve santrifüj içine yıkama tampon çözeltisi 1 L. NOT: DNaz tedavi RNA izolasyonu sonrası ters transkripsiyon veya çoğaltım işlemi ise tavsiye edilir. </li><li> Sağ adım 1.8 sonra genomik DNA sindiremez. NOT: Önerilen DNaz kiti Malzeme Listesinde mevcuttur. Önerilen kit içerir: DNaz I stok solüsyonu ve Buffer. </li><li> 35 ul tampon, 5 uL DNaz I stok solüsyonu karıştırılarak DNaz inkübasyon karışım hazırlayın. yavaşça ters çevirerek karıştırın. </li><li> saflaştırma kolonudur zarı içine, doğrudan 40 uL DNase inkübasyon karışımı pipetle. 15 dakika oda sıcaklığında inkübe edin. </li><li> Pipet 40 mcL Yıkama arıtma sütun zarı içine Tampon 1 ve daha sonra 15 saniye süreyle 8,000 xg&#39;de santrifüj. </li><li> 8,000 x g&#39;de 1 dakika için arıtma kolonu ve santrifüj içine pipetle 100 uL yıkama tampon çözeltisi 2. </li><li> saflaştırma kolonudur ve santrifüj içine bir 100 uL yıkama tamponu 2 Pipet16,000 x g&#39;de 2 dakika süre ile. </li><li> Yeni bir 0.5 mL mikrosantrifüj tüpüne saflaştırma sütunu aktarın. doğrudan arıtma sütunun zar üzerine Seyreltme Tampon Pipet 11 mcL. elüsyon tamponu dağıtım sırasında membran içine, Seyreltme Tampon maddesi maksimum absorpsiyonu garantilemek üzere, hafifçe membran yüzeyine pipet ucu dokunun. </li><li> Oda sıcaklığında 1 dakika boyunca sütun inkübe edin. sütununda, Seyreltme Tampon maddesi dağıtma 1000 xg&#39;de 1 dakika için sütun santrifüj ve ardından RNA elüte etmek için 16,000 x g&#39;de 1 dakika boyunca dönerler. </li><li> RNA kalitesini ve miktarını 19 değerlendirmek için üreticinin talimatlarına göre Bioanalyzer alet kullanın. </li><li> kullanılana kadar -80 ° C&#39;de RNA numunesi saklayın. </li></ol> 2. Mikroarray Analizi RNA Amplifikasyon ve Etiketleme NOT: RNA amplifikasyonu ve etiketleme işlemleri ile çalışan ilk kez kullanıcı için, RNA alo başak-beş ng kullanımıGerçek Arna örnekleri ile ng RNA amplifikasyonu ve melezleşme kalite kontrol ölçümü izlemek için. Başak RNA karışımı önceden belirlenen oranlarda on nitro sentezlendi, poliadenile transkript içerir. Prosedür düzgün yapıldığında, etiketli transkript özellikle dizilerde tamamlayıcı kontrol sondaları sadece melezleşme ve veri asgari öz- veya çapraz melezleşme ile kalite kontrol güvencesi izlemek için taranabilir. Dizi ve veri analizi taradıktan sonra, kontrol şiddetleri içinde-çivili ve normalizasyon prosedürü ile ilgili daha fazla detay açıklama önceki yayınlarda 20, 21 bölümüne bakınız. Aşağıdaki yordam rutin mikroarray kullanıcılara verilir. <ol><li> 11 uL - 10 toplam hacmi 100 pg kaliteli RNA örnek (RNA Bütünlük sayısı (RIN) değeri en az&gt; 7.0) hazırlayın. </li><li> amplifikasyon kiti edememek RNA örnekleri artırın(Küçük pg 100 gibi), piko ölçekli örnekleri ile çalışır. NOT: Tavsiye edilen amplifikasyon kiti bilgisi Malzeme Listesinde mevcuttur ve herhangi bir değişiklik yapılmadan üreticinin talimatlarına uyar. </li><li> amplifiye Arna büyüklüğü aralığındaki profilini değerlendirmek için bir floresan bazlı kantitatif kullanın. </li><li> amplifiye Arna konsantrasyonunu ölçmek için (260 ve 280 nm &#39;de absorbans göre) spektrofotometre cihazı kullanın. </li><li> Etiketleme için hazır olana kadar -80 ° C &#39;de büyütülmüş ARNA saklayın. </li><li> ULS Arna etiketleme kiti kullanım kılavuzuna göre floresan etiketleme için güçlendirilmiş RNA 2 ug kullanın. Not: 647 boya kolayca ozon tarafından parçalanır foto-degradasyon ve Siyanin 647 boya duyarlı siyanin 547 ve siyanin için, etiketleme prosedürü ışık az miktarda ve bir ozon içermeyen ortamda gerçekleşir. </li><li> Ozon seviyesi kadar ozon kutusuna (Şekil 1A) açın 0.001 ppm. </li><li&gt; siyanin 547 veya siyanin 647, etiketleme tamponu 2 uL 2 ul ekleyerek ozon kutusuna (Şekil 1B) içindeki tepkime setup fazlası, ve daha sonra Karanlıkodanızda altında RNaz içermeyen su ile 20 ul&#39;lik nihai bir hacme kadar ayarlayın. ışık kaynağına karanlık oda filtre ekleyin. </li><li> İyice karıştırın ve 15 dakika boyunca 85 ° C &#39;de bir PCR inkübe edin. en az 1 dakika boyunca buz üzerinde Örnekleri. Cy-boya etiketli güçlendirilmiş RNA temizlemek için, (1,9 ila 1.11) DNaz tedavi ile ilgili adımlar dışında, RNA ekstraksiyon kiti ile devam etmeden önce tüplerin içeriğini toplamak için aşağı doğru döndürün. </li><li> ölçülmesi ve etiketli büyütülmüş RNA&#39;nın konsantrasyonu belirlemek ve kullanımdan önce 3 gün -80 ° C&#39;de en fazla etiketli ARNA tutmak için spektrofotometre cihazı kullanın. </li></ol> 3. Mikroarray Hibridizasyon ve yıkama Not: Aşağıdaki önemli adımlar İki renkli Microarray- tabanlı Gen İfadesi Analizi Protokolüne göre adapte edilir (versiyon6.5, Mayıs 2010). <ol><li> Her cyanine 547- ve Siyanin 647 etiketli Arna ng 825 eşit miktarda ekleyin ve 25x parçalanma ve 10x engelleme tampon ile karıştırın. </li><li> 15 dakika boyunca 60 ° C&#39;de ısıtın ve 1 dakika boyunca buz üzerinde soğutulur. </li><li> 2x hibridizasyon tamponu eşit miktarda ekleyin ve iyice karıştırın. </li><li> 13.000 rpm&#39;de santrifüj, sonra karışım hibridizasyon için hazır hale gelir. </li><li> Dizi slayt üzerine karışımdan 100 uL yüklemek ve melezleme odasının içine slayt mühür. </li><li> Fırında 10 rpm&#39;de dönen 65 ° C&#39;de 17 saat melezleme fırını içine monte bölmeyi yükleyin. </li><li> stabilizasyon daldırma ve 30 saniye için çözüm kurutulmasıyla ozon koruma tedavisi ile protokole göre diziler yıkayın. </li><li> toz parçacıkları ile çözümünden diziler çıkarın ve dizi yüzeyi kuru olduğundan emin olun </li></ol> 4. Tarama Mikroarray slayt <ol><li> karanlık bir yerde diziler tutun ve TARAMAn açmaker (15 dk bekleme süresi) kullanmak için hazır olana kadar. </li><li> tarayıcıya, sol dizi barkod yükleyin ve taramaya başlayın. yazılım kullanım kılavuzuna göre nokta analiz yapmak ve (GPR) dosya biçimi olarak verileri kaydetmek. </li></ol> 5. İstatistiksel Analiz <ol><li> FlexArray yazılımı http://genomequebec.mcgill.ca/FlexArray/license.php &quot;ithal ham veri&quot; üzerine tıklayın ve FlexArray içine GPR dosyaları yüklemek indirin. </li><li> Gerekirse talimat takip ederek normalleşme ve istatistiksel analiz. Not: Daha önce 22 açıklandığı gibi bu çalışmada olumlu nokta seçimi için eşik hesaplayın. </li><li> tüm melezleştirilmiş noktalar ve arka plan sinyalleri görüntülemek için FlexArray arsa izleyiciden kılavuzda açılır seçin. Not: mikrodizi hibridizasyon sonrası kontrol çivili benzer bir görüntü önceki çalışmada 8&#39;de görülebilir. </li><li> seçinFlexArray gelen diziler normalleşme süreci arasında bir persantil aşağıdaki dizinin içinde lös normalleşme buna kılavuzu açılır. </li><li> Bir Excel içine FlexArray tüm normalleştirilmiş verileri dışa daha fazla kullanım için dosya. </li></ol> 6. Biyoinformatik Analizi Sığır embriyo özgü transkriptleri prob dizilerinin orijinal açıklama (BESTv1) mikrotertip önce 8 tarif edilmiştir. Bu çalışmada, 20.403 benzersiz Gen Semboller (GS) (elde edildi <a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_1.xlsx">Dosya1 Supplement</a> EmbryoGENE Laboratuar Bilgi Yönetim Sistemi (LIMS) ELMA (http://elma.embryo-gene.ca) yoluyla). 6765 benzersiz genlerin (listesi <a href="http://ecsource.jove.com/files/ftp_upload/53754/Supplement_file_2.xlsx">file2 Ek</a> ) seçilmiş ve mikroarray normalleşme sürecinden sonra tüm olumlu sinyaller (Adım 5 tekabül oldu0,5) sığır Günlük 7 embriyolar gen ifadesi profilinden. Olumlu bir sinyal eşik A değerinin sinyal yoğunluğu gelen geçirgen yayında 16 üzerinden hesaplanmıştır. <ol><li> Sınıflandırma Sistemi 23, 24 (Evrimsel ilişkiler yoluyla protein analizi) PANTHER fonksiyonel analizi gerçekleştirmek için aşağıdaki linki (http://www.pantherdb.org/about.jsp) açın. </li><li> İstatistiksel overrepresentation testi kullanılarak embriyonik gelişim sırasında belirli biyolojik süreci tanımlamak ve referans listesine 25 olarak Bos taurus varsayılan ayarı (veritabanındaki tüm genler) kullanmak için 6765 embriyo özgü GS listesini yükleyin. Not: Bu istatistiksel olarak fazla veya az ifade edilen bir binom testi kullanılarak olan GO açısından gelişimsel ilgili süreçlerin belirlenmesini sağlar. </li><li> Yazılım varsayılan olarak &lt;0.05 Set P-değeri eşik. Veriler indirilebilir ve expo olabilirDaha fazla seçim 16 Excel dosyasına rted. </li></ol>

<ol>
	<li>Royal, M. D., Smith, R. F., Friggens, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fertility+in+dairy+cows:+bridging+the+gaps.">Fertility in dairy cows: bridging the gaps.</a> Animal. 2 (08), 1101-1103 (2008).</li><li>Diskin, M. G., Murphy, J. J., Sreenan, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryo+survival+in+dairy+cows+managed+under+pastoral+conditions.">Embryo survival in dairy cows managed under pastoral conditions.</a> Anim. Reprod. Sci. 96 (3-4), 297-311 (2006).</li><li>Wrenzycki, C., 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=Effects+of+culture+system+and+protein+supplementation+on+mRNA+expression+in+pre-implantation+bovine+embryos.">Effects of culture system and protein supplementation on mRNA expression in pre-implantation bovine embryos.</a> Hum. Reprod. 16 (5), 893-901 (2001).</li><li>Menezo, Y. J., Herubel, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+and+bovine+models+for+human+IVF.">Mouse and bovine models for human IVF.</a> Reprod. Biomed. Online. 4 (2), 170-175 (2002).</li><li>Schena, M., Shalon, D., Davis, R. W., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+monitoring+of+gene+expression+patterns+with+a+complementary+DNA+microarray.">Quantitative monitoring of gene expression patterns with a complementary DNA microarray.</a> Science. 270 (5235), 467-470 (1995).</li><li>Patterson, T. 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=Performance+comparison+of+one-color+and+two-color+platforms+within+the+MicroArray+Quality+Control+(MAQC)+project.">Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.</a> Nat. Biotechnol. 24 (9), 1140-1150 (2006).</li><li>Rhodes, D. R., Chinnaiyan, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+analysis+of+the+cancer+transcriptome.">Integrative analysis of the cancer transcriptome.</a> Nat. Genetics. 37, 31-37 (2005).</li><li>Robert, C., 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=Combining+resources+to+obtain+a+comprehensive+survey+of+the+bovine+embryo+transcriptome+through+deep+sequencing+and+microarrays.">Combining resources to obtain a comprehensive survey of the bovine embryo transcriptome through deep sequencing and microarrays.</a> Mol. Reprod. Dev. 78 (9), 651-664 (2011).</li><li>Nygaard, V., Hovig, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Options+available+for+profiling+small+samples:+a+review+of+sample+amplification+technology+when+combined+with+microarray+profiling.">Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling.</a> Nucleic Acids Res. 34 (3), 996-1014 (2006).</li><li>Kurn, N., Chen, P., Heath, J. D., Kopf-Sill, A., Stephens, K. M., Wang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+isothermal,+linear+nucleic+acid+amplification+systems+for+highly+multiplexed+applications.">Novel isothermal, linear nucleic acid amplification systems for highly multiplexed applications.</a> Clin Chem. 51 (10), 1973-1981 (2005).</li><li>Van Gelder, R. N., von Zastrow, M. E., Yool, A., Dement, W. C., Barchas, J. D., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amplified+RNA+synthesized+from+limited+quantities+of+heterogeneous+cDNA.">Amplified RNA synthesized from limited quantities of heterogeneous cDNA.</a> Proc. Natl. Acad. Sci. 87 (5), 1663-1667 (1990).</li><li>Phillips, J., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antisense+RNA+Amplification:+A+linear+amplification+method+for+analyzing+the+mRNA+population+from+single+living+cells.">Antisense RNA Amplification: A linear amplification method for analyzing the mRNA population from single living cells.</a> Methods. 10 (3), 283-288 (1996).</li><li>Gilbert, I., Scantland, S., Dufort, I., Gordynska, O., Labbe, A., Sirard, M. A., Robert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+monitoring+of+aRNA+production+during+T7+amplification+to+prevent+the+loss+of+sample+representation+during+microarray+hybridization+sample+preparation.">Real-time monitoring of aRNA production during T7 amplification to prevent the loss of sample representation during microarray hybridization sample preparation.</a> Nucleic Acids Res. 37 (8), 65 (2009).</li><li>Gijlswijk, R. P., Talman, E. G., Jansse, P. J., Snoeijers, S. S., Killian, J., Tanke, H. J., Heetebrij, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+Linkage+System:+versatile+nucleic+acid+labeling+technique.">Universal Linkage System: versatile nucleic acid labeling technique.</a> Expert Re. Mol. Diagn. 1 (1), 81-91 (2001).</li><li>Gilbert, I., Scantland, S., Sylvestre, E. L., Dufort, I., Sirard, M. A., Robert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Providing+a+stable+methodological+basis+for+comparing+transcript+abundance+of+developing+embryos+using+microarrays.">Providing a stable methodological basis for comparing transcript abundance of developing embryos using microarrays.</a> Mol. Hum. Reprod. 16 (8), 601-616 (2010).</li><li>Tsoi, 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=Development+of+a+porcine+(Sus+scofa)+embryo-specific+microarray:+array+annotation+and+validation.">Development of a porcine (Sus scofa) embryo-specific microarray: array annotation and validation.</a> BMC Genomics. 13, 370 (2012).</li><li>Salehi, R., 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=Superovulatory+response+and+embryo+production+in+Holstein+cows+fed+diets+enriched+in+oleic,+linoleic+or+&#945;-linolenic+acid.">Superovulatory response and embryo production in Holstein cows fed diets enriched in oleic, linoleic or &#945;-linolenic acid.</a> Reprod. Fertil. Dev. 26 (1), 218-218 (2013).</li><li>Thangavelu, G., Colazo, M. G., Ambrose, D. J., Oba, M., Okine, E. K., Dyck, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diets+enriched+in+unsaturated+fatty+acids+enhance+early+embryonic+development+in+lactating+Holstein+cows.">Diets enriched in unsaturated fatty acids enhance early embryonic development in lactating Holstein cows.</a> Theriogenology. 68 (7), 949-957 (2007).</li><li>. Agilent 2100 Bioanalyzer User's Guide Available from: <a target="_blank" href="https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack)">https://www.agilent.com/cs/library/usermanuals/Public/G2946-90004_Vespucci_UG_eBook_(NoSecPack)</a> (2016)</li><li>Kerr, K. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+analysis+of+benchmark+datasets+for+Agilent+two-color+microarray.">Extended analysis of benchmark datasets for Agilent two-color microarray.</a> BMC Bioinformatics. 8, 371 (2007).</li><li>Zhu, Q., Miecznikowski, J. C., Halfon, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+wholly+defined+Agilent+microarray+spike-in+dataset.">A wholly defined Agilent microarray spike-in dataset.</a> Bioinformatics. 27 (9), 1284-1289 (2011).</li><li>Vallee, M., Gravel, C., Palin, M. F., Reghenas, H., Stothard, P., Wishart, D. S., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+novel+and+known+oocyte-specific+genes+using+complementary+DNA+subtraction+and+microarray+analysis+in+three+different+species.">Identification of novel and known oocyte-specific genes using complementary DNA subtraction and microarray analysis in three different species.</a> Biol. Reprod. 73 (1), 63-71 (2005).</li><li>Thomas, P. D., 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=PANTHER:+a+library+of+protein+families+and+subfamilies+indexed+by+function.">PANTHER: a library of protein families and subfamilies indexed by function.</a> Genome Res. 13 (9), 2129-2141 (2003).</li><li>Mi, H., 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=The+PANTHER+database+of+protein+families,+subfamilies,+functions+and+pathways.">The PANTHER database of protein families, subfamilies, functions and pathways.</a> Nucleic Acids Res. 33, 284-288 (2005).</li><li>Mi, H., Muruganujan, A., Casagrande, J. T., Thomas, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Large-scale+gene+function+analysis+with+the+PANTHER+classification+system.">Large-scale gene function analysis with the PANTHER classification system.</a> Nat. Protoc. 8, 1551-1566 (2013).</li><li>Ross, P. J., Wang, K., Kocabas, A., Cibelli, 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=Housekeeping+gene+transcript+abundance+in+bovine+fertilized+and+cloned+embryos.">Housekeeping gene transcript abundance in bovine fertilized and cloned embryos.</a> Cell Reprogram. 12 (6), 709-717 (2010).</li><li>Gilbert, I., 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=The+dynamics+of+gene+products+fluctuation+during+bovine+pre-hatching+development.">The dynamics of gene products fluctuation during bovine pre-hatching development.</a> Mol. Reprod. Dev. 76, 762-772 (2009).</li><li>Vallee, 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=Revealing+the+bovine+embryo+transcript+profiles+during+early+in+vivo+embryonic+development.">Revealing the bovine embryo transcript profiles during early in vivo embryonic development.</a> Reproduction. 138 (1), 95-105 (2009).</li><li>Dafforn, 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=Linear+mRNA+amplification+from+as+little+as+5+ng+total+RNA+for+global+gene+expression+analysis.">Linear mRNA amplification from as little as 5 ng total RNA for global gene expression analysis.</a> Biotechniques. 37 (5), 854-857 (2004).</li><li>Beaujean, N., Jammes, H., Jouneau, A., Dufort, I., Rovert, C., Sirard, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+Reprogramming.">Nuclear Reprogramming.</a> Studying Bovine Early Embryo Transcriptome by Microarray. , (2015).</li><li>Patterson, T. 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=Performance+comparison+of+one-color+and+two-color+platforms+within+the+MicroArray+Quality+Control+(MAQC)+project.">Performance comparison of one-color and two-color platforms within the MicroArray Quality Control (MAQC) project.</a> Nat. Biotechnol. 24 (9), 1140-1150 (2006).</li></ol>

The authors have nothing to disclose.

İlk sorun gen ifadesinin çalışmak için yüksek kaliteli RNA yeterli miktarda almıyor Günlük 7 sığır embriyolarının kullanarak mikroarray analizi gerçekleştirmek için. Geleneksel fenol / kloroform RNA ekstraksiyonu ve etanol çökeltme yöntemi, düşük verim ve RNA amplifikasyon reaksiyonu inhibe olası artık fenol elde Gün 7 embriyo için uygun değildir. Bunun yerine, standart sütun tabanlı bir yöntem, toplam RNA izole etmek ve daha sonra konsantrasyonunu arttırmak için en az elüsyon tamponuyl...

Yüksek verimli sütçü sığırlarda erken embriyonik kayıp süt endüstrisi 1, 2 ana zorluklardan biridir. Sığır nedeniyle benzer gelişimsel süreç 3, 4 insan preimplantasyon embriyo gelişimini incelemek için ilginç bir model haline gelmiştir. Ancak, daha fazla araştırma sığır erken embriyonik gelişiminde rol oynayan genler hakkında daha iyi bir anlayış olması gerekir. İlk mikrodizi teknolojisi bu yana yirmi yıl 1995 5 geliştirilen sonra daha sofistike prob üretim teknolojisi azaltılmış baskı hataları içinde ve farklı mikroarray platformları 6 arasında dizi yongalarının değişkenlik gelişimi. Geliştirilmiş mikrodizi teknolojisi erken embriyo q, son zamanlarda klinik araştırmalar 7 ve bu teknolojinin yaygın bir uygulama ile sonuçlanmıştıruality değerlendirmesi 8. mikroarray teknolojisi için gerekli malzeme büyük miktarda mikroarray teknolojisi başlangıçta böyle erken embriyonik gelişme olarak araştırma alanları bir dizi giremedi ana nedeni olduğunu. Daha yakın zamanda, RNA amplifikasyon yöntemleri doğrusal RNA Başlangıç Maddesi 9 alt nanogram arasında mikrogram seviyesine kadar RNA amplifikasyonu için geliştirilmiştir. Piyasada bulunan birçok ticari RNA amplifikasyon kitleri vardır; Ancak, daha popüler iyi gelişmiş kitleri Ribo-Tek Primer İzotermal Amplification 10 ile ilgili ve T7 organizatörü 11 yöntemleri sürülür. En popüler antisens RNA amplifikasyonu 5 &#39;ucunda 12 bir T7 promotörü bağlantılı bir oligo dT primeri ile in vitro transkripsiyonu ile kullanır. Bu teknoloji doğrusal amplifikasyon f sonra temsili bir anti-duyu transkript en muhafaza veriyorveya diziler melezleme 13. Bu yöntem, sığır embriyo 8 ekstrakte edilen toplam RNA pikogram seviyesini yükseltmek için adapte edilmiştir. Evrensel Bağlantı Sistemi (ULS) doğrudan guanin 14 N7 pozisyonunda bir koordinatif bağ oluşturarak, platin bağlı flüoresan boya ya siyanin 547 veya cyanine 647 ile DNA veya RNA güçlendirilmiş içeren etiketleme yöntemidir. Bu yöntem, Arna enzimatik yöntem 15 tarafından üretilen tadil edilmiş aminoallyl göre değişiklik olmadan Arna amplifiye daha stabil oluşturmak için embriyolar araştırma uyarlanmıştır. Tek boya ve iki boyalar etiketleme Her iki yöntem mikroarray Evrensel Bağlantı Sistemi kullanılarak adapte edilmiştir. Büyük bir mikroarray karşılaştırmaları çalışma tek ve iki renkli dizisi platformları 6 arasındaki veri kalitesi iyi bir korelasyon olduğunu oldu. Son zamanlarda, hem T7 yükseltici sürücün, antisens RNA amplifikasyonu ve uls etiketleme yöntemleri mikrodizi hibridizasyon 8, 16 için Arna malzeme etiketli yüksek kalitede yeterli miktarda üretmek için daha güvenilir bir protokol sağlamaktır geliştirilmiştir. Bu nedenle, bu çalışmada örnek olarak 7. Gün sığır embriyoları kullanılarak iki renkli mikroarray yer alan veri analizi RNA ekstraksiyon önemli adımlardan bazıları göstermek için bir protokol sağlar.

<table><tbody><tr><td>PicoPure RNA Isolation Kit</td><td>Applied Biosystems</td><td>KIT0204</td><td></td></tr><tr><td>RNase-Free DNase Set (50)</td><td>Qiagen</td><td>79254</td><td></td></tr><tr><td>Agilent RNA 6000 Pico Kit</td><td>Agilent Technologies</td><td>5067-1513</td><td></td></tr><tr><td>Arcturus RiboAmp HS PLUS Kit</td><td>Applied Biosystems</td><td>KIT0505</td><td></td></tr><tr><td>2100 Bioanalyzer Instruments</td><td>Agilent Technologies</td><td>G2940CA</td><td></td></tr><tr><td>RNA Screen Tape</td><td>Agilent Technologies</td><td>5067-5576</td><td></td></tr><tr><td>ULS Fluorescent Labeling Kit</td><td>Kreatech Diagnostics</td><td>EA-021</td><td></td></tr><tr><td>Custom Gene Expression Microarrays</td><td>Agilent Technologies</td><td>G2514F</td><td></td></tr><tr><td>Agilent Gene Expression wash buffer 1</td><td>Agilent Technologies</td><td>Part #5188-5325</td><td></td></tr><tr><td>Agilent Gene Expression wash buffer 2</td><td>Agilent Technologies</td><td>Part #5188-5326</td><td></td></tr><tr><td>2x Hi-RPM Hybridization buffer</td><td>Agilent Technologies</td><td>&amp;nbsp;Part #5190-0403</td><td></td></tr><tr><td>25x Fragment buffer</td><td>Agilent Technologies</td><td>Part #5185-5974</td><td></td></tr><tr><td>10x GE Blocking Agent</td><td>Agilent Technologies</td><td>Part #5188-5281</td><td></td></tr><tr><td>Stabilization and drying solution</td><td>Agilent Technologies</td><td>&amp;nbsp;Part #5185-5979</td><td></td></tr><tr><td>Gasket slides enabled by Agilent SureHyb techonolgy</td><td>Agilent Technologies</td><td>G2524-60012</td><td>Pack of 20 gasket slides, 4 microarrays/slide</td></tr><tr><td>Two-Color RNA Spike-In Kit</td><td>Agilent Technologies</td><td>&amp;nbsp;Cat# 5188-5279</td><td></td></tr><tr><td>GenePix 4000B array scanner</td><td>Molecular Devices</td><td>GENEPIX 4000B-U</td><td></td></tr><tr><td>Ozone Free Box</td><td>BioTray</td><td>OFB_100x200</td><td></td></tr><tr><td>GAL file</td><td>Agilent Technologies</td><td>-</td><td></td></tr></tbody></table>

transcriptome profiling of in-vivo produced bovine pre-implantation embryos using two-color microarray platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

7. Gün sığır embriyolarının toplam RNA ve amplifiye Arna temsili bir sonuç Şekil 3&#39;te gösterilen ve Tablo 1 &#39;de özetlenmiştir. RNA bütünlüğü ve profil RNA ekstraksiyonu sonrasında değerlendirilebilir. RNA kalitesinin değerlendirilmesi Bioanalyzer cihazı (Şekil 3A) ve daha yüksek 7.0 amplifikasyonu (Tablo 1) için kullanılacak nit...

Watch this Scientific Journal Video about Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform at JoVE.com

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptome Profil<em&gt; In-vivo</emİki renkli Mikroarray Platformu Kullanma&gt; Üretilen Sığır pre-implantasyon Embriyolar

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

transcriptome-profiling-vivo-produced-bovine-pre-implantation-embryos

Research

JoVE Journal

Developmental Biology

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

7.7K Görüntüleme.  University of Alberta. Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Video: Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

Floresan protein ifadesindeki<em&gt; Branchiostoma lanceolatum</em&gt; Döllenmemiş Oositlerinin içine mRNA Enjeksiyon

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Gelişim Biyolojisi

Laser-assisted Cytoplasmic Microinjection in Livestock Zygotes

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can create genetic modifications that will be present in every cell of an adult organism. It can also be used to deliver siRNA, mRNAs or blocking antibodies to study gene function in preimplantation embryos. The conventional technique for microinjecting embryos used in rodents consists of a very thin micropipette that directly penetrates the plasma membrane when advanced into the embryo. When this technique is applied to livestock animals it usually results in low efficiency. This is mainly because in contrast to mice and rats, bovine, ovine, and porcine zygotes have a very dark cytoplasm and a highly elastic plasma membrane that makes visualization during injection and penetration of the plasma membrane hard to achieve. In this protocol, we describe a suitable microinjection method for the delivery of solutions into the cytoplasm of cattle zygotes that has proved to be successful for sheep and pig embryos as well. First, a laser is used to create a hole in the zona pellucida. Then a blunt-end glass micropipette is introduced through the hole and advanced until the tip of the needle reaches about 3/4 into the embryo. Then, the plasma membrane is broken by aspiration of cytoplasmic content inside the needle. Finally, the aspirated cytoplasmic content followed by the solution of interest is injected back into the embryonic cytoplasm. This protocol has been successfully used for the delivery of different solutions into bovine and ovine zygotes with 100% efficiency, minimal lysis, and normal blastocysts development rates.

This protocol shows how to perform cytoplasmic microinjection in farm animal zygotes. This technique can be used to deliver any solution into the one-cell embryo such as genome editing tools to generate knockout animals.

Lazer destekli Hayvancılık Zigotlann Sitoplazmik Mikroenjeksiyon

Cytoplasmic microinjection into one-cell embryos is a very powerful technique. As an example, it enables the delivery of genome editing tools that can ...

Genetik

Accurate and Phenol Free DNA Sexing of Day 30 Porcine Embryos by PCR

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, the ability to determine an embryo&#39;s sex is necessary in many experiments particularly regarding early development. The present protocol demonstrates an inexpensive, rapid and non-toxic preparation of pig genomic DNA for use with PCR. Day 30 embryos must be humanely collected according to the guidelines established by Institutional Animal Policy and Welfare Committees for the present protocol. The preparation of the whole embryo for this PCR based sexing technique simply involves grinding the frozen embryo to a fine powder using a pre-chilled mortar and pestle. PCR-quality DNA is released from a small amount of embryo powder by applying a hot incubation in an alkaline lysis reagent. Next, the DNA solution is mixed with neutralization buffer and used directly for PCR. Two primer pairs are generated to detect specific sex determining region of the Y- chromosome (SRY) and ZFX region of the X- chromosome with high accuracy and specificity. The same protocol can be applied to other elongated embryos (Day 10 to Day 14) earlier than Day 30. Also, this protocol can be carried with 96-welled plates when screening a large number of embryos, making it feasible for automation and high-throughput sex typing.

This protocol describes an accurate, inexpensive, rapid and non-toxic method to determine the sex of Day 30 porcine embryo using PCR method after grinding an embryo into powder without phenol chloroform extraction and DNA column purification.

PCR ile Gün 30 Domuz Embriyolar Doğru ve Fenol Ücretsiz DNA cinsiyet tayini

Research into prenatal programming in the pig has shown that the sex of the developing embryo or fetus can influence the developmental outcome. Therefore, ...

Functional Cloning Using a Xenopus Oocyte Expression System

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in abundance, may not be secreted but tethered to the signaling cell(s), or may require the presence of binding partners or upstream regulatory molecules. Thus in a search for gene products capable of eliciting an early lens-inductive response in competent ectoderm, we utilized an expression cloning system that would allow identification of paracrine or juxtacrine factors as well as transcriptional or other regulatory proteins. Pools of mRNA were injected into Xenopus oocytes, and responding tissue placed directly on the oocytes and co-cultured. Following functional cloning of ldb1 from a neural plate stage cDNA library based on its ability to elicit the expression of the early lens placode marker foxe3 in lens-competent animal cap ectoderm, we characterized the mRNA expression pattern, and assayed developmental progression following overexpression or knockdown of ldb1. This system is suitable in a very wide variety of contexts where identification of an inducer or its upstream regulatory molecules is sought using a functional response in competent tissue.

Functional Cloning Using a Xenopus Oocyte Expression System

We describe a Xenopus oocyte and animal cap system for the expression cloning of genes capable of inducing a response in competent ectoderm, and discuss techniques for the subsequent analysis of such genes. This system is useful in the functional identification of a wide range of gene products.

Bir kullanılması Fonksiyonel Klonlama<em&gt; Xenopus</em&gt; Oosit ekspresyon sistemi

Identification of genes responsible for embryonic induction poses a number of challenges; to name a few, secreted molecules of interest may be low in ...

Quantitative Analysis of Protein Expression to Study Lineage Specification in Mouse Preimplantation Embryos

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for embryo collection, immunofluorescence, imaging on a confocal microscope, and image segmentation and analysis are described. This method allows quantitation of the expression of multiple nuclear markers and the spatial (XYZ) coordinates of all cells in the embryo. It takes advantage of MINS, an image segmentation software tool specifically developed for the analysis of confocal images of preimplantation embryos and embryonic stem cell (ESC) colonies. MINS carries out unsupervised nuclear segmentation across the X, Y and Z dimensions, and produces information on cell position in three-dimensional space, as well as nuclear fluorescence levels for all channels with minimal user input. While this protocol has been optimized for the analysis of images of preimplantation stage mouse embryos, it can easily be adapted to the analysis of any other samples exhibiting a good signal-to-noise ratio and where high nuclear density poses a hurdle to image segmentation (e.g., expression analysis of embryonic stem cell (ESC) colonies, differentiating cells in culture, embryos of other species or stages, etc.).

This protocol presents a method to perform quantitative, single-cell in situ analysis of protein expression to study lineage specification in mouse preimplantation embryos. The procedures necessary for collection of blastocysts, whole-mount immunofluorescent detection of proteins, imaging of samples on a confocal microscope, and nuclear segmentation and image analysis are described.

Protein İfade Kantitatif Analiz Fare Preimplantasyon Embriyolar Lineage Şartname Eğitim için

This protocol presents a method to perform quantitative, single-cell in situ analyses of protein expression to study lineage specification in mouse ...

Loss- and Gain-of-function Approach to Investigate Early Cell Fate Determinants in Preimplantation Mouse Embryos

Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene modification in preimplantation embryos provides a means to study the underlying mechanisms of genes implicated in, for instance, cellular differentiation into the trophectoderm (TE) and the inner cell mass (ICM). Here, we describe a protocol that examines the role of neogenin as an authentic receptor for initial cell fate determination in preimplantation mouse embryos. First, we discuss the experimental manipulations that were used to produce gain and loss of neogenin function by microinjecting neogenin cDNA and shRNA; the effectiveness of this approach was confirmed by a strong correlation between the pair-wise expression levels of either red fluorescent protein (RFP) or green fluorescent protein (GFP) and the immunocytochemical quantification of neogenin expression. Secondly, overexpression of neogenin in preimplantation mouse embryos leads to normal ICM development while neogenin knockdown causes the ICM to develop abnormally, implying that neogenin could be a receptor that relays extracellular cues to drive blastomeres to early cell fates. Given the success of this detailed protocol in investigating the function of a novel embryonic developmental stage-specific receptor, we propose that it has the potential to aid in exploration and identification of other stage-specific genes during embryogenesis.

The goal of this protocol is to describe a loss- and gain-of function method that is applicable to identify neogenin as a stage-specific receptor that leads to trophectoderm and inner cell mass differentiation in preimplantation mouse embryos.

Preimplantasyon Fare Embriyolar Erken Hücre Kader Belirleyicileri Araştırma kaybı: ve Kazanç fonksiyon-Yaklaşımı

 Gene silencing and overexpression techniques are instrumental for the identification of genes involved in embryonic development. Direct target gene ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

C. elegans Embriyonik Gelişimi Analiz Etmek Için Yarı-yüksek-throughput Görüntüleme Yöntemi ve Veri Görselleştirme Araç Seti

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One ...

Multiplexed Single Cell mRNA Sequencing Analysis of Mouse Embryonic Cells

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental biology. It has been successfully used to identify rare cell populations, discover novel marker genes, and decode spatial and temporal developmental information. The single cell method has also evolved from the microfluidic based Fluidigm C1 technology to the droplet-based solutions in the last two to three years. Here we used the heart as an example to demonstrate how to profile the mouse embryonic tissue cells using the droplet based scRNA-Seq method. In addition, we have integrated two strategies into the workflow to profile multiple samples in a single experiment. Using one of the integrated methods, we have simultaneously profiled more than 9,000 cells from eight heart samples. These methods will be valuable to the developmental biology field by providing a cost-effective way to simultaneously profile single cells from different genetic backgrounds, developmental stages, or anatomical locations.

Here we presented a multiplexed single cell mRNA sequencing method to profile gene expression in mouse embryonic tissues. The droplet-based single cell mRNA sequencing (scRNA-Seq) method in combination with multiplexing strategies can profile single cells from multiple samples simultaneously, which significantly reduces reagent costs and minimizes experimental batch effects.

Fare Embriyonik Hücrelerinin Çok katlı Tek Hücreli mRNA Sıralama Analizi

Single cell mRNA sequencing has made significant progress in the last several years and has become an important tool in the field of developmental ...

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3&#39; untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3&#39; UTR reporter as an example.

This protocol describes a reporter assay to study the regulation of mRNA translation in single oocytes during in vitro maturation.

Tek Bir Oocyte Reporter Tahlil Kullanarak In vitro Olgunlaşma Sırasında Maternal mRNA Çeviri Programını Tanımlama

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that ...

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

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

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

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

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

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

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