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>

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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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>&#160;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>&#160;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>&#160;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>&#160;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

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Fotoçevrim sonra Floresans Bozunma kullanarak Oturma balığı Embriyolar protein stabilitesi ölçümü (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Gelişim Biyolojisi

Contrast Imaging in Mouse Embryos Using High-frequency Ultrasound

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted applications, facilitate the detection and measure of vascular biomarkers at the molecular level. Within the mouse embryo, this noninvasive technique may be used to uncover basic mechanisms underlying vascular development in the early mouse circulatory system and in genetic models of cardiovascular disease. The mouse embryo also presents as an excellent model for studying the adhesion of microbubbles to angiogenic targets (including vascular endothelial growth factor receptor 2 (VEGFR2) or &#945;v&#946;3) and for assessing the quantitative nature of molecular ultrasound. We therefore developed a method to introduce ultrasound contrast agents into the vasculature of living, isolated embryos. This allows freedom in terms of injection control and positioning, reproducibility of the imaging plane without obstruction and motion, and simplified image analysis and quantification. Late gestational stage (embryonic day (E)16.6 and E17.5) murine embryos were isolated from the uterus, gently exteriorized from the yolk sac and microbubble contrast agents were injected into veins accessible on the chorionic surface of the placental disc. Nonlinear contrast ultrasound imaging was then employed to collect a number of basic perfusion parameters (peak enhancement, wash-in rate and time to peak) and quantify targeted microbubble binding in an endoglin mouse model. We show the successful circulation of microbubbles within living embryos and the utility of this approach in characterizing embryonic vasculature and microbubble behavior.

Here, we present a protocol to inject ultrasound microbubble contrast agents into living, isolated late-gestation stage murine embryos. This method enables the study of perfusion parameters and of vascular molecular markers within the embryo using contrast-enhanced high-frequency ultrasound imaging.

Yüksek frekanslı Ultrason Kullanımı Fare Embriyolar Kontrast Görüntüleme

Ultrasound contrast-enhanced imaging can convey essential quantitative information regarding tissue vascularity and perfusion and, in targeted ...

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Timus Karakterizasyonu kullanma Fare Embriyo atalarıdır Settling<em&gt; In Vivo</em&gt; Ve<em&gt; In Vitro</em&gt; Tahliller

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Analiz<em&gt; İn Vivo</em&gt; Hücre Göç Zebra balığı Embriyolar hücre nakli ve Time-lapse Görüntüleme kullanılarak

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

This video article details a straightforward in vivo methodology that can be used to systematically and efficiently characterize components of complex signaling pathways and regulatory networks in many invertebrate embryos.

Sadelik Gücü: olarak deniz kestanesi Embriyolar<em&gt; Vivo</em&gt; Karmaşık Hücre-hücre Sinyal Ağ Etkileşimleri incelenmesi için Gelişim Modelleri

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

Kimerik Zebra balığı Embriyolar Bireysel Retina progenitörlerin Transgenik Gen İfade Vivo Görüntüleme Hücre otonom olmayan Etkiler Eğitim için

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Fast and Efficient Expression of Multiple Proteins in Avian Embryos Using mRNA Electroporation

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. The high transfection efficiency permits at least 4 distinct mRNAs to be co-transfected with ~87% efficiency. Most of the electroporated mRNAs are degraded during the first 2 h post-electroporation, permitting time-sensitive experiments to be carried out in the developing embryo. Finally, we describe how to dynamically image live embryos electroporated with mRNAs that encode various subcellular targeted fluorescent proteins.

We report messenger RNA (mRNA) electroporation as a method that permits fast and efficient expression of multiple proteins in the quail embryo model system. This method can be used to fluorescently label cells and record their in vivo movements by time-lapse microscopy shortly after electroporation.

MRNA Electroporation kullanarak Avian embriyoları çoklu proteinlerin hızlı ve verimli Ifade

We report that mRNA electroporation permits fluorescent proteins to label cells in living quail embryos more quickly and broadly than DNA electroporation. ...

Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more than 50 different pathological conditions, such as rheumatoid arthritis, oculopathy, cardiovascular diseases, and tumor metastasis. During angiogenesis drug development, it is crucial to use in vitro assay systems with appropriate cell types and proper conditions to reflect the physiologic angiogenesis process. To overcome limitations of current in vitro angiogenesis assay systems using mainly endothelial cells, we developed a 3-dimensional (3D) co-culture spheroid sprouting assay system. Co-culture spheroids were produced by two human vascular cell precursors, endothelial colony forming cells (ECFCs) and mesenchymal stem cells (MSCs) with a ratio of 5 to 1. ECFCs+MSCs spheroids were embedded into type I collagen matrix to mimic the in vivo extracellular environment. A real-time cell recorder was utilized to continuously observe the progression of angiogenic sprouting from spheroids for 24 h. Live cell fluorescent labeling technique was also applied to tract the localization of each cell type during sprout formation. Angiogenic potential was quantified by counting the number of sprouts and measuring the cumulative length of sprouts generated from the individual spheroids. Five randomly-selected spheroids were analyzed per experimental group. Comparison experiments demonstrated that ECFCs+MSCs spheroids showed greater sprout number and cumulative sprout length compared with ECFCs-only spheroids. Bevacizumab, an FDA-approved angiogenesis inhibitor, was tested with the newly-developed co-culture spheroid assay system to verify its potential to screen anti-angiogenic drugs. The IC50 value for ECFCs+MSCs spheroids compared to the ECFCs-only spheroids was closer to the effective plasma concentration of bevacizumab obtained from the xenograft tumor mouse model. The present study suggests that the 3D ECFCs+MSCs spheroid angiogenesis assay system is relevant to physiologic angiogenesis, and can predict an effective plasma concentration in advance of animal experiments.

Three-dimensional co-culture spheroid angiogenesis assay system is designed to mimic the physiologic angiogenesis. Co-culture spheroids are formed by two human vascular cell precursors, ECFCs and MSCs, and embedded in collagen gel. The new system is effective for evaluating angiogenic modulators, and provides more relevant information to the in vivo study.

Endotel Kolonisi Oluşturan Hücreler ve Mezenkimal Kök Hücreler tarafından Oluşturulan Eş Kültür Sfaoidler Kullanılarak Üç Boyutlu Anjiyogenez Tsay Sistemi

Studies in the field of angiogenesis have been aggressively growing in the last few decades with the recognition that angiogenesis is a hallmark of more ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Kafa Uzayı Gaz Kromatografisi Kullanan ZebraBalığı Embriyolarında Etanol Düzeylerinin Ölçülmesi

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

Nitel ve Nicel Histolojik Yöntemlerle Fare Embriyolarında Konjenital Kalp Defektlerinin Analizi

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

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