Uyuşturucu Merkezi Ulusal Enstitüsü (P50 DA 011.806) ve Amerika Birleşik Devletleri Ordusu Milli Savunma Bakanlığı (W81XWH-07-1-0183) tarafından desteklenmiştir.

1. Özel miRNA microarrays yazdırma <ol><li> Mikroarray bir üniversite veya bir şirket mikroarray çekirdek tesisi hizmetleri kullanarak slaytlar yazdırın. Mikroarray imalat kalitesi mikroarray deneyinin başarısı için en önemli faktörlerden biridir. Mümkünse birkaç hizmetleri deneyin. İdeal olarak, bir seferde 50-100 slayt yazdırmak, ve tüm slaytları iyi ayrılmış, tek tek noktalar ile aynı bakacağız. </li><li> Mikroarray destek için, biz EKSİKLİK II kaplı slaytlar kullanın. </li><li> MiRNA probları için, NCode Multi-Türlerin Mikroarray Probe V2 Set miRNA kullanın. </li><li> 10 mcM 3 x SSC oligonükleotidler çözülür ve quadruply slaytları yazdırmak. GAAPS II slaytlar için talimatlara göre ultraviyole ışınlama slaytlar sabitleyin. Elmas kalemle basılı problar ile alan ve yan işaretlemek için bir slayt etiketleyin. Mağaza at 22 ° C </li></ol> 2. Örnek hazırlama <ol><li> Herhangi bir yöntemveya reaktif sürece küçük RNA&#39;lar korur, RNA izole etmek için kullanılan olabilir. Biz genellikle Trizol (Invitrogen), A 260nm ve A 280nm okumaları ile ölçülen RNA hazırlıkları yeterli miktar ve kalitede, toplam RNA ayıklamak için kullanın . </li><li> RNA etiketleme, 1 x tamponu (50 mM HEPES, pH 7.8, 20 mM MgCl 2, 10 mg / ml BSA,% 10 5&#39;-PCU-DY547-3 &#39;6 0.5 mikrogram ile toplam RNA karışımı ~ 25 mg için DMSO) içeren ~ 20 adet T4 RNA ligaz 1, ~ 10 mM DTT ve ATP için 10 x T4 RNA ligaz 1 tampon ~ 0,02-0,03 son hacmi ile desteklenebilir. </li><li> 2-24 saat boyunca bir buzdolabı içinde buz üzerinde devam etmek için etiketleme İzin. 0,3 M sodyum asetat ve etanol 3 cilt RNA çöktürün. Verimli ligasyonu ve yağış için, üzerinden 2 mg / ml total RNA çözülür ve ~ 20 ul toplam ligasyonu hacmi tutmak. <ol><li> Az RNA varsa, giriş RNA ve 5&#39;-PCU-DY547-3 &#39;Küçülmüş olabilir miktarı &lt;/ Li&gt; <li> RNA çok seyreltik ise, yağış yardım etmek için maya tRNA gibi taşıyıcı ekleyin. </li></ol></li><li> Referans DNA oligonükleotidler memeli miRNA&#39;lar Ulysis Alexa Fluor 647 Nükleik Asit Etiketleme Seti 6 hibridizasyon işlemi için bir kontrol olarak kullanarak bir alt kümesine karşılık gelen toplam 1 mcg birleştirin ve etiket . CentriSep bir sütunu kullanarak işaretli DNA arındırmak ve -20 ° C&#39;de karanlıkta kaydetmek </li></ol> 3. Mikroarray hibridizasyon <ol><li> Ilk kez slayt kullanmak için, 37 3 x SSC,% 0.1 SDS, 30-60 dakika süreyle% 0,2 BSA filtrelenmiş bir çözüm slaytları önceden melezleşme ° C Batmak su birkaç kez, sonra izopropanol. Az 5 dakika boyunca 22 100 g slayt adaptör ° C ile bir santrifüj kullanarak slaytları Kuru </li></ol> Not: Biz mikroarray hibridizasyon gerçekleştirmek için Corning mikroarray hibridizasyon odaları ve Lifterslips Erie Bilimsel mSeries kullanın. <ol start="2"><li> Li durulayınhava kurumadan su fterslips, daha sonra etanol,. Mikroarray hibridizasyon odasının tabanı içinde bir slayt ve slayt bir lifterslip üstüne yerleştirin. Lifterslip slayt iletişime beyaz şeritler ile prob alanı kapsayacaktır. </li><li> Karanlıkta, etiketli RNA çöktürülmüş aşağı doğru döndürün. % 70 etanol ile bir kez yıkayın ve hava pelet kuru. Pelet kırmızımsı olmalıdır. </li><li> 400 mM Na 2 HPO 4, pH 7.0,% 0.8 BSA,% 5 SDS,% 12 saflaştırılmış 1/15-1/100 ul formamid 6, RNA örnek başına etiketli referans DNA (2.4) içeren bir melezleşme çözüm hazırlayın. Eklendi referans DNA miktarı farklı oligonükleotidler (2.4) kaç etiketlenir bağlıdır. 100&#39;ün üzerinde varsa, daha az seyreltme gereklidir. </li><li> RNA pelet ~ 60 ul hibridizasyon solüsyonu ile iyice eritin. </li><li> Corning mikroarray hibridizasyon odasının tabanı nemlendirici kuyuların her biri 20 ul su ekleyin. </li><li> T ekleslayt etiketli RNA ve referans DNA karışımı. Ince bir pipetlemeyin ucu kullanın lifterslip kenarına hafifçe dokunmak ve çözüm emme yoluyla lifterslip ve slayt arasındaki boşluk girmek için izin verir. </li><li> Hibridizasyon odasının tabanı üzerinde kapak yerine metal klipler, daha sonra Odanın mühür. </li><li> Corning oda kaset ile gelen plastik torba içinde bütün odasına koyun. </li><li> Islatılmış kağıt havlu ile bir kap içinde oda kaset (ler) yerleştirin; plastik wrap ile kabın kapağı ve 37 iç ° C ~ 24 saat boyunca nemli, hücre kültürü inkübatör. Bu önlemler (3.6, 3.9, 3.10), hibridizasyon çözüm inkübasyon sırasında kurumasına olmadığını garanti eder. </li></ol> 4. Post-hibridizasyon işleme <ol><li> Oda kasetleri tek tek sökün. 22 2 x SSC Batmak bir slayt ve onun lifterslip ° C Lifterslip doğal slayt kapalı düşecek. 0.8 x SSC yerleştirin. Yıkamaiki kez, daha sonra 0.8 x SSC slaytlar 0,4 x SSC, toplam 1-2 dakika içinde üç kez. Slaytları slayt adaptör ile bir santrifüj kullanarak kurutun. Lifterslips su ile yıkayın ve daha sonra yeniden kullanmak için kaydedin. </li><li> Slaytlar uygun herhangi bir tarayıcı, örneğin, Perkin Elmer ScanArray 5000 veya Moleküler Cihazlar Axon GenePix 4000B mikroarray tarayıcı ile tarayın. Ilgili görüntü dosyalarını elde etmek için dalga boyları yaklaşık 547 nm ile 647 nm tarayın. </li><li> Girişi, 4.2 &#39;den resim dosyaları) piksel yoğunlukları ölçmek için bu BlueFuse gibi programlar kullanın. Daha fazla dikkate mikrodizinlerinde anormal noktalar hariç programı ile tek tek noktalar kontrol edin. Bu anormal lekeler genellikle kötü slayt baskı veya slayt işleme hibridizasyon sonra ortaya çıkar. </li><li> Veri analizi ve sunumu için, GeneSpring ve Excel gibi programları kullanın. Mikroarray veri normalleşmesi için Genel hususlar geçerli olan bu yazının kapsamı dışındadır. </li><li> Bir kez tatmin edici sinyaller af elde edilirter 4.3), 7 için yeniden mikroarray slaytlar şerit. 10-20 dakika sonra önceden ısıtılmış 62 bir boyama çanak, 1 mM NaOH ve 0,1 x SSC ° C batırmayın, su kaydırağı durulayın. Inkübasyon kez tekrarlayın. 22 nazik sallayarak 60 dakika kadar su birkaç kez slaytlar ° C yıkayın Santrifüj kullanılarak slaytlar, ve 22 mağaza ° C kurulayın </li><li> Rejenere slaytlar kullanmak için, onları tekrar ön melezleşme gerekli değildir. Sadece izopropanol tarafından takip suda yıkayın ve slaytlar sağ hibridizasyon önce kuru. </li></ol> 5. Temsilcisi Sonuçlar: Biz büyük ölçüde küresel miRNA ifade binlerce örnek profil açıklanan prosedürleri takip, yani, pek çok farklı koşullar altında insan örnek zebrafish izole RNA&#39;lar. Şekil 1 çok hassas ve güçlü bir hibridizasyon sinyalleri göstermek için bir mikroarray taranmış bir görüntü gösterir kaydırın. Pearson correlatimükemmel tekrarlanabilirlik gösteren ~ 0.99 7, teknik arasındaki katsayıları mikroarray hibridizasyon, çoğaltır. <img alt="Şekil 1" src="/files/ftp_upload/3250/3250fig1.jpg" /> Hibridizasyon sonra taranan miRNA mikroarray slayt 1 Kompozit görüntü Şekil. Kırmızı noktalar, sarı lekeler, DNA ve RNA aynı sondaları tarafından döllemeler iken, referans DNA DY547 etiketli RNA örnek yeşil noktalar, döllemeler sonuçlandı.

<ol>
	<li>Ambros, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+functions+of+animal+microRNAs.">The functions of animal microRNAs.</a> Nature. 431, 350-355 (2004).</li><li>Friedman, R. C., Farh, K. K., Burge, C. B., Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92-105 (2009).</li><li>Chekulaeva, M., Filipowicz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+miRNA-mediated+post-transcriptional+regulation+in+animal+cells.">Mechanisms of miRNA-mediated post-transcriptional regulation in animal cells.</a> Curr. Opin. Cell Biol. 21, 452-460 (2009).</li><li>Farazi, T. A., Spitzer, J. I., Morozov, P., Tuschl, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRNAs+in+human+cancer.">miRNAs in human cancer.</a> J. Pathol. 223, 102-115 (2011).</li><li>Small, E. M., Olson, E. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pervasive+roles+of+microRNAs+in+cardiovascular+biology.">Pervasive roles of microRNAs in cardiovascular biology.</a> Nature. 469, 336-342 (2011).</li><li>Thomson, J. M., Parker, J., Perou, C. M., Hammond, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+custom+microarray+platform+for+analysis+of+microRNA+gene+expression.">A custom microarray platform for analysis of microRNA gene expression.</a> Nat. Methods. 1, 47-53 (2004).</li><li>Zhang, X., Xu, W., Tan, J., Zeng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stripping+custom+microRNA+microarrays+and+the+lessons+learned+about+probe:slide+interactions.">Stripping custom microRNA microarrays and the lessons learned about probe:slide interactions.</a> Anal. Biochem. 386, 222-227 (2009).</li><li>Griffiths-Jones, S., Saini, H. K., van Dongen, S., Enright, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miRBase:+tools+for+microRNA+genomics.">miRBase: tools for microRNA genomics.</a> Nucl. Acids. Res. 36, D154-D158 (2008).</li><li>Landgraf, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mammalian+microRNA+expression+atlas+based+on+small+RNA+library+sequencing.">A mammalian microRNA expression atlas based on small RNA library sequencing.</a> Cell. 129, 1401-1414 (2007).</li><li>Chiang, H. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+microRNAs:+experimental+evaluation+of+novel+and+previously+annotated+genes.">Mammalian microRNAs: experimental evaluation of novel and previously annotated genes.</a> Genes. Dev. 24, 992-1009 (2010).</li></ol>

Çıkar çatışması ilan etti.

Mikroarray derin sıralama teknolojilerindeki son gelişmelere rağmen, DNA ve RNA yüksek verimlilik analizi için uygun bir seçenek kalır. Derin bir sıralama ile karşılaştırıldığında, mikroarray deneyler ucuzdur ve tipik bir moleküler biyoloji laboratuvarı deney ve esneklik sağlar ve zaman kazandırır veri analizi, in-house, en gerçekleştirebilirsiniz. Gelecekte, muhtemelen mikroarray&#39;ler uygundur genler, örneğin setleri yoğun sorgulamak için, bir genom veya miRNA&#39;lar transkripsiyon faktö...

<table border="1"><tbody><tr><th> Öğeler </th><th> Satıcı </th><th> Katalog numarası </th><th> Yorumlar </th></tr><tr><td> NCode Multi-Türlerin Mikroarray Probe Set V2 miRNA </td><td> Invitrogen </td><td> MIRMPS201 </td><td> MiRBase Sürüm 9.0 (Ekim 2006) göre tasarlanmıştır. ~ Değiştirilmeden 1140, 34-44 nt uzunluğunda oligonükleotidler, solucan, sinek, zebrafish, fare, sıçan, ve insan miRNA&#39;lar probları olarak ve iç kontrol probları gibi snoRNAs gibi bir dizi içerir. MiRNA probları dolayısıyla olgun miRNA&#39;lar ~ 44 nt boyutu tamamlayıcı dizilerinin çiftlerin. Analizi için bir ilgi belirli bir gen (ler) den miRNA&#39;lar odaklanmak. </td></tr><tr><td> Trizol </td><td> Invitrogen </td><td> 15596018 </td><td> Toplam RNA örnekleri hazırlamak ve ölçmek için daha hızlı ve daha kolay ve mRNA analizi gibi downstream uygulamalar için uygun olmasına rağmen Ayrıca, etiketleme için küçük RNA fraksiyonu zenginleştirmiş. </td></tr><tr><td> T4 RNA ligaz 1 </td><td> Yeni İngiltere Biolabs </td><td> M0204L </td><td></td></tr><tr><td> Ulysis Alexa Fluor 647 Nükleik Asit Etiketleme Kiti </td><td> Invitrogen </td><td> U21660 </td><td> Bu kit veya benzer ürünlerin yanı sıra deneysel RNA örnekleri ya da bir kontrol RNA (yerine kontrol DNA) etiket kullanılabilir. </td></tr><tr><td> 5&#39;-PCU-DY547-3 &#39; </td><td> Dharmacon </td><td> Özel yapılmış </td><td> Küçük RNA fraksiyonu benzer ligasyonu ile etiketli olabilir. </td></tr><tr><td> CentriSep sütunlar </td><td> Princeton Ayrımları </td><td> CS-901 </td><td></td></tr><tr><td> GAPSII kaplı slayt </td><td> Corning </td><td> 40004 </td><td> Slayt Diğer tür de kullanılıyor olabilir. </td></tr><tr><td> Mikroarray hibridizasyon odaları </td><td> Corning </td><td> 2551 ya da 40.080 </td><td> Hibridizasyon odaları ve lamelleri başka türlü de çalışması gerekir. ~ 2 saat piyasada bulunan hibridizasyon makineleri kullanma, belirgin örneğin hibridizasyon zamanı azaltabilir. </td></tr><tr><td> Lifterslips </td><td> ThErmo / Erie Bilimsel </td><td> 25X60I-M5439-001-LS </td><td></td></tr><tr><td> BlueFuse </td><td> BlueGenome </td><td></td><td></td></tr><tr><td> GeneSpring </td><td> Agilent </td><td></td><td></td></tr></tbody></table>

performing custom microrna microarray experiments

microRNA (miRNA&#39;lar), yaygın olarak ökaryotlarda 1 olarak ifade edilir ~ 22 nükleotidler (nt) uzun RNA molekülleri büyük bir aileyiz . Kompleks genomların öncelikle bir çok sayıda post-transkripsiyonel hedef genlerin 2, 3 ekspresyonunu inhibe kullarak en az yüzlerce kodlayın. miRNA&#39;lar biyolojik süreçlerin 1 geniş bir kontrol eder. Buna ek olarak, değişen miRNA ifadesi, bu tür kanserleri gibi insan hastalıkları ile ilişkili olduğu ve miRNA&#39;lar hastalıkları ve prognozu 4, 5 biyobelirteçleri olarak hizmet edebilir. Bu nedenle, ifade ve pek çok farklı koşullar altında miRNA&#39;lar fonksiyonları anlamak önemlidir. Real-time PCR, mikroarray ve derin sıralama: Üç büyük yaklaşımlar profil miRNA ifade istihdam edilmiştir. MiRNA mikroarray tekniği yüksek verimli olma avantajı, genellikle daha az pahalı, deney ve analiz adımları carr olabilirüniversiteler, tıp fakülteleri ve ilişkili hastaneler, bir moleküler biyoloji laboratuvarı ied. Burada, özel miRNA mikroarray deneyler için bir yöntem açıklanmaktadır. MiRNA miRNA mikroarray&#39;ler prob seti üretmek için cam slaytlar basılacaktır. RNA küçük RNA türleri koruyan bir yöntem ya da reaktif kullanılarak izole edilmiş, ve ardından floresan bir boya ile etiketlenmiş. Kontrol olarak, miRNA&#39;lar bir alt kümesi karşılık gelen referans DNA oligonükleotidler de farklı bir floresan boya ile işaretlenmiştir. Referans DNA slayt ve melezleşme kalitesini göstermek için hizmet verecek ve aynı zamanda veri normalleşmesi için kullanılacaktır. RNA ve DNA karışık ve veritabanı miRNA&#39;lar en problar içeren bir mikroarray slayt melezleşmiştir. Yıkadıktan sonra, slayt görüntüler elde etmek için taranmış ve şiddetleri sayısal tek tek noktalar. Bu ham sinyalleri, ileri işlenmiş ve ilgili miRNA&#39;lar ifade veri olarak analiz edilecektir. Microarray slaytlar microarrays maliyetini azaltmak ve tutarlılık mikroarray deneyler geliştirmek için elimden ve rejenere olabilir. Aynı usul ve esaslar özel mikroarray deneylerin diğer türleri için geçerlidir.

Watch this Scientific Journal Video about Performing Custom MicroRNA Microarray Experiments at JoVE.com

Performing Custom MicroRNA Microarray Experiments

Özel microRNA mikroarray deneyler basit bir prosedür açıklanmıştır. Adımlar, RNA, etiketleme RNA ve referans DNA izole mikroarray&#39;ler için örnekleri literatürde, mikroarray&#39;ler tarama ve niceleme hibridizasyon sinyalleri analiz içerir.

Özel MicroRNA Mikroarray deneyler gerçekleştirme

microRNAs (miRNAs) are a large family of &tilde; 22 nucleotides (nt) long RNA molecules that are widely expressed in eukaryotes 1.  Complex genomes encode ...

performing-custom-microrna-microarray-experiments

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

19.5K Görüntüleme.  University of Minnesota. Özel microRNA mikroarray deneyler basit bir prosedür açıklanmıştır. Adımlar, RNA, etiketleme RNA ve referans DNA izole mikroarray'ler için örnekleri literatürde, mikroarray'ler tarama ve niceleme hibridizasyon sinyalleri analiz içerir.

Video: Performing Custom MicroRNA Microarray Experiments

Gibberella zeae Ascospore Production and Collection for Microarray Experiments.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain quality. F. graminearum also causes stalk and ear rots of maize and is a producer of mycotoxins such as the trichothecenes that contaminate grain and are harmful to humans and livestock (Goswami and Kistler, 2004).

The fungus produces two types of spores. Ascospores, the propagules resulting from sexual reproduction, are the main source of primary infection.  These spores are forcibly discharged from mature perithecia and dispersed by wind (Francl et al 1999). Secondary infections are mainly caused by macroconidia which are produced by asexual means on the plant surface. To study the developmental processes of ascospores in this fungus, a procedure for their collection in large quantity under sterile conditions was required. Our protocol was filmed in order to generate the highest level of information for understanding and reproducibility; crucial aspects when full genome gene expression profiles are generated and interpreted. In particular, the variability of ascospore germination and biological activity are dependent on the prior manipulation of the material. The use of video for documenting every step in ascospore production is proposed in order to increase standardization, complying with the increasingly stringent requirements for microarray analysis. The procedure requires only standard laboratory equipment. Steps are shown to prevent contamination and favor time synchronization of ascospores.

To study the developmental processes of ascospores in Gibberella zeae, a procedure for collection under sterile conditions is filmed in order to generate the highest level of information for protocol description. This should facilitate the reproducibility of the experiment, a crucial aspect when full genome expression profile tests are implemented.

Gibberella zeae Ascospore Üretim ve Tahsilat Mikroarray deneyler için.

Fusarium graminearum Schwabe (teleomorph Gibberella zeae) is a plant pathogen causing scab disease on wheat and barley that reduces crop yield and grain ...

Biyoloji

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Elektrofizyoloji Deneyler Yapay Bilayers hazırlanması

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

İşleme Loblolly Çam PtGen2 cDNA Mikroarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Global Gene Expression Analysis Using a Zebrafish Oligonucleotide Microarray Platform

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray technology is used in numerous biological disciplines in a variety of applications including global gene expression analysis in relation to developmental stage, to a disease state, and in toxic responses. Herein, we include a demonstration of global gene expression analysis using a comprehensive zebrafish-specific oligonucleotide microarray platform. The zebrafish expression microarray platform contains 385,000 probes, 60 base pairs in length, interrogating 37,157 targets with up to 12 probes per target. For this platform, all cDNA and genomic information available for the zebrafish was collected from various genomic databases including Ensembl (http://www.ensembl.org), VEGA (http://vega.sanger.ac.uk), UCSC (http://genome.ucsc.edu), and ZFIN (http://www.zfin.org). As a result this expression array provides complete coverage of the current zebrafish transcriptome. The zebrafish expression microarray was printed by Roche NimbleGen (Madison, WI). This technical demonstration includes the fluorescent labeling of a cDNA product, hybridization of the labeled cDNA product to the microarray platform, and array scanning for signal acquisition using the one color analysis strategy.  

Gene microarrays are powerful tools in gene expression profiling at a genome-wide level. This technology has application in a variety of biological disciplines including developmental biology and toxicology. In this video, we detail a protocol for global gene expression analysis using a comprehensive oligonucleotide microarray platform for the zebrafish.

Zebra balığı Oligonükleotid Mikroarray Platformu kullanarak Global Gen İfadesi Analizi

Gene microarray technology permits quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. Gene microarray ...

Measurement Of Neuromagnetic Brain Function In Pre-school Children With Custom Sized MEG

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain generates electric fields  - that can be recorded using electroencephalography (EEG)- and their concomitant magnetic fields  - detected by MEG. MEG signals are detected by specialized sensors known as superconducting quantum interference devices (SQUIDs). Superconducting sensors require cooling with liquid helium at -270 &deg;C. They are contained inside a vacumm-insulated helmet called a dewar, which is filled with liquid. SQUIDS are placed in fixed positions inside the helmet dewar in the helium coolant, and a subject's head is placed inside the helmet dewar for MEG measurements. The helmet dewar must be sized to satisfy opposing constraints. Clearly, it must be large enough to fit most or all of the heads in the population that will be studied. However, the helmet must also be small enough to keep most of the SQUID sensors within range of the tiny cerebral fields that they are to measure. Conventional whole-head MEG systems are designed to accommodate more than 90% of adult heads. However adult systems are not well suited for measuring brain function in pre-school chidren whose heads have a radius several cm smaller than adults. The KIT-Macquarie Brain Research Laboratory at Macquarie University uses a MEG system custom sized to fit the heads of pre-school children. This child system has 64 first-order axial gradiometers with a 50 mm baseline[2] and is contained inside a magnetically-shielded room (MSR) together with a conventional adult-sized MEG system [3,4]. There are three main advantages of the customized helmet dewar for studying children. First, the smaller radius of the sensor configuration brings the SQUID sensors into range of the neuromagnetic signals of children's heads. Second, the smaller helmet allows full insertion of a child's head into the dewar. Full insertion is prevented in adult dewar helmets because of the smaller crown to shoulder distance in children. These two factors are fundamental in recording brain activity using MEG because neuromagnetic signals attenuate rapidly with distance. Third, the customized child helmet aids in the symmetric positioning of the head and limits the freedom of movement of the child's head within the dewar. When used with a protocol that aligns the requirements of data collection with the motivational and behavioral capacities of children, these features significantly facilitate setup, positioning, and measurement of MEG signals.

The advent of MEG systems sized for young children opens important new opportunities to study brain development. The new system, together with a protocol that aligns experimental requirements with the capacities of children, can be used to study cognitive and language processes in healthy, awake children aged three to six.

Özel Ölçekli MEG ile Okul Öncesi Çocuk Neuromagnetic Beyin Fonksiyonu Ölçümleri

Magnetoencephalography is a technique that detects magnetic fields associated with cortical activity [1]. The electrophysiological activity of the brain ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

Mikroarray Analizi için Saccharomyces cerevisiae</em

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

Genome Editing with CompoZr Custom Zinc Finger Nucleases (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such as chemical-based mutagenesis or random integration of DNA sequences confer indiscriminate genetic changes in an overall inefficient manner and require incorporation of undesirable synthetic sequences or use of aberrant culture conditions, potentially confusing biological study. By contrast, transient ZFN expression in a cell can facilitate precise, heritable gene editing in a highly efficient manner without the need for administration of chemicals or integration of synthetic transgenes.
Zinc finger nucleases (ZFNs) are enzymes which bind and cut distinct sequences of double-stranded DNA (dsDNA). A functional CompoZr ZFN unit consists of two individual monomeric proteins that bind a DNA "half-site" of approximately 15-18 nucleotides (see Figure 1). When two ZFN monomers "home" to their adjacent target sites the DNA-cleavage domains dimerize and create a double-strand break (DSB) in the DNA.1 Introduction of ZFN-mediated DSBs in the genome lays a foundation for highly efficient genome editing. Imperfect repair of DSBs in a cell via the non-homologous end-joining (NHEJ) DNA repair pathway can result in small insertions and deletions (indels). Creation of indels within the gene coding sequence of a cell can result in frameshift and subsequent functional knockout of a gene locus at high efficiency.2 While this protocol describes the use of ZFNs to create a gene knockout, integration of transgenes may also be conducted via homology-directed repair at the ZFN cut site.
The CompoZr Custom ZFN Service represents a systematic, comprehensive, and well-characterized approach to targeted gene editing for the scientific community with ZFN technology. Sigma scientists work closely with investigators to 1) perform due diligence analysis including analysis of relevant gene structure, biology, and model system pursuant to the project goals, 2) apply this knowledge to develop a sound targeting strategy, 3) then design, build, and functionally validate ZFNs for activity in a relevant cell line. The investigator receives positive control genomic DNA and primers, and ready-to-use ZFN reagents supplied in both plasmid DNA and in-vitro transcribed mRNA format. These reagents may then be delivered for transient expression in the investigator&#x2019;s cell line or cell type of choice. Samples are then tested for gene editing at the locus of interest by standard molecular biology techniques including PCR amplification, enzymatic digest, and electrophoresis. After positive signal for gene editing is detected in the initial population, cells are single-cell cloned and genotyped for identification of mutant clones/alleles.

Genome Editing with CompoZr Custom Zinc Finger Nucleases ZFNs

The CompoZr Custom Zinc-Finger Nuclease (ZFN) Service enables precise genome editing in any organism or cell line at any locus defined by the user. This article describes the process for the design, manufacture, validation and implementation of the CompoZr Custom ZFN Service.

CompoZr Özel Çinko Parmak Nükleazlar ile Genom Düzenleme (ZFNs)

Genome editing is a powerful technique that can be used to elucidate gene function and the genetic basis of disease. Traditional gene editing methods such ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

Kan ve Kültür Medya türetilen Exosomes saflaştırılması ve microRNA Profil

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; In situ</em&gt; Formalin Sabit Böbrek Dokular Hibridizasyon

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Utilizing Custom-designed Galvanotaxis Chambers to Study Directional Migration of Prostate Cells

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and epithelial wound healing. Applying a direct current electric field to cultured cells in vitro induces directional cell migration, or galvanotaxis. The 2-dimensional galvanotaxis method we demonstrate here is modified with custom-made poly(vinyl chloride) (PVC) chambers, glass surface, platinum electrodes and the use of a motorized stage on which the cells are imaged. The PVC chambers and platinum electrodes exhibit low cytotoxicity and are affordable and re-useable. The glass surface and the motorized microscope stage improve quality of images and allow possible modifications to the glass surface and treatments to the cells. We filmed the galvanotaxis of two non-tumorigenic, SV40-immortalized prostate cell lines, pRNS-1-1 and PNT2. These two cell lines show similar migration speeds and both migrate toward the cathode, but they do show a different degree of directionality in galvanotaxis. The results obtained via this protocol suggest that the pRNS-1-1 and the PNT2 cell lines may have different intrinsic features that govern their directional migratory responses.

We present a method to apply a physiological electric field to migrating, immortalized prostate cells in a custom-made galvanotaxis chamber. Using this method, we demonstrate that 2 lines of non-tumorigenic prostate cells demonstrate different degrees of migration directionality in the field.

Prostat Hücrelerinin Yönlü Göç Eğitim için özel tasarlanmış galvanotaksis Chambers kullanılması

The physiological electric field serves specific biological functions, such as directing cell migration in embryo development, neuronal outgrowth and ...

Özel MicroRNA Mikroarray deneyler gerçekleştirme

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