Dr. Joe Naim, Matt Bryman Michael Bordo Ujwal Bagal, Huizhe Jin, ve Amanda Bouffier: Biz, yardım toplama çam dokuların mümkün olmazdı olmadan aşağıdaki kişilere teşekkür etmek istiyorum.

Bölüm 1: Ağaç Hasat Ağaç ve yıkılan sonra, dikkatli ve mümkün olduğunca hızlı çalışması için önemlidir. Her biri farklı bir doku tipi hasat edilir örnek malzemenin herhangi bir çapraz kontaminasyonu engellemek için, onlar hemen kendi sıvı azot kaplar içine yerleştirilmiş olmalıdır. Bu RNA izolasyonu protokol ölçeklenebilir. <ol><li> Yaklaşık 0.5-0.75 m uzunluğa ve testereli bir skor ile iki veya üç yerlerde kabuğu cıvata uzunlamasına eksen cıvata Kes. </li><li> Kabuğu, kök tamamen ayrılıncaya kadar ahşap ayrı ve kabuğu bölümü geri çekerken keski çalışmaya devam başlayana kadar bir ahşap keski kullanarak, gol bölgenin kenarında çalışır. </li><li> İkincil ksilem kabuğu soyulmuş cıvata uzunlamasına ekseni boyunca kazıma straight edge, tek taraflı jilet ile hasat edilir. Doku kontaminasyonu en aza indirmek için lateks eldiven giyin. </li><li> Phleom kabuğu bölümlerin iç tarafında bulunan, kolayca soyma elle hasat edilir. </li><li> Reaksiyon ahşap zemin saygı ile kendi orijinal yönünü göstermek için sprey boya ile işaretleme sonra alt uzuvlarda toplanıyor. </li><li> Uzuvlar cıvata içine 1-2 metre kesilmiş ve boylamasına ekseni boyunca yaklaşık iki tür reaksiyon ahşap yerlere göre ters taraftan gol: ters ahşap (bacak üst tarafındaki) ve sıkıştırma ahşap (alt ekstremite tarafı). Kabuğu, sadece her tür reaksiyon ahşap kaplama kabuğu bir anda kaldırılmış olması dışında, yukarıda açıklandığı gibi uzuv ayrılır. İkincil ksilem ve floem, yukarıda açıklanan ve sıvı azot yerleştirilmiş olarak tahsil edilir. </li><li> Iğneler, sürgün ipuçları ve genç koniler gibi diğer örnekleri, elle hasat ya da gerektiği gibi budama makasları kullanarak olabilir. </li><li> Toplanan numuneler, sıvı nitrojen içinde dondurulmuş işaretli torbaları veya uygun kaplara yerleştirilir ve laboratuvara gönderilmek Geri dönmek için kuru buz içinde paketlenmiş olmalıdır. </li></ol> Bölüm 2: Numune Dondurucu Mill İşleme <ol><li> Spex 6850 dondurucu değirmen rezervuar sıvı nitrojen ile doldurun ve taşlama şişeleri birine etkisi çubuğunu yerleştirin ve önceden soğuk sıvı azot ile doldurun. Sıvı nitrojen dökün ve flakon örnek eklemek. </li><li> Aşırı sıvı nitrojen Tambur kalır ve sonunda kapağı Odanın mühür oturan önce bu aşırı azot gazı tamamen Bacalı emin olun. </li><li> Taşlama flakon taşıyıcı ve yakın içine yerleştirin. </li><li> 14 bir oran ayarda iki kür için 1 dakika / devir Mill odunsu örnekleri. </li><li> Önceden soğutulmuş ve küçük bir miktar sıvı azot içeren bir behere öğütülmüş numune (odun unu) kaldırmak için bir sıvı azot soğutulmuş spatula kullanın. </li><li> Gerekli -80 ° C kadar bir saklama kabı ve yerine sıvı azot / numune şerbeti dökün. </li></ol> Bölüm 3: RNA izolasyonu, 1. Gün <ol><li> RNA İzolasyon Tampon 20ml 50ml kapaklı Falcon tüp içine yerleştirin ve sonra 400 mcL β-mercaptoethanol, girdap, kısaca ısı 60 ° C su banyosunda ekleyin. </li><li> Her tüpe 4 gr dondurulmuş doku. Cap ve 10 saniye boyunca vorteks tarafından takip elle kuvvetlice çalkalanır. </li><li> Tüpüne eşit miktarda kloroform ekleyin ve karıştırın hafifçe ters. Dönen bir tekerlek üzerinde tüp yerleştirin ve 5 dakika boyunca karıştırma sonu aşırı uç izin. </li><li> 12.000 (17.200 xg&#39;de), 5 dakika boyunca SS34 sabit açılı rotor devir az 50 ml Oak Ridge tüp ve santrifüj karışımı süzün. </li><li> Taze Oak Ridge tüp sulu faz transfer ve kloroform yarım hacmi ekleyin. </li><li> 1 dakika ve 5 dakika boyunca dönen bir tekerlek karıştırmaya devam Vortex. 12.000 rpm (17.200 xg), 5 dakika boyunca SS34 sabit açılı rotor santrifüjleyin. </li><li> Sulu faz taze Oak Ridge tüpüne aktarın ve kloroform yarım hacmi ekleyin. 1 dakika karıştırın. SS34 10 dakika boyunca sabit açılı rotor 12.000 rpm (17.200 xg) santrifüjleyin. </li><li> Sulu faz 50 ml yüksek hızda polipropilen tüpe aktarın. SS34 20 dakika boyunca sabit açılı rotor 10.000 rpm (11,900 xg) santrifüjleyin. </li><li> Durusu 50 ml Falcon tüp içine süpernatant ve süpernatant çeyrek hacmi 10 M LiCl çözüm ekleyin. 4 Kısaca vorteks ve yeri ° C, gece yağış için. </li></ol> Bölüm 4: RNA izolasyonu, 2. Gün <ol><li> Isı SSTE başlamadan önce 65 ° C su banyosunda tampon. </li><li> 4 SS34 sabit açılı rotor 11.000 rpm (14.450 xg) 30 dakika süreyle santrifüj bir polipropilen boru ve pelet RNA LiCl çöktürülmüş örnek dökün ° C. </li><li> Santrifüj sonra,dökün ve sıvı atmak ve yaklaşık 1 dakika boyunca Kimwipes tüpler ters. Herhangi bir kalan süpernatant Pipet. </li><li> Isıtmalı SSTE örnekleri iyice karıştırmak için bir pipet kullanarak 800 mcL her pelletini tekrar. 2 mL Ambion RNAse ücretsiz mikrofuge&#39;de tüpler yeniden süspanse örnek aktarın. </li><li> Isı örnekleri 65 ° C,, tam olarak yeniden süspanse sağlamak için kuvvetli bir vorteks takip 2 dakika boyunca. </li><li> Her numune tüpü, fenol-kloroform, pH8 bir eşit miktarda ekleyin. 14.000 rpm&#39;de 3 dakika 30 saniye sonra Vorteks her bir örnek ve bir mikrofuge&#39;ye Spin </li><li> Yeni bir 2mL Ambion RNAse ücretsiz mikrofuge&#39;de borular, sulu faz transfer ve kloroform eşit miktarda ekleyin. Vorteks her 30 saniye ve 3 dakika 14.000 rpm&#39;de mikrofuge&#39;ye spin örnek. </li><li> Faz-Lock Jeli tüpler daha önce 2 dakika 11.000 rpm&#39;de microfuging tarafından hazırlanan sulu faz aktarın. Kloroform yarım hacmi ekleyin ve hafifçe karıştırın pipetle. </li><li> 5 dakika 11.000 rpm&#39;de santrifüj ve taze 2mL Ambion RNAse ücretsiz mikrofuge&#39;de borular, sulu faz transfer. </li></ol> Bölüm 5: RNA Yağış ve Etanol Yıkama <ol><li> 50 mcL 3.0 M sodyum asetat, pH 4.8 ve sulu faz 1 ml% 100 etanol ekleyin. Girdap ve çökelti -80 ° C de 30 dakika veya gece -20 ° C </li><li> Mikrofuge&#39;de örnekleri 30 dakika boyunca 14.000 4 dak ° C, ve dikkatle süpernatantı aspire. Pelet rahatsız etmeyin. </li><li> 1 ml -20 ° C% 70 (v / v) etanol ile RNA pelet yıkayın. 1 dakika 14.000 rpm&#39;de mikrofuge&#39;de ve etanol aspirat. </li><li> 5.3 adımı tekrarlayın. </li><li> 3-5 dakika bankta tüpler ve hava-kuru granül şapkasını çıkarmak. </li></ol> Bölüm 6: Tekrar Süspansiyonu ve Miktar <ol><li> 100 mcL DEPC arıtılmış su her pelletini tekrar. Vorteks Mix ve tüpler buz üzerine yerleştirmeden önce 2 dakika 65 ° C&#39;de inkübe edin. Örnek tabanda sağlamak için gerekirse tekrarlayın. </li><li> Istenen ve spektrofotometrik kantitatif, 10mM Tris, pH 7.5 1:10 seyreltme 50 mcL örnekleri gibi Havuzu. 260/280 ve 260/230 Absorbans oranları genellikle sırasıyla 2.1 ve 2.2 &#39;den daha fazladır. 60-120μg / g dondurulmuş doku, RNA izolasyonu getirileri aralığı. </li><li> TAE tampon 1.5-2.5 mg RNA% 1 agaroz jel üzerinde çalışıyor örnekleri analiz edin. Daha kritik örnekleri için, üreticinin talimatlarına göre bir Agilent 2100 Bioanalyzer kullanarak RNA analiz edin. </li></ol> Temsilcisi Sonuçlar: Odunsu örnekleri ile farklı kozalaklı dokuların aralığı RNA izolasyonu verim 60-120 mikrogram / g dondurulmuş doku genellikle 70-80g / g dondurulmuş doku çevresindeki verimli. 260/280 ve 260/230 tanı oranları genellikle 2.1 &#39;den daha fazladır ve RNA örneği ise, önemli bir fenolik veya karbonhidrat kirlenme ücretsiz olduğunu iyi göstergeleridir. EtBr boyama agaroz jel elektroforezi ile analiz Örnekleri 25S, 18S ve 5S RNA (Şekil 1) için üç ayrı grup göstermelidir. Agaroz jel üzerinde 18S 28S stokiyometri tespiti biraz öznel ve jel, boyama, ve örnek yük koşulları çalışan gibi faktörlerin hepsi bir etkisi olabilir. Genel olarak, 28S 18S oranı&gt; 1.5 olarak görünecektir. Ancak, bazı kozalaklı örnekleri daha düşük oranları var. Örneğin, Agilent 2100 analizi aşağıdaki stokiyometri 1.2:1 için yakın farklı doku tiplerinin kozalaklı örnekleri çeşitli örneklerini gördük. Böylece, belirgin bir düşük 28S 18S agaroz jel elektroforezi ile stokiyometri mutlaka düşük kaliteli örnek göstermek değildir. Yaprak, ateş ve koni örnekleri genellikle ek farklı bantları chloroplastic ribozomal RNA&#39;lar nedeniyle mevcut olacak. Iyi örnek veya yüksek molekül kütlesi bantları (8-10 kb) göç etmeyen EtBr lekeli malzemeleri genellikle genomik DNA kontaminasyonu (Şekil 2) gösterir. 5S bant ve azaltılmış ribozomal bant boyama ya da belirgin bir 18S&gt; 28S stokiyometri RNA bozulması meydana gelmiştir (Şekil 3) iyi bir gösterge ya da aşağıda çevresinde Düşük moleküler kütlesi bulaşıyor. Agilent 2100 Elektroferogram analiz, taban çizgisi 18S ve 28S sahip 28S ribozomal RNA&#39;lar ilgili önemli zirveleri ile düşük ve nispeten pürüzsüz olmalıdır: 1.2 &#39;den 2.0&#39; a her yerde değişen 18S oranları. Agilent RNA Bütünlük sayısı (RIN) değeri RNA kalitesini daha iyi bir belirleyicisi olabilir ve RNA mikroarray hedeflerinin hazırlanması için kullanılacak en az 7 veya daha büyük olmalıdır. 1.4 eşit 18S 18S: Şekil 5 RNA hazırlık 28S ortaya koyduğu gibi çok yüksek bir bazal ve fark bozulması olan fakir bir ksilem gösterirken oranı ve bir RIN değeri 8.6: Şekil 4, bir 28S ile ksilem RNA tipik bir Agilent 2100 Elektroferogram gösterir ra0.65 ve 3.4 RIN değeri eşit tio. <img src="/files/ftp_upload/1751/1751fig1.jpg" alt="Şekil 1" /> Şekil 1, yüksek kaliteli çam RNA agaroz jel analizi . Lane 1, NEB 1 kb standart, Lane 2 Loblolly çam ikincil ksilem karakteristik ribozomal 28S, 18S ve 5S bantları ve belirgin 28S ile tipik bir toplam RNA izolasyonu gösterir: 18S oranı&gt; 1. <img src="/files/ftp_upload/1751/1751fig2.jpg" alt="Şekil 2" /> Şekil 2 çam RNA örnek agaroz jel analizi genomik DNA ile kontamine olmuş . Lane 1 NEB 1 kb standart 2, Lane, genomik DNA kontaminasyonu gösteren ksilem RNA örnek. <img src="/files/ftp_upload/1751/1751fig3.jpg" alt="Şekil 3" /> Şekil 3 genomik DNA ile RNA bozulma ve kirlenme gösteren çam örnek agaroz jel analizi. Lane 1, NEB 1 kb standart, Lane2, genomik DNA kontaminasyonu ve şiddetli RNA bozulma gösteren ksilem RNA örnek. <img src="/files/ftp_upload/1751/1751fig4.jpg" alt="Şekil 4" /> Şekil 4 ksilem total RNA Agilent 2100 Elektroferogram Loblolly çam ksilem açıklanan yöntemi kullanılarak izole edilmiştir. 28S: 18S oranı 1.4 eşittir, RIN değeri 8.6 eşittir <img src="/files/ftp_upload/1751/1751fig5.jpg" alt="Şekil 5" /> Şekil 5 Agilent 2100, ciddi bozulma ve genomik kirlenme gösteren apikal uç total RNA Elektroferogram . 28S 18S oranı 0.65 eşittir, RIN değeri 3.4 eşittir

<ol>
	<li>Chang, S., Puryear, J., Cairney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

Kozalaklı ağaç türlerinin yüksek kaliteli bir RNA Alınması odunsu dokularında bulunan fenolik ve polisakkarit bileşiklerin yüksek düzeyde verilen zor bir görev olabilir. Chang ve arkadaşları tarafından geliştirilen protokol ile başlayarak. (1), biz daha titiz çıkarma bulundu ve tutarlı kozalaklı türlerin geniş bir yelpazede örneklenmiş çeşitli odunsu ve odunsu olmayan dokular çok yüksek kalitede toplam RNA izolasyonu yol adımları temizlemek gerekir. Bu video modifiye RNA izolasyon protokol...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
</tr>
<tr>
<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
</tr>
<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
		</table>
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an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

Dokular özellikle Loblolly çam gibi kozalaklı türler, Pinaceae ailesine mensup olanlar, (den izole<em&gt; Pinus taeda</em&gt; L.), yüksek konsantrasyonlarda RNA arıtma ile müdahale fenolik bileşikler ve polisakkaritler içerir. Bu türlerin yüksek kaliteli RNA izolasyonu mikroarray ve diğer genomik analizler için yeterli kalitede RNA izole etmek için birden fazla organik çıkarma adımları oluşan bir RNA izolasyonu protokolü ve istihdam alanında titiz doku toplama yöntemleri gerektirir. Alan toplanan Loblolly çam örnekleri zor olabilir, ama büyük ölçüde standart doku ve RNA izolasyonu işlemleri için çeşitli değişiklikler sonuçlarını iyileştirmek yüksek kaliteli RNA izolasyonu. Örnekleri, özellikle büyük ölçekli hasat sırasında, düzgün toplanan ve alandan taşınan eğer genel RNA bozulması ölçüde artırır. Toplam RNA verim örnekleri odunsu dokulara gelen özellikle, izolasyon RNA önce sıvı azot dondurucu değirmen ezerek örnekleri ile önemli ölçüde artış olabilir. Bu öncelikle, fenolik bileşikler, ve her ikisi de, en kozalaklı türlerin odunsu dokular alıntılar yüksek düzeyde mevcut polisakkaritler gibi oksitleyici ajanların varlığı nedeniyle. Kaldırılmaz, bu kirleticiler, RNA bozulması, bu sonucu düşük verim ve düşük kaliteli bir RNA örnek olarak sorunları, önde gelen üzerinde taşıyabilir. Bulaşma, fenolik bileşikler, polisakkaritler, yanı sıra da azaltmak veya cDNA sentezi için yaygın olarak kullanılan ters transkriptaz veya diğer polimerazları aktivite hatta tamamen ortadan kaldırabilir. Araştırmacılar bekliyoruz eğer Özellikle, RNA, cDNA kütüphaneleri nesil çift iplikli cDNA sentezi PCR veya qPCR Kullanıcı için tek iplikli cDNA sentezi, veya mikroarray hedef malzeme sentezi için Şablonu olarak kullanılmak üzere kaderinde en yüksek kalitede olması gerekir optimum sonuçlar elde etmek için. Diğer birçok bitki türünün yaygın istihdam RNA izolasyon teknikleri kozalaklı örneklerinden bu kirleri çıkarmak ve böylece aşağı manipülasyonlar için uygun toplam RNA örnekleri verim yok yeteneklerini genellikle yetersizdir. Bu videoda, floem hasadı, ikincil ksilem ve reaksiyon ahşap ksilem, kırk yaşındaki ağacın kesilmesi ile başlayan kozalaklı dokuların alan koleksiyon için yöntemler göstermektedir. Ayrıca, tutarlı bir sonraki enzimatik manipülasyonlar için yüksek kaliteli bir RNA vermiştir RNA izolasyonu protokol göstermektedir.

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Loblolly çam ksilem ve floem de dahil olmak üzere birçok bitki dokularında, (<em&gt; Pinus taeda</em&gt; L.), fenolik ve RNA arıtma müdahale polisakkaritler yüksek düzeyde içerir. Bu sunum alanında yetişen doku ve mikroarray&#39;ler ve diğer genomik analizler için yeterli kalitede RNA izolasyonu hasat teknikleri tartışır.

RNA İzolasyonu Loblolly Çam Daha İyi Bir Yöntem ( S. taeda L.) ve Diğer Conifer Türler

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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Research

JoVE Journal

Biology

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

13.7K Görüntüleme.  University of Georgia (UGA). Loblolly çam ksilem ve floem de dahil olmak üzere birçok bitki dokularında, ( Pinus taeda L.), fenolik ve RNA arıtma müdahale polisakkaritler yüksek düzeyde içerir. Bu sunum alanında yetişen doku ve mikroarray'ler ve diğer genomik analizler için yeterli kalitede RNA izolasyonu hasat teknikleri tartışır.

Video: An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

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 ...

Biyoloji

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Gen İfadesi Analizi Embriyonik Zebra balığı ve cDNA Sentezi RNA İzolasyon

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases, profilleme ve Diğer S Adenosil- L</subYakalama Bileşik Kütle Spektrometresi (CCMS)> homosistein bağlayıcı Proteinler

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Kinesin İzolasyonu ve Saflaştırılması gelen Drosophila Embriyolar

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Dan Ekstrasellüler Akışkan Vampiric İzolasyonu<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Uçuş Performans Doğru ve hızlı ölçüm için geliştirilmiş Yöntemi<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Fare pankreasından alınan RNA İzolasyonu: A Ribonuclease zengin Doku

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hibrid-Cut: Dirençli Bir Bitki Doku Örnekleri Bir Geliştirilmiş Kesit Yöntemi

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Stoma bantlı teyp-kabuğu: Guard hücre numune hazırlama için geliştirilmiş bir yöntem

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Fare Primer Hepatosit İzolasyonu için Geliştirilmiş Zaman ve İş Gücü Verimli Protokolü

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...