Bu çalışma Fransız Ulusal Araştırma Ajansı (ANR-14-CE02-0010) tarafından desteklenmiştir. Biz yazının eleştirel okuma Dr Benjamin Field ve Dr. Elodie Lanet teşekkür ederim. Biz video düzenleme ile yaptığı yardım için Bay Michel Terese teşekkür ederim.

% 20 ila 50 (ağırlık / hacim) sukroz dereceleri hazırlanması 1. Not: Gradyan 13,2 ml&#39;lik bir ultra santrifüj tüpüne sükroz 4 kat (% 50,% 35 ve% 20 2 kat) imal edilmiştir. Bizim tecrübelerimize göre, iki ayrı katmanlar halinde% 20 sukroz dökme ölçüde Polizom hazırlıkları kalitesini artırır. <ol><li> stok çözümleri hazırlayın. tüm çözümler RNAse ve DNAse serbest olduğundan emin olun. <ol><li> 400 mM Tris-HCI pH 8.4, 200 mM KCI, 100 mM MgCl2: 10X tuz solüsyonu. </li><li> 200 ml 1X tuz çözeltisi içinde sakrozun 137 g çözülür: 2 M sukroz çözeltisi hazırlayın. </li></ol></li><li> gradyanlar hazırlamak için, Tablo 1 &#39;de tarif edildiği gibi, tuz çözeltisi içinde sukroz çözeltisi ile seyreltilir. Verilen miktarlar altı geçişlerini içindir. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Nihai sakaroz konsantrasyonu </td><td> sukroz 2M (mi) </td><td> Tuz çözeltisi 1X (mi) </td><td> Nihai Vol. (mi) </td></tr><tr><td> % 50 </td><td> 8.8 </td><td> 3.2 </td><td> 12 </td></tr><tr><td> % 35 </td><td> 12.9 </td><td> 12.1 </td><td> 25 </td></tr><tr><td> % 20 </td><td> 7.4 </td><td> 17.6 </td><td> 25 </td></tr><tr><td> % 20 </td><td> 5.8 </td><td> 14.2 </td><td> 20 </td></tr></tbody></table> Tablo Sukroz Çözüm 1. dilüsyonlar altı geçişlerini hazırlamak. <ol start="3"><li> Tablo 2&#39;deki gibi katmanları dökün. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> sukroz katmanıdır </td><td> % 50 </td><td> % 35 </td><td> % 20 </td><td> % 20 </td></tr><tr><td> Vol. (Mi) </td><td> 1.85 </td><td> 3.65 </td><td> 3.65 </td><td> 1.35 </td></tr></tbody></table> Katman başına sukroz çözeltisi Tablo 2. Cilt. <ol start="4"><li> Her katman döküldükten sonra, bir -40 ° C tüpler tutmak veya -80 ° C derin dondurucuda tam donma kadar bir sonraki sakaroz katmanı eklemeden önce. Donma, genellikle -40 ° C de 2 saat sonra elde edilir, ancak bir sonraki tabaka dökülmeden önce yaklaşık 6 saat bekleyen önerilir. Son 20% tabaka donduruldu ve deney gününde taze olarak ilave edilebilir. Not: Bir sonraki bir eklemeden önce her bir katmanı Donma tabakaların herhangi karıştırma veya rahatsızlık önler. Gradyanlar bir -40 ° C&#39;de saklanır ya edilebilir -80 ° C sıcaklıkta dondurucuya en az altı ay süreyle. </li><li> Zaten bitmiş değilse, deney günü degradeler son% 20 katman ekleyin. Sonra, degradeler soğuk bir odada ya da bir buzdolabı çözülme sağlar. </li></ol> Sitosolik özleri 2. Hazırlık Not: tavsiyesinebiyolojik numune başına iki gradyanları kullanılarak sona erer. 300 mg 6 günlük, Arabidopsis thaliana fide çalışırken iki gradyan hazırlanması için bitki malzemesi uygun bir miktardır. az translationally aktif dokularda çalışırken, bitki materyali miktarı 600 mg kadar artırılabilir. <ol><li> ½ Murashige ve Skoog yetiştirilen Hasat altı gün eski fideler 8 orta sıvı nitrojen içinde hızlı dondurulması ile% 1 sükroz veya eşdeğer medya ile desteklenmiş </li><li> sıvı nitrojen ile önceden soğutulmuş havan ve tokmak taşlama bitki malzemesi. </li><li> önceden soğutulmuş tartım tabağına toz malzemenin 300 mg tartılır. Numunenin çözülme önlemek için hızlı bir şekilde bu adımı gerçekleştirin. Not: -80 ° C derin dondurucuda en fazla bir hafta dondurulmuş örnekleri tutun. </li><li> Dallanmış önceden soğutulmuş polisom tamponu 2.4 ml (Tuz Çözeltisi 4X, 5.26 mM EGTA,% 0.5 (h / h) oktilfenoksi poli (etilenoksi) etanol, 50 μg.ml -1 sikloheksimid, ekleme 50 μg.ml 1kloramfenikol) ve pipet ile karıştırılarak homojenize. iki adet 1,5 ml tüpler içine aktarın. ısınma örnekleri önlemek için hızlı bir şekilde çalışın. </li><li> bir mikrosantrifüj 4 ° C&#39;de 15 dakika boyunca 16,000 x g&#39;de santrifüjleyin yıkıntıları pelet haline getirildi. Not: Her zaman, RNA bozulmasını önlemek 4 ° C&#39;de örnekleri tutmak için, RNaz / DNaz ücretsiz koşullarda RNaz / DNaz ücretsiz çözümler ve çalışmayı kullanma. Heparin RNA korumasını geliştirmek için, 300 μg.ml -1 nihai konsantrasyona kadar, polisom tampon eklenebilir. Ancak, aşağı analizi engelleyebilir heparin beri, bu lityum klorür yağış yerine etanol yağış (bkz not 4.7) gerçekleştirerek RNA yağış adımı sırasında kaldırılması gerekir. </li></ol> 3. Polizom Profil <ol><li> Dikkatle pelet bozmadan süpernatant pipetle. Bitki parçaları pipetle varsa, adımı 2.5 tekrarlayın. hafifçe pipettin tarafından degrade üstünde yüzer yükleyinsabit bir akışı tüpün yan duvar üzerine g. Her 1.5 ml tüp için bir degrade kullanın. </li><li> (A SW41 rotor kullanıldığında, örneğin 32.000 rpm) bir ultrasantrifüjdeki, 4 ° C&#39;de 2 saat 45 dakika süre ile 175,000 x g&#39;de kepçe ve santrifüj soğutulmuş aktarın. </li><li> Şekil 1&#39;de açıklandığı gibi degrade toplama sistemi kurmak. UV küvet 1 mm yol uzunluğuna sahiptir. </li></ol><img alt="Şekil 1" src="/files/ftp_upload/54231/54231fig1.jpg" /> Şekil 1. Gradient toplama sistemi. UV küvet degrade altına inen bir cam kılcal tüp polivinil klorür boru ile bağlanır. degrade bir peristaltik pompa sistemi sayesinde yoluyla ilerler. OD 260 sürekli okunur ve 2 ml&#39;lik fraksiyonlar toplanır. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig1large.jpg" target="_blank">Büyük halini görmek için tıklayınız</a>bu figür. <ol start="4"><li> Oda sıcaklığına fraksiyon toplayıcısı ayarlayın ve 2 mi fraksiyon koleksiyonu sağlayacak bir hızda döner bandı ayarlayın. Önceden soğutulmuş toplama tüpleri kullanın. </li><li> gradyan toplama sistemine polivinil klorür boru ile bağlı bir cam kılcal tüp kullanarak alttan üste doğru kesirler toplayın. polivinil klorür boru ve cam kılcal boru arasındaki bağlantıyı sabitlemek için plastik bir parafin filmi kullanın. </li><li> degrade yukarıya doğru 260 nm&#39;de absorbansı okumak. Tüm gradyanı elde edildiğinde, RNA ekstraksiyonu önce buz üzerinde toplama tüpleri. </li></ol> 4. RNA Ekstraksiyon <ol><li> aşağıdaki gibi, santrifüj tüpleri şapkalı 50 ml iki geçişlerini toplanan kesirler, havuz: Polizom: fraksiyonlar 1-3 (12 mi) Monosomes: fraksiyon 4 (4 mi) Süpernatant: fraksiyonlar 5 ve 6 (8 mi) </li><li> Her fraksiyon için, 1 vol ekleyin. 8 M guanidin hidroklorür,50 ug akril taşıyıcısı ve 1.5 hac. izopropanol. Not: Akrilik taşıyıcı alkol çökeltme sırasında, nükleik asitlerin geri kazanımını arttırmak için coprecipitant olarak kullanılan lineer akrilamid vardır. </li><li> tüpler ters çevirerek karıştırınız ve -20 ° C &#39;de O / N hızlandırabilir. </li><li> bir ultrasantrifüjdeki 4 ° C&#39;de 1 saat, (a SW32 rotor içinde 32.000 rpm) 175,000 x g&#39;de santrifüjleyin. Çökeltme aşaması, ultra-santrifüj ile uyumlu olmayan bir tüp içinde gerçekleştirilir, uygun bir tüp aktarın. </li><li> Süpernatant atılır ve 200 mL RNaz içermeyen TE tamponu (10 mM Tris-HCI, pH 7,5-1 mM EDTA) içinde pelet çözülür. yeni bir 1.5 ml&#39;lik tübe transfer edin. </li><li> 1 vol ekleyerek RNA ayıklayın. suyla doyurulmuş fenol (pH 6.6) ve 1 hacim. kloroform: izoamil alkol (24: 1). kuvvetlice karıştırın. 4 ° C&#39;de 20 dakika boyunca 15,000 x g&#39;de santrifüjleyin. yeni bir 1.5 ml&#39;lik tübe sulu faz transfer. Not: asit fenol (pH 4.5), DNA kontaminasyonu en aza indirmek için de kullanılabilir. </li><li>1/10 vol ekleyerek RNA hızlandırabilir. 3 M sodyum asetat ve 3 hac. % 100 etanol eklenmiştir. -20 ° C&#39;de 20 dakika veya O / N -80 ° C&#39;de çökelmesine izin verin. 4 ° C&#39;de 15 dakika boyunca 15,000 x g&#39;de santrifüjleyin. Not: polisom tamponu (bakınız Not 2.5) içinde heparin kullanılarak, uygun bir şekilde alt analizi 9 müdahale edebilecek heparin arındırıldı etanol çökeltme yerine lityum klorür (LiCİ) çökelmesini gerçekleştirin. Ekstraksiyon işleminden sonra, 4 ° C&#39;de 20 ° C veya O / N 30 dakika çöktürülmüştür 2.5 M bir son konsantrasyona kadar LiCI ekleyin. 4 ° C&#39;de 15 dakika boyunca 15,000 x g&#39;de santrifüjleyin. </li><li> 500 ul% 75 etanol ile pelet yıkayın. Hava pelet kurutulur ve 30 ul TE tampon maddesi içinde çözülür. Bir RNazsız% 1.2 agaroz jeli (100V, 15 dakika) ya da kapiller elektroforez yöntemlerle elektroforez RNA kalitesini değerlendirmek. </li></ol> 5. Veri Analizi <ol><li> Bir grafik ve veri analizi yazılımı İhracat ham veriler. NotŞekil 2A ve B de gösterildiği gibi, ham profil niteliksel bilgileri vermek: görülebileceği polisom tepe sayısı monosome tepe yüksekliği ve serbest ribozom alt birimlerine karşılık gelen bir omuz varlığı. </li><li> örnekler arasındaki profillerin karşılaştırılması izin profilleri Birleştirme. Her numune için monosome zirve X değerini belirlemek için ekran okuyucu aracını kullanın ve eğrilerin başında ekstra veri noktaları kaldırarak monosome doruklarına hizalayın. eğrinin en alçak noktası belirlemek ve Y değeri (ekran okuyucu aracını kullanarak) belirler. &quot;Set sütunları değer&quot; fonksiyonunu kullanarak 0&#39;a en alçak noktası Y değerini ayarlayın. </li><li> Ham bulunmaktadır (Şekil 3C) için eğri altındaki alanların integrasyonu ile belirlenmişlerdir polisomlann / monosomes oranını belirler. alanın sınır entegre edilecek tanımlamak için veri seçici aracını kullanın. Sonra entegre fonksiyonu (Analiz-Matematik) kullanın. </li><li> normalleştirmekmonosome tepe zirvesine Y değerine göre bir eğrinin tüm veri noktalarını bölerek monosome zirve eğrileri (ekran okuyucu aracını kullanarak belirlenir). sütunu seçin, ardından Analiz-Matematik altında, Normale seçin ve &quot;belirli bir değere bölünmesi&quot; yöntemleri seçin. Bu adım, polizom göreli seviyesi (Şekil 3D) karşılaştırılmasına olanak sağlar. </li></ol><img alt="şekil 2" src="/files/ftp_upload/54231/54231fig2.jpg" /> Şekil 2. Örnek polisom profilleri., Arabidopsis thaliana, vahşi tip (wt, ekotip col-0) ve mutant fideleri uzun gün fotoperiyodların (16 saat ışık, 8 saat karanlık) altında yarım Murashige ve Skoog ortamı üzerine, altı gün süre ile büyütülmüştür. A: wt fideler ham Polizom profilleri. B: mutant fideler ham Polizom profilleri. C: eğri D altındaki alanın entegrasyonu ile polizom ve monosomes yüzdesinin belirlenmesi: Polizom profiles monosome zirveye normalize. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

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The authors have nothing to disclose

The protocol we present here is an easy and cheap method for generating polysome profiles and isolating mRNAs associated with polysomes, single ribosomes or free of ribosomes. A wide range of different polysome fractionation methods is described in the literature. The method we have described here has been optimized to keep only the necessary compounds and has been adapted for plant material. In particular, we reduced the amount of detergent11 and added chloramphenicol to the buffer to fix the chloroplastic ribosomes to the mRNA (as cycloheximide does for the cytosolic ribosomes)12 . We have also reduced the total ultracentrifugation time7.
To obtain high quality polysome profiles, it is essential to use freshly collected plant material and to perform all steps at 4&#176;C. When using tissues that are poorly translationally active, more plant material can be loaded on the gradient (up to 600 mg).
Analysis of polysome profiles can provide insights into both the overall translational status of cells13 and the translational status of a specific mRNA. We have used RNAs isolated by this method for different applications. Using the RNAs for microarrays allowed us to identify a class of cadmium stress response genes for which transcription and translation are uncoupled 14. We also used this method to identify small RNAs associated with polysomal fractions. This identification was made by northern blotting15 of RNA extracted from polysomal fractions, and provided biochemical evidence for a translational component in the miRNA pathway in plants. In another study, a cis-NAT RNA was identified by quantitative RT-PCR. This cis-NAT is associated to the phosphate homeostasis and promote translation of the PHO1;2 transcript 16. 
The main limits of the polysome profiling approach are the lack of information concerning both the position of the ribosome on the mRNA and its progression along the mRNA. Ribosome profiling has emerged to address these limitations 17 and has been successfully used on plant tissues18. Ribosome profiling provides a global measurement of translation by taking advantage of the advances in sequencing technology. Nevertheless, as for any sequencing based assays, the quality of the results depends on the mapping of the sequences to the genome, therefore focusing on the small ribosome-protected fragments makes it difficult to deconvolute repetitive sequences. Moreover, the digestion of RNA not protected by the ribosomes leads to the loss of regulatory information contained in the 3' and 5' UTRs. It is then impossible to distinguish transcript variants that have different 3' or 5' UTRs and show different levels of translation19. The polysome profiling method described here is rapid and does not require specific technical skills. Altogether, these two methods represent complementary approaches to study translation regulation.

Protein sentezi, bütün hücreler 1 çok önemli ve enerjik olarak masraflı bir işlemdir. Her şeyden önce, hücreler için makine, ribozom üretiminde enerji yatırım gereklidir. Örneğin bir aktif bölünmesi maya hücre Dakikada kadar 2.000 olarak ribozomlar üretir. Böyle bir üretimi toplam transkripsiyonel aktivitenin% 60 ve hücrenin 2 toplam birleştirme etkinliğinin% 90 kadar gerektirmektedir. Ayrıca, enerji amino asitler, aminoasil-tRNA ve peptid bağlarının sentezi için gereklidir. Bitkilerde, ATP 3 4.5 5.9 moleküllerden bir peptid zinciri maliyetlerine bir amino asit eklenerek. Nedenle, protein mRNA&#39;nın çeviri bunun değişen çevre koşullarına ile ilgili söz konusu, özellikle düzenlemenin önemli bir yer olması şaşırtıcı değildir. Ribozom bir mRNA&#39;nın birliğidir çeviri başlangıç ​​aşaması, düzenlenmesi ana hedefidirçeviri 4. Bir tercüme düzenlemenin bir sonucu olarak ve diğer transkripsiyon sonrası düzenleme adım olarak, protein konsantrasyonuna varyasyonları sadece% 40 mRNA bolluğu 5,6 ile açıklanabilir. Böylece, toplam mRNA çalışma protein bolluğu hakkında nispeten zayıf bilgi verir. Öte yandan, ribozom ile mRNA&#39;nın dernek çeviri katılanların mRNA erişim sağlayan protein bolluğu içine daha iyi fikir verir. Aktif tercüme mRNAlar polisomlann adlandırılan yapılar çeşitli ribozom ile ilişkilidir. Tersine, kötü çevrilmiş mRNA&#39;ların tek Ribozom (monosome) ile ilişkili olacaktır. Sonuç olarak, bir mRNA&#39;nın translasyon durumu ribozom 7 ile ilişkisi izleyerek değerlendirilebilir. Bu protokol altı gün eski Arabidopsis thaliana fide, RNA sonraki izolasyonu polizom izolasyonunu ve sonuçların analizini açıklar. polysomes ve monosomes bir sükroz yoğunluk gradyanı ile ayrılmıştır. Gradyanlar altı fraksiyon halinde toplanır. Polisomlar, monosomes ve ribozomlar ile ilişkili olmayan serbest 60S ve 40S ribozomal alt birimleri ve mRNA içeren hafif kısmını (bundan sonra adlandırılan yüzer madde): kesirler Bazı üç iyi ayrılmış kesirler elde etmek için bir araya getirilir. Küresel için etkinliği ve polisomlann profilleri karşılaştırarak eğri altında kalan alan entegrasyonu tarafından belirlenen bir polisom / monosome oranı üretilmesiyle tahmin edilebilir. mRNA ve proteinlerin daha sonra farklı fraksiyonlardan çıkarılır ve RT-PCR, qRT-PCR, Northern blot, mikrodizi, Western blot veya proteomik ile analiz için kullanılmıştır. Bu protokol, diğer bitkiler ve dokular için onaylanmıştır. Bu protokol gerçekleştirmek için gerekli ekipman yaygın çoğu laboratuvarlarda bulunur: bir degrade üreticisi için gerek yoktur. Bir sonraki önlemek eklemeden önce her katmanı Donmatabakaların herhangi bir karışımını ya da rahatsızlık dan. Resim boru delicisi gradyanı içinde bir cam kılcal tüp daldırma ile elde edilebilmektedir gradyan toplanması için kullanılır. Bu nedenle, yüksek maliyetli ultrasantrifüj borular hasarsız kalır ve birçok kez yeniden kullanılabilmektedir. Topluca, bu mevcut protokol Polizom profil için kolay ve ucuz bir yöntemdir yapar.

<table border="0" cellpadding="0" cellspacing="0" width="735">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 13.2 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331372</td>
			<td style="width:261px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge tube, thinwall, polyallomer - 38.5 ml</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">326823</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Glass capillary tube</td>
			<td style="width:109px;">Drummond Scientific</td>
			<td style="width:132px;">1-000-1000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge&#160;</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">Optima series</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW41</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">331362</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Ultracentrifuge Rotor SW32</td>
			<td style="width:109px;">Beckman Coulter</td>
			<td style="width:132px;">369650</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Peristaltic pump</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Tygon R3607 polyvinyl chloride tubing&#160;</td>
			<td>Fisher Scientific</td>
			<td>070534-22</td>
			<td>Polyvinyl chloride tubing, 2.29 mm&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Fraction collector Model 2110</td>
			<td style="width:109px;">Bio-Rad</td>
			<td style="width:132px;">731-8120</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV cuvette</td>
			<td style="width:109px;">Hellma</td>
			<td style="width:132px;">170.700-QS</td>
			<td>Quartz flow-through cuvette</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">UV Spectrophotometer</td>
			<td style="width:109px;">Varian</td>
			<td style="width:132px;">Cary50</td>
			<td>Read every 0.0125 sec</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">All chemicals</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td>Use only Molecular Biology Grade</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Murashige and Skoog Basal Salt Mixture (MS)</td>
			<td>Sigma-Aldrich</td>
			<td>M5524</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Rnase-Free water</td>
			<td style="width:109px;">Any</td>
			<td style="width:132px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">Petri Dishes</td>
			<td>Fisher Scientific</td>
			<td>10083251&#160;</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:233px;">Octylphenoxy poly(ethyleneoxy)ethanol, branched (Nonidet P40)</td>
			<td style="width:109px;">Euromedex</td>
			<td style="width:132px;">UN3500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:233px;">Linear acrylamide (acryl carrier)</td>
			<td style="width:109px;">ThermoFischer scientific</td>
			<td style="width:132px;">AM9520</td>
			<td>RNA precipitation carrier</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:233px;">OriginPro 8</td>
			<td style="width:109px;">OriginLab</td>
			<td style="width:132px;"></td>
			<td>Analysis software</td>
		</tr>
	</tbody>
</table>

an easy method for plant polysome profiling

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the analysis of translational regulation. The method described here is an easy and economical way for fractionating polysomes from various plant tissues. A sucrose gradient is made without the need for a gradient maker by sequentially freezing each layer. Cytosolic extracts are then prepared in a buffer containing cycloheximide and chloramphenicol to immobilize the cytosolic and chloroplastic ribosomes to mRNA and are loaded onto the sucrose gradient. After centrifugation, six fractions are directly collected from the bottom to the top of the gradient, without piercing the ultracentrifugation tube. During collection, the absorbance at 260 nm is read continuously to generate a polysome profile that gives a snapshot of global translational activity. Fractions are then pooled to prepare three different mRNA populations: the polysomes, mRNAs bound to several ribosomes; the monosomes, mRNAs bound to one ribosome; and mRNAs that are not bound to ribosomes. mRNAs are then extracted. This protocol has been validated for different plants and tissues including Arabidopsis thaliana seedlings and adult plants, Nicotiana benthamiana, Solanum lycopersicum, and Oryza sativa leaves.

Literatürde, polisom profilleri genellikle örneğin, yukarıdan aşağıya doğru, gradyanlar toplanır şeklinin bir sonucu olarak, ağır fraksiyonuna hafif fraksiyondan gösterilmiştir. Burada anlatılan protokolde geçişlerini alttan üste doğru toplanır, biz göstermek profilleri ağır fraksiyon (Polisomlar) ile başlar ve hafif fraksiyon (serbest ribozom alt birimlerinin ve RNA&#39;lar) (Şekil 2A) gidin. Daha sonra, altı, 2 ml fraksiyonlar her gradyanı toplama ancak polisom içeriğine ilişkin daha ayrıntılı bir analizi yapılacak varsa küçük fraksiyonlar toplanabilir. Birleştirme ve monosome tepe (Şekil 2B) eğrileri normalize farklı çizgiler ya da büyüme koşullarından profillerin karşılaştırılmasına olanak sağlar. Bu da, bağımsız başlatma düzeyi polizom nispi miktarı hakkında bilgi verir. Bir başka bir yolu daprofilleri nalyze, eğri altında kalan alan hesaplamak ve böylece, tek bir monosomes ya polizom (Şekil 2C) ile ya bağlantılı mRNA&#39;nın görece miktarı belirleyebilir. Bu oran, bir bitki büyüme koşulları için geçerlidir. Bununla birlikte, bu yaklaşım, zayıf translasyon aktif dokulardaki durumunda ilgili olmayabilir. Biz A. için bu yöntemi kullandık thaliana bütün fidanları, genç ve yaşlı rozet yanı N. gibi benthamiana (Şekil 3C), S. lycopersicum (Şekil 3E) ve O. sativa yaprağı (Şekil 3D). profil şekli büyüme koşulları, bitki yaş ve analiz dokularda bağlıdır. Burada A. kullanılan Altı gün eski fidan thaliana. Bu aşamada, translasyon etkinliği yüksektir ve profil iyi şekilli tepe (Şekil 2A ve B) gösterir. Bu da böyledirA. thaliana çiçekler (Şekil 3A) kullanılır. 4 haftalık A. kullanırken thaliana rozetler (Şekil 3B), numuneler çoğunlukla hücreler bölünmeye değil nerede tam gelişmiş yetişkin bırakır içerirler. Bu nedenle, polizom ve monosomes toplam miktarı düşüktür. profil az, ama yine de şekillendirilmiş Polizom doruklarına gösterir. monosome zirve yanında, omuz ücretsiz 60S ribozomal alt birimden büyük miktarını gösterir. bitki veya dokuların diğer tür, Polizom zirveleri zorlukla görülebilir. 30 gün yaşlı O. kullanırken bu durum sativa yaprağı (Şekil 3D). dönüştürülmesiyle ilgi mRNA miktarı, (hiç bir polisom pik profilinde görülebilmektedir) çok düşük olduğu zaman bile, monosome tepe varlığı parçalanması doğru yapılır ve mRNA&#39;ların daha ileri analiz için fraksiyondan ekstre edilebilir olduğunu gösterir . ekstre RNA kalitesini kalitesini agaroz jel elektroforezi ile değerlendirilir ( <str<html> ong&gt; Şekil 4). 25S ve 18S sitozolik ribozomal RNA jel üzerinde açıkça görünür olmalıdır. RNA yeşil dokularda 10 çıkarılır zaman kloroplast ribozomal RNA karşılık gelen alt bantları da görünür olmalıdır. <img alt="Şekil 3," src="/files/ftp_upload/54231/54231fig3.jpg" /> Şekil farklı bitki materyali ve türlerden 3. Polizom profilleri. (A) Arabidopsis thaliana çiçekler (300mg), (B) Arabidopsis thaliana 4 haftalık rozet (600mg), (C) Nicotiana benthamiana (40 gün eski kısa gün yetiştirilen bitkiler genç yaprakları - 300mg), (D) Oryza sativa ( 30 gün eski bitkilerin yaprakları - 300mg), (E) Solanum likopersikum (35 gün eski kısa gün yetiştirilen bitkiler genç yaprakları - 300mg). monosome tepe oklar ile belirtilmiştir.iles / ftp_upload / 54231 / 54231fig3large.jpg &quot;target =&quot; _ blank &quot;&gt; bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız. <img alt="Şekil 4," src="/files/ftp_upload/54231/54231fig4.jpg" /> . (- 15 dak 100V) Şekil RNA kalitesinin 4. Değerlendirme RNA belirtilen kısımların bir% 1.2 agaroz jel üzerine yüklendi ve elektroforezi ile ayrıldı (500 6 gün yaşlı, Arabidopsis thaliana fideleri tomurcuklarından ekstre ng). Sitosolik (25S ve 18S) rRNA ok başları ve kloroplast bir braket ile (23S ve 16S) rRNA ile gösterilir. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54231/54231fig4large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about An Easy Method for Plant Polysome Profiling at JoVE.com

An Easy Method for Plant Polysome Profiling

This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Bitki Polizom Profiling için bir Kolay Yöntem

Translation of mRNA to protein is a fundamental and highly regulated biological process. Polysome profiling is considered as a gold standard for the ...

an-easy-method-for-plant-polysome-profiling

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K Görüntüleme.  Aix-Marseille Université. This protocol describes an easy method to extract and fractionate transcripts from plant tissues on the basis of the number of bound ribosomes. It allows a global estimate of translation activity and the determination of the translational status of specific mRNAs.

Video: An Easy Method for Plant Polysome Profiling

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

Ekstrasellüler polisakkaritler Oligo Kitle Profil (Olimp)

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Biyoloji

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

İzotop Etiketleme kütle spektrometresi kullanarak Tiyol Redoks Proteom profil

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Polizom profilleme Stres Durumlardaki Çeviri başlatılması Analizi

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

Tüm montaj ovüllerindeki in Kromatin Modifikasyonu ve Nükleer Mimarlık Kantitatif, Tek hücreli Analizi için Etkin Bir Yöntem<em&gt; Arabidopsis</em

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Assessment of Selective mRNA Translation in Mammalian Cells by Polysome Profiling

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown to allow to selective translation of specific mRNAs, which typically encode for proteins required for a particular stress response. Identification of these mRNAs, as well as the characterization of regulatory mechanisms responsible for selective translation has been at the forefront of molecular biology for some time. Polysome profiling is a cornerstone method in these studies. The goal of polysome profiling is to capture mRNA translation by immobilizing actively translating ribosomes on different transcripts and separate the resulting polyribosomes by ultracentrifugation on a sucrose gradient, thus allowing for a distinction between highly translated transcripts and poorly translated ones. These can then be further characterized by traditional biochemical and molecular biology methods. Importantly, combining polysome profiling with high throughput genomic approaches allows for a large scale analysis of translational regulation.

The ability of cells to adapt to stress is crucial for their survival. Regulation of mRNA translation is one such adaptation strategy, providing for rapid regulation of the proteome. Here, we provide a standardized polysome profiling protocol to identify specific mRNAs that are selectively translated under stress conditions.

Polizom Profil Yöntemi ile memeli hücrelerine selektif mRNA dönüşümüyle Değerlendirilmesi

Regulation of protein synthesis represents a key control point in cellular response to stress. In particular, discreet RNA regulatory elements were shown ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

Bireysel Hücre Tiplerinin transkripsiyonel Profiling için bir alet olarak lazer destekli mikrodiseksiyon (LAM)

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

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 ...

Standardized Method for High-throughput Sterilization of Arabidopsis Seeds

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of microbial contaminants present on the seed surface. Currently, Arabidopsis seeds are sterilized using two distinct sterilization techniques in conditions that differ slightly between labs and have not been standardized, often resulting in only partially effective sterilization or in excessive seed mortality. Most of these methods are also not easily scalable to a large number of seed lines of diverse genotypes. As technologies for high-throughput analysis of Arabidopsis continue to proliferate, standardized techniques for sterilizing large numbers of seeds of different genotypes are becoming essential for conducting these types of experiments. The response of a number of Arabidopsis lines to two different sterilization techniques was evaluated based on seed germination rate and the level of seed contamination with microbes and other pathogens. The treatments included different concentrations of sterilizing agents and times of exposure, combined to determine optimal conditions for Arabidopsis seed sterilization. Optimized protocols have been developed for two different sterilization methods: bleach (liquid-phase) and chlorine (Cl2) gas (vapor-phase), both resulting in high seed germination rates and minimal microbial contamination. The utility of these protocols was illustrated through the testing of both wild type and mutant seeds with a range of germination potentials. Our results show that seeds can be effectively sterilized using either method without excessive seed mortality, although detrimental effects of sterilization were observed for seeds with lower than optimal germination potential. In addition, an equation was developed to enable researchers to apply the standardized chlorine gas sterilization conditions to airtight containers of different sizes. The protocols described here allow easy, efficient, and inexpensive seed sterilization for a large number of Arabidopsis lines.

The objective of this study was to determine the effects of bleach and chlorine gas sterilization on seed germination of a range of Arabidopsis genotypes grown on sterile media. Optimized sterilization protocols have been developed to prevent the growth of microbial contaminants while providing satisfactory seed survival.

Yüksek işlem hacmi sterilizasyon Arabidopsis tohum için standart yöntem

Arabidopsis thaliana (Arabidopsis) seedlings often need to be grown on sterile media. This requires prior seed sterilization to prevent the growth of ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this scenario, the search for new antifungal drugs is necessary. The protocol reported here details a method to screen peptides for their antifungal properties. It is based on the broth microdilution susceptibility test from the Clinical and Laboratory Standards Institute (CLSI) M27-A3 guidelines with modifications to suit the research of antimicrobial peptides as potential new antifungals. This protocol describes a functional assay to evaluate the activity of antifungal compounds and may be easily modified to suit any particular class of molecules under investigation. Since the assays are performed in 96-well plates using small volumes, a large-scale screening can be completed in a short amount of time, especially if carried out in an automation setting. This procedure illustrates how a standardized and adjustable clinical protocol can help the bench-work pursuit of new molecules to improve the therapy of fungal diseases.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.