Yazarların Viksø-Nielsen (Novozymes) selülolitik enzimlerin hediye için teşekkür etmek istiyorum. Bu çalışma, 7.ÇP YENİLEME ve BBSRC projeleri BB/G016178 ve BB/G016194 tarafından finanse edildi.

1. Örneklerinin hazırlanması: Biz normalde otsu ve odunsu bitkilerin ya sapları ile çalışır. Otsu malzemelerin durumda biz olgunluk bitkiler büyümeye ve tohum oluşumu yaşlanması tamamlandıktan sonra sonra biz kuru sapları toplamak. Sapları 4 mm parça halinde doğranmış ve üç öğütme topları ile 2 ml flakon yerleştirilir. Odunsu bir malzeme söz konusu olduğunda, örneklerin bir ders ahşap dosya ile kaba talaş zemin ve daha sonra daha fazla işlem için bir bilyalı değirmen yerleştirilir. Örnekleri taşlama / istasyonu (Şekil 1) biçimlendirme içinde bir rafa yerleştirilir. Bu istasyon sırayla biler karışımları ve her bir örnek ağırlığındadır. Örnek başına gerekli tekrarlar, belirli bir parçacık boyutu için içsel değişkenlik belirlenir ön deneyler bir dizi zemin malzemesi test ederek kurulmuştur. 2 ml flakon altta deldi ve malzemeler seçilmiş iyi flakonun titreşim dağıtılmıştır. Titreşimli kol, toz doğru dağıtım için izin dengesi bir geri besleme tarafından kontrol edilir. Robot standart bir analiz / 4 mg yayar ve 4 tekrarlar durumlarda şartlarda ihtiyaç vardır. Bu 20 bitki örneklerinin / plaka analiz edilmesini sağlıyor. 2. Tedavi öncesi Numunelerin biçimlendirilmiş sonra, 96-iyi plakalar, otomatik bir sıvı taşıma sistemi (Şekil 2) tarafından işlenir. Burada hafif bir tedavi öncesi asit veya alkali çözeltiler 350 mcL ekleyerek ve uyarlanmış bir blok üzerine tabak ısıtma bitki materyali yapılır. Analizi sırasında buharlaşmasını önlemek için, 96 kuyucuğu bir silikon mat ile mühürlenir. Sıcaklığı ve süresi, tedavi öncesi çalışılan malzemeye göre değişiklik yapılabilir. Maksimum sıcaklık, ancak 100 ° C Sert ön işlemleri test etmek için alternatif bir prosedür, çizgi dışı malzemeler ön işlemden ve pretreatmet süreci ortadan kaldırmak için. 3. Hidroliz <ol><li> Malzemelerin ön tedavi edildikten sonra, robot, tedavi öncesi çözüm ve yıkar, tüm kuyuları, 5 kez 850 mcL 25 mM Asetat Tamponu otomatik olarak kaldırır. Çalkalama, tedavi öncesi bir çözüm tampon ekleyerek ve daha sonra örnek katıların yerleşmelerine izin verme sonra toplam hacminin% 50 kaldırarak tarafından yürütülmektedir. Aspirasyon yüksekliği iyi alt katıların aspire önlemek için sıvı kütlesinin yarısı; bu yaklaşımı kullanarak katıların ihmal kaybı. Bu yıkar sonra iyi pH 4.5 ve malzeme enzimatik hidroliz için hazırdır. </li><li> Sellülazlar ve hemicellulases bir çözüm içeren bir enzim karışımı robot tarafından eklenir. Her kuyuya, 850 mcL enzim karışımı sürülür ve 50 ° C sallayarak inkübatör plaka taşınır. Standart hidroliz 8 saat için yapılır, ama bu sefer Deneyin amacı adapte edilebilir. Otlar için kullanılan standart bir enzim yükleme malzeme 7 FPU / g. </li></ol> 4. Indirgen şeker tespiti Hidroliz sonra yayımlanan indirgen şeker tayini, modifiye edilmiş bir 3-metil-2-benzothiazolinone hydrozone (MTBH) yöntemi 3 kullanılarak gerçekleştirildi . Otomatikleştirmek için en kolay ve protein gibi bileşikler girişime en az hassas olması nedeniyle MBTH en uygun yöntem olarak seçilmiştir. , Doğru, biyokütle hidrolizatların konsantrasyonlarında şekerler ölçmek öyle ki, Biz standart bir optik 96 plaka için uygundur 250 mcL nihai hacmi ile robot platformu kullanmak için bu son derece duyarlı bir yöntem değiştirdi. Altındaki tüm adımları otomatik olarak yapılır ve her saccharification reaksiyon için indirgen şekerler üç bağımsız tespitler yapılmıştır. <ol><li> Hidrolizat 30 mcL derin kuyu plaka alınan ve 25 mM Sodyum Asetat tampon etekli PCR 96 plaka 45 mcL ile karıştırılır. </li><li> (25) 1N NaOH mcL ve 0.43 mg / ml MBTH ve 0.14 mg / ml DTT içeren bir çözüm 50 ul eklendi. </li><li> Karıştırıldıktan sonra, PCR plaka 60 ısıtılmış ° C 20 dakika 96 kuyulara tam olarak aynı miktarda ısı sağlayan bir PCR veya benzeri bir cihaz. </li><li> Ortaya çıkan reaksiyon optik plaka transfer edildi. </li><li> Oksitleyici reaktif (% 0.2 FeNH4 (SO4) 2,% 0.2 SÜLFAMİK asit ve% 0.1 HCI) 100 ul ekleyin. En az 1 saat süreyle iyice karıştırınız ve geliştirmek için bırakın </li><li> Her plakası da 50 nmol, 100 nmol ve 150 nmol glukoz standart reaksiyonlar içermelidir. Optik plakaları 620 nm&#39;de okunur. </li></ol> 5. Temsilcisi Sonuçlar: Şekil 3 ve 4 farklı türde analiz örnekleri sunulmaktadır. Otomatik platformu kullanarak tüm sonuçlar elde edildi. Şekil 3 tekrarselülolitik enzimlerin artan miktarlarda toprak kavak 8 saat inkubasyon tarafından yayımlanan indirgen şeker artış sunar. Şekil 4, asit ve alkali tedavi öncesi kavak numuneler üzerinde etkileri sunar. NaOH ön arıtma daha verimli olduğunu hidrolizi sırasında serbest bırakılması şekerler kolaylaştırmada seyreltilmiş H2SO4 tedavi öncesi. Hidroliz sonra ölçülen şekerlerin miktarı, tedavi öncesi 1M daha yüksek NaOH konsantrasyonları kullanılarak yapıldığında azalır. <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="Şekil 1" /> Şekil 1 öğütme ve biyokütle 96 iyi biçimlendirme Robotik istasyonu <img src="/files/ftp_upload/3240/3240fig2.jpg" alt="Şekil 2" /> Şekil 2: Yüksek Verim saccharification analizi için sıvı taşıma istasyonu <img src="/files/ftp_upload/3240/3240fig3.jpg" alt="Şekil 3" /> Şekil 3 kavak örnekleri şeker benzerleri azaltarak serbest bırakılması farklı bir enzim yükleri Etkisi <img src="/files/ftp_upload/3240/3240fig4.jpg" alt="Şekil 4" /> NaOH ön işlemleri aynı sıcaklıkta ve saatte asit ön arıtma daha sonra serbest H2SO4 farklı yüzdeler ile Pretreament 90 ° C de 30 dakika sonra serbest Şekil 4 saccharification kavak örneklerinin farklı ön işlemleri Etkisi A. Şeker B. Şeker .

<ol>
	<li>Carpita, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Biogenesis+of+the+Cell+Walls+of+Grasses.">Structure and Biogenesis of the Cell Walls of Grasses.</a> Annu Rev Plant Physiol Plant Mol Biol. 47, 445-476 (1996).</li><li>Gomez, L. D., Steele-King, C. G., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustainable+liquid+biofuels+from+biomass:+the+writing's+on+the+walls.">Sustainable liquid biofuels from biomass: the writing's on the walls.</a> New Phytol. 178, 473-485 (2008).</li><li>Anthon, G. E., Barrett, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+reducing+sugars+with+3-methyl-2-benzothiazolinonehydrazone.">Determination of reducing sugars with 3-methyl-2-benzothiazolinonehydrazone.</a> Anal Biochem. 305, 287-289 (2002).</li><li>Gomez, L. D., Whitehead, C., Barakate, A., Halpin, C., McQueen-Mason, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+saccharification+assay+for+determination+of+digestibility+in+plant+materials.">Automated saccharification assay for determination of digestibility in plant materials.</a> Biotechnol Biofuels. 3, 23-23 (2010).</li></ol>

Variations of the standard saccharification protocol can be used in the same platform for determining the activity of cellulolytic enzymes (i.e. by variation of the enzyme concentrations used in a plate as well as using paper as substrate); comparison of the efficiency of several enzyme mixtures on a specific material; time course for saccharification; etc.
A standard saccharification protocol to compare the saccharification potential in different plant materials involves an eight hour hydrolysis (Figures 3 and 4). Most of the plant materials analysed requires four replicates. Under these conditions, the platform can process 80 samples/day. This analysis is being used to screen large populations of barley, maize, and brachypodium in order to establish variability in saccharification potential and the genes involved in its determination4.

<table border="1"><tbody><tr><th> Reaktifin Adı </th><th> Şirket </th><th> Katalog numarası </th></tr><tr><td> Taşlama ve tartım robotu </td><td> Labman Otomasyon </td><td></td></tr><tr><td> Kapaklar ile 2 ml mikro tüp </td><td> Sarstedt Ltd </td><td> 72,694 </td></tr><tr><td> 5 mm paslanmaz çelik boncuk </td><td> Qiagen Ltd </td><td> 69989 </td></tr><tr><td> 1.2ml Abgene kare iyi depolama plakalar </td><td> Fisher Scientific Ltd. </td><td> TUL-050-050C </td></tr><tr><td> 96 kare kuyucuğu whatman kapağı mat </td><td> Fisher Scientific Ltd. </td><td> 7704-0104 </td></tr><tr><td> Sıvı hanling robot </td><td> Tecan Grup Ltd. </td><td> Özgürlük Evo 200 </td></tr><tr><td> Sülfürik asit </td><td> Fisher Scientific Ltd. </td><td> S/9231/PB17 </td></tr><tr><td> Sodyum hidroksit </td><td> Fisher Scientific Ltd. </td><td> BPE359-500 </td></tr><tr><td> Sodyum asetat </td><td> Sigma-Aldrich </td><td> S8750-500G </td></tr><tr><td> Asetik asit </td><td> Fisher Scientific Ltd. </td><td> A/0420/PB17 </td></tr><tr><td> Novozyme 188 </td><td> Novozyme </td><td> DCN00214 </td></tr><tr><td> Celluclast 1.5L </td><td> Novozyme </td><td> CCN03122 </td></tr><tr><td> 96 iyi PCR etek plakaları </td><td> Sarstedt Ltd </td><td> 72.1980.202 </td></tr><tr><td> D-glikoz </td><td> Fisher Scientific Ltd. </td><td> G/0450/53 </td></tr><tr><td> 3-Metil-2-benzothiazolinone Hidrazon hidroklorür hidrat </td><td> Sigma-Aldrich </td><td> 129.739-25G </td></tr><tr><td> DL-dithiothreitol </td><td> Sigma-Aldrich </td><td> D9163-1G </td></tr><tr><td> Corning mikroplaka 96 sıra düz dipli </td><td> Fisher Scientific Ltd. </td><td> TKT-521-050H </td></tr><tr><td> Amonyum demir (III) sülfat dodekahidratı </td><td> Sigma-Aldrich </td><td> F1668-250G </td></tr><tr><td> SÜLFAMİK asit </td><td> Sigma-Aldrich </td><td> 242.772-500G </td></tr><tr><td> Hidroklorik asit </td><td> Fisher Scientific Ltd. </td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

Bitki lignocellulosic biyokütle makyaj polisakkaritler daha sonra bir biorefinery kurulmasında kullanılabilir şekerlerin bir dizi üretmek için ayrılabilir. Bu hammaddeler, mevcut fosil yakıt bağımlılığını yerine, sürdürülebilir ve karbon nötr bir yeni sanayi platformu, oluşturacaktır. Lignocellulosic malzemeleri gözlenen yapı sökümüne dinlemezlik bitki hücre duvarları çeşitli içsel özelliklerine göre üretilir. Kristal selüloz xylans ve arabinoxylans olarak matris polisakkaritler gömülü ve 1 sindirmek için de zor olan fenolik polimer lignin, tüm yapı kaplı. Saccharification 2 değişkenliği değerlendirmek için saccharification için hücre duvarlarının önemli darboğazlar ve aynı zamanda ekran mutant ve üreme nüfusu keşfetmek gerekir sindirilme bitki materyali geliştirmek için. Bu görevler, yüksek kapasiteli bir yaklaşım gerektirir ve burada 96-plaka formatında saccharification analizi gerçekleştirebilir analitik bir platform sunuyoruz. Bu platform, çeşitli bitki türlerinin büyük popülasyonlarının lignoselüloz sindirilme tarama izin vermek için geliştirilmiştir. Biz çok sayıda otomatik bir sistem hızla değerlendirilmelidir izin vermek için nazik hazırlama, kısmi enzimatik hidroliz ve şeker tayini için reaksiyon hacmi küçülttüm. Bu otomatik bir platform, tekrarlanabilir bir şekilde standartlaştırılmış bir parçacık boyutu bitki materyali azaltmak için kontrollü şartlar altında topu freze gerçekleştirirken miligram miktarda biyokütle ile çalışır. Zemin numuneleri sonra, otomatik biçimlendirme robot, 96 derin kuyu plaka (Şekil 1) karşılık gelen kuyu içine malzeme belirtilen ve kaydedilen miktarda dağıtır. Normalde 4 analiz için çoğaltır 4 kuyu içine aynı malzeme dağıtım. Plakalar istenilen düzende bitki materyali ile doldurulur sonra, bir sıvı taşıma istasyonu (Şekil 2) elle taşınır. Bu istasyondan numune seyreltik asit veya alkali ya da hafif bir tedavi öncesi tabi tutulur ve 90 ° C&#39;ye kadar sıcaklıklarda inkübe Tedavi öncesi çözüm daha sonra kaldırılır ve örnekleri için uygun pH hidroliz dönmek için tamponu ile durulanır. Örnekler daha sonra 50 değişken uzunlukta bir zaman için bir enzim karışımı ° C ile inkübe Bir kısım hidrolizat alınan ve indirgen şekerler MBTH kolorimetrik yöntemi ile otomatik olarak belirlenir.

Watch this Scientific Journal Video about High-throughput Saccharification Assay for Lignocellulosic Materials at JoVE.com

High-throughput Saccharification Assay for Lignocellulosic Materials

Bitki biyokütle örnekleri çok sayıda saccharification potansiyeli belirlemek için basit ve hızlı bir yöntem açıklanmıştır. Bu analiz için otomatik bir platform 96 kuyucuğu ve yayımlanan şekerler hazırlama, hidroliz ve miktar daha sonraki performans analizi için bitki biyokütle hazırlık içerir.

Lignocellulosic Malzemeler için Yüksek verim Saccharification Testi

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be 
used in establishing ...

high-throughput-saccharification-assay-for-lignocellulosic-materials

Research

JoVE Journal

Biology

13.2K Görüntüleme.  University of York. Bitki biyokütle örnekleri çok sayıda saccharification potansiyeli belirlemek için basit ve hızlı bir yöntem açıklanmıştır. Bu analiz için otomatik bir platform 96 kuyucuğu ve yayımlanan şekerler hazırlama, hidroliz ve miktar daha sonraki performans analizi için bitki biyokütle hazırlık içerir.

Video: High-throughput Saccharification Assay for Lignocellulosic Materials

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

Yüksek verim Maya Plazmid overekspresyonu Ekran

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Biyoloji

RNA Secondary Structure Prediction Using High-throughput SHAPE

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed "high-throughput selective 2' hydroxyl acylation analyzed by primer extension", or SHAPE, allows prediction of RNA secondary structure with single nucleotide resolution. This approach utilizes chemical probing agents that preferentially acylate single stranded or flexible regions of RNA in aqueous solution. Sites of chemical modification are detected by reverse transcription of the modified RNA, and the products of this reaction are fractionated by automated capillary electrophoresis (CE). Since reverse transcriptase pauses at those RNA nucleotides modified by the SHAPE reagents, the resulting cDNA library indirectly maps those ribonucleotides that are single stranded in the context of the folded RNA. Using ShapeFinder software, the electropherograms produced by automated CE are processed and converted into nucleotide reactivity tables that are themselves converted into pseudo-energy constraints used in the RNAStructure (v5.3) prediction algorithm. The two-dimensional RNA structures obtained by combining SHAPE probing with in silico RNA secondary structure prediction have been found to be far more accurate than structures obtained using either method alone.

High-throughput selective 2' hydroxyl acylation analyzed by primer extension (SHAPE) utilizes a novel chemical probing technology, reverse transcription, capillary electrophoresis and secondary structure prediction software to determine the structures of RNAs from several hundred to several thousand nucleotides at single nucleotide resolution.

Yüksek verim SHAPE kullanarak RNA İkincil Yapısı Tahmin

Understanding the function of RNA involved in biological processes requires a thorough knowledge of RNA structure. Toward this end, the methodology dubbed ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Bir ev yapımı Çift kızdırma Lusıferaz Testi kullanarak yüksek verim Fonksiyonel Taraması

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

A High Throughput MHC II Binding Assay for Quantitative Analysis of Peptide Epitopes

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design1,2. Here, a peptide-MHC II binding assay is scaled to 384-well format. The scaled down protocol reduces reagent costs by 75% and is higher throughput than previously described 96-well protocols1,3-5. Specifically, the experimental design permits robust and reproducible analysis of up to 15 peptides against one MHC II allele per 384-well ELISA plate. Using a single liquid handling robot, this method allows one researcher to analyze approximately ninety test peptides in triplicate over a range of eight concentrations and four MHC II allele types in less than 48 hr. Others working in the fields of protein deimmunization or vaccine design and development may find the protocol to be useful in facilitating their own work. In particular, the step-by-step instructions and the visual format of JoVE should allow other users to quickly and easily establish this methodology in their own labs.

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or design. &#160;Here, a peptide-MHC II binding assay scaled to 384-well plates is described. This cost effective format should prove useful in the fields of protein deimmunization and vaccine design and development.

Peptid Epitopların Kantitatif Analiz için bir yüksek veri akışı MHC II Bağlanma Deneyi

Biochemical assays with recombinant human MHC II molecules can provide rapid, quantitative insights into immunogenic epitope identification, deletion, or ...

High-throughput Titration of Luciferase-expressing Recombinant Viruses

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with viruses that do not form plaques. As an alternative to plaque assays, we have developed a high-throughput titration method that allows for the simultaneous titration of a high volume of samples in a single day. This approach involves infection of the samples with a Firefly luciferase tagged virus, transfer of the infected samples onto an appropriate permissive cell line, subsequent addition of luciferin, reading of plates in order to obtain luminescence readings, and finally the conversion from luminescence to viral titers. The assessment of cytotoxicity using a metabolic viability dye can be easily incorporated in the workflow in parallel and provide valuable information in the context of a drug screen. This technique provides a reliable, high-throughput method to determine viral titers as an alternative to a standard plaque assay.

This article presents a high-throughput luciferase expression-based method of titrating various RNA and DNA viruses using automated and manual liquid handlers.

Lusiferaz ifade eden rekombinant virüsler yüksek verimlilik Titrasyon

Standard plaque assays to determine infectious viral titers can be time consuming, are not amenable to a high volume of samples, and cannot be done with ...

Recovery of Adult Zebrafish Hearts for High-throughput Applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.

Yüksek verim Uygulamaları Yetişkin Zebra Hearts Kurtarma

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Post-transkripsiyonel ayarlı genlerin kimyasal Modülatörler için yüksek verimli tarama

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

3&#39;LIFE Test kullanma Yüksek throughput miRNA Hedefleri tespiti

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag Analizi - A Yüksek Verimli, Sc2.0 Genotiplendirme için Real Time PCR Testi

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of energy homeostasis and obesity. The small size of zebrafish allows for assays on embryos to be conducted in a 96- or 384-well plate format, Morpholino and CRISPR based technologies promote ease of genetic manipulation, and drug treatment by bath application is viable. Moreover, zebrafish are ideal for forward genetic screens allowing for novel gene discovery. Given the relative novelty of zebrafish as a model for obesity, it is necessary to develop tools that fully exploit these benefits. Herein, we describe a method to measure energy expenditure in thousands of embryonic zebrafish simultaneously. We have developed a whole animal microplate platform in which we use 96-well plates to isolate individual fish and we assess cumulative NADH2 production using the commercially available cell culture viability reagent alamarBlue. In poikilotherms the relationship between NADH2 production and energy expenditure is tightly linked. This energy expenditure assay creates the potential to rapidly screen pharmacological or genetic manipulations that directly alter energy expenditure or alter the response to an applied drug (e.g. insulin sensitizers).

High Throughput Danio Rerio Energy Expenditure Assay

Zebrafish are an important model organism for the study of energy homeostasis. By utilizing a NADH2 sensitive redox indicator, alamar Blue, we have developed an assay that measures the metabolic rate of zebrafish larvae in a 96 well plate format and can be applied to drug or gene discovery.

Yüksek Verimli<em&gt; Danio rerio</em&gt; Enerji Harcaması Deneyi

Zebrafish are an important model organism with inherent advantages that have the potential to make zebrafish a widely applied model for the study of ...