Gli autori desiderano ringraziare A Viksø-Nielsen (Novozymes) per il dono degli enzimi cellulosolitici. Questo lavoro è stato finanziato dal 7 ° PQ RINNOVO e dai progetti BBSRC BB/G016178 e BB/G016194.

1. Preparazione dei campioni Di solito lavoro con steli da entrambi erbacee o piante legnose. Nel caso di materiali erbacee che crescono le piante a maturità, e dopo lo sviluppo di semi che raccogliamo steli secchi, una volta senescenza è completa. Gli steli sono tagliate in 4 segmenti mm e collocati in 2 flaconi ml con tre palle di fresatura. Nel caso di materiali legnosi, i campioni sono terreno di segatura grossolana con un file in legno corso e quindi posto in un mulino a sfere per ulteriori elaborazioni. I campioni sono posti in un rack all&#39;interno della rettifica / formattazione stazione (Figura 1). Questa stazione macina in sequenza, si mescola e pesa ogni campione. Il numero di ripetizioni necessarie per campione è stabilita testando il materiale a terra in una serie di esperimenti preliminari in cui si determina la variabilità intrinseca di una certa dimensione delle particelle. Il 2 flaconi ml sono forate nella parte inferiore e materiali sono dispensati dalle vibrazioni del flaconcino il pozzetto selezionato. Il braccio vibrante è controllato da un feedback da parte l&#39;equilibrio che consenta l&#39;erogazione accurata della polvere. Il robot dispensa 4 mg / e in un&#39;analisi standard e 4 ripetizioni sono necessari nella maggior parte dei casi. Ciò consente di 20 campioni vegetali / piastra da analizzare. 2. Pretrattamento Una volta che i campioni sono formattati, le piastre a 96 pozzetti vengono elaborati da un sistema automatizzato di gestione dei liquidi (Figura 2). Ecco un pretrattamento mite viene eseguita sul materiale vegetale con l&#39;aggiunta di 350 ml di soluzioni acide o alcaline e il riscaldamento della piastra su un blocco adattato. Per evitare l&#39;evaporazione durante l&#39;analisi, il 96 pozzetti sono sigillati con silicone opaco. La temperatura e l&#39;ora del pre-trattamento può essere modificato a seconda dei materiali oggetto di studio. La temperatura massima, comunque, è di 100 ° C. Una procedura alternativa per i test pre-trattamenti più dura è quella di pretrattare i materiali off line ed eliminare il pretreatmet dal processo. 3. Idrolisi <ol><li> Dopo che i materiali sono pretrattati, il robot rimuove automaticamente la soluzione di pretrattamento, e lavaggi, tutti i pozzi, 5 volte con 850 microlitri 25 mM tampone acetato. I risciacqui vengono effettuate con l&#39;aggiunta di tampone alla soluzione pre-trattamento e, successivamente, la rimozione del 50% del volume totale dopo aver lasciato i solidi nel campione di stabilirsi. L&#39;altezza della aspirazione è fissato a metà della massa liquida al fine di evitare l&#39;aspirazione dei solidi dal fondo del pozzo, vi è la perdita trascurabile di solidi utilizzando questo approccio. Dopo questi lavaggi del pH nel pozzo è 4.5 e il materiale è pronto per idrolisi enzimatica. </li><li> Una miscela di enzimi che contiene una soluzione di cellulasi e emicellulasi viene aggiunto dal robot. In ciascun pozzetto, 850 ml di miscela di enzimi è erogato e la piastra viene spostato in un 50 ° C agitando incubatore. L&#39;idrolisi standard viene eseguita per 8 ore, ma questa volta può essere adattata allo scopo dell&#39;esperimento. Il carico enzima standard utilizzato per erbe FPU è di 7 / g di materiale. </li></ol> 4. Rilevamento di zuccheri riduttori Determinazione degli zuccheri riduttori rilasciato dopo idrolisi è stata effettuata utilizzando una versione modificata del 3-metil-2-benzotiazolinone hydrozone (MTBH) metodo 3. MBTH è stato selezionato come il metodo più adatto perché era il più facile da automatizzare e meno soggetti a interferenze da composti come le proteine. Abbiamo modificato questo metodo altamente sensibile per l&#39;uso sulla piattaforma robotica in modo che possa quantificare con precisione gli zuccheri alle concentrazioni presenti in idrolizzati biomassa, con un volume finale di 250 microlitri che è adatto a uno standard ottico 96 pozzetti. Tutte le fasi di seguito vengono eseguite automaticamente e tre determinazioni indipendenti dalla zuccheri riducenti sono state effettuate per ogni reazione saccarificazione. <ol><li> 30 ml di idrolizzato è stato preso dal piatto profondo e ben miscelati con 45 ml di 25 mM tampone acetato di sodio in una minigonna PCR 96 pozzetti. </li><li> 25 ml di NaOH 1N e 50 ml di una soluzione contenente 0,43 mg / ml MBTH e 0,14 mg / ml DTT sono stati aggiunti. </li><li> Dopo la miscelazione, la piastra PCR è stata riscaldata a 60 ° C per 20 minuti in un termociclatore o un dispositivo simile che fornisce esattamente la stessa quantità di calore a tutti i 96 pozzetti. </li><li> La reazione che ne deriva è stato trasferito a una piastra ottica. </li><li> Aggiungere 100 ml di reagente ossidante (0,2% FeNH4 (SO4) 2, 0,2% di acido solfammico e 0,1% di HCl). Mescolare bene e lasciare a sviluppare per almeno 1 h. </li><li> Ogni piatto dovrebbe contenere anche le reazioni standard di 50 nmol, 100 nmol e 150 nmol glucosio. Le piastre ottici vengono letti a 620 nm. </li></ol> 5. Rappresentante dei risultati: Esempi di diversi tipi di analisi sono presentati in figura 3 e 4. Tutti i risultati sono stati ottenuti utilizzando la piattaforma automatizzata. Figura 3 ripresenta l&#39;aumento di zuccheri riduttori rilasciata da 8 incubazioni h da pioppo terra con quantità crescenti di enzimi cellulosolitici. La figura 4 presenta gli effetti di acidi e alcalini pretrattamento su campioni di pioppo. Il pretrattamento NaOH è più efficiente che il pretrattamento H2SO4 diluito nel facilitare il rilascio di zuccheri durante l&#39;idrolisi. La quantità di zuccheri misurati dopo idrolisi diminuisce quando il pre-trattamento viene eseguito utilizzando le concentrazioni di NaOH superiore a 1M. <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="Figura 1" /> Figura 1. Stazione robotica per la macinazione e 96 e la formattazione delle biomasse <img src="/files/ftp_upload/3240/3240fig2.jpg" alt="Figura 2" /> Figura 2. Stazione di gestione dei liquidi per l&#39;analisi ad alta saccarificazione Throughput <img src="/files/ftp_upload/3240/3240fig3.jpg" alt="Figura 3" /> Figura 3. Effetto di carichi diversi enzimi sul rilascio di equivalenti di riduzione zucchero da campioni di pioppo <img src="/files/ftp_upload/3240/3240fig4.jpg" alt="Figura 4" /> Figura 4. Effetto dei pre-trattamenti diversi sulla saccarificazione di campioni di pioppo. Zuccheri A. rilasciato dopo pretrattamento con diverse percentuali di H2SO4 a 90 ° C per 30 minuti. Zuccheri B. rilasciato dopo pretrattamenti NaOH alla stessa temperatura e il tempo che il pretrattamento acido .

<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> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Macinazione e peso del robot </td><td> Labman Automation </td><td></td></tr><tr><td> 2 ml micro tubo con tappi </td><td> Sarstedt Ltd </td><td> 72,694 </td></tr><tr><td> 5 pedine mm in acciaio inox </td><td> Qiagen Ltd </td><td> 69989 </td></tr><tr><td> 1,2 ml Abgene piazza pozzetti di stoccaggio </td><td> Fisher Scientific Ltd </td><td> TUL-050-050C </td></tr><tr><td> Whatman tappo tappetino per 96 piatti quadrati e </td><td> Fisher Scientific Ltd </td><td> 7704-0104 </td></tr><tr><td> Hanling robot liquidi </td><td> Tecan Group Ltd. </td><td> Freedom Evo 200 </td></tr><tr><td> Acido solforico </td><td> Fisher Scientific Ltd </td><td> S/9231/PB17 </td></tr><tr><td> Idrossido di sodio </td><td> Fisher Scientific Ltd </td><td> BPE359-500 </td></tr><tr><td> Di sodio acetato </td><td> Sigma-Aldrich </td><td> S8750-500G </td></tr><tr><td> L&#39;acido acetico </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 pozzetti gonna PCR pieno </td><td> Sarstedt Ltd </td><td> 72.1980.202 </td></tr><tr><td> D-glucosio </td><td> Fisher Scientific Ltd </td><td> G/0450/53 </td></tr><tr><td> 3-metil-2-benzotiazolinone idrazone cloridrato idrato </td><td> Sigma-Aldrich </td><td> 129739-25G </td></tr><tr><td> DL-ditiotreitolo </td><td> Sigma-Aldrich </td><td> D9163-1G </td></tr><tr><td> Corning micropiastre 96 pozzetti fondo piatto e </td><td> Fisher Scientific Ltd </td><td> TKT-521-050H </td></tr><tr><td> Ammonio ferro (III) solfato dodecaidrato </td><td> Sigma-Aldrich </td><td> F1668-250G </td></tr><tr><td> Sulfamic acido </td><td> Sigma-Aldrich </td><td> 242772-500G </td></tr><tr><td> Acido cloridrico </td><td> Fisher Scientific Ltd </td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

Polisaccaridi che costituiscono la biomassa lignocellulosica impianto può essere suddiviso per produrre una gamma di zuccheri che successivamente può essere utilizzato nella creazione di una bioraffineria. Queste materie prime costituirebbero una nuova piattaforma industriale, che sia sostenibile e carbon neutral, per sostituire l&#39;attuale dipendenza dai combustibili fossili. La riluttanza di decostruzione osservati in materiali lignocellulosici è prodotto da diverse proprietà intrinseche delle pareti delle cellule vegetali. Cellulosa cristallina è incorporato in matrice di polisaccaridi come xylans e arabinoxilani, e tutta la struttura è incassata dal fenolici lignina polimero, che è anche difficile da digerire 1. Al fine di migliorare la digeribilità dei materiali vegetali abbiamo bisogno di scoprire le principali strozzature per la saccarificazione delle pareti cellulari e anche mutanti schermo e popolazioni nidificanti di valutare la variabilità in saccarificazione 2. Queste attività richiedono un approccio un elevato throughput e qui vi presentiamo una piattaforma di analisi che possono effettuare analisi saccarificazione in un formato a 96 pozzetti della piastra. Questa piattaforma è stata sviluppata per consentire la proiezione di digeribilità lignocellulosa di grandi popolazioni di specie vegetali diverse. Abbiamo ridimensionato volumi di reazione per il pretrattamento dolce, parziale idrolisi enzimatica e determinazione lo zucchero, per consentire un gran numero di valutare rapidamente in un sistema automatizzato. Questa piattaforma automatizzata lavora con quantità dell&#39;ordine di milligrammi di biomassa, l&#39;esecuzione di fresatura palla in condizioni controllate per ridurre i materiali vegetali di una granulometria standardizzata in modo riproducibile. Una volta che i campioni sono a terra, il robot formattazione automatica dispensa determinata quantità di materiale e registrato nei pozzetti corrispondenti della piastra da 96 pozzi profondi (Figura 1). Normalmente, distribuire lo stesso materiale in 4 pozzi di avere 4 repliche per l&#39;analisi. Una volta che i piatti sono pieni di materiale vegetale nel layout desiderato, vengono spostati manualmente ad una stazione di manipolazione dei liquidi (Figura 2). In questa stazione i campioni sono sottoposti ad un pretrattamento mite sia con acido diluito o alcalini e incubate a temperature fino a 90 ° C. La soluzione di pretrattamento viene successivamente rimossa ed i campioni sono sciacquati con tampone per tornare ad un pH adatto per idrolisi. I campioni vengono quindi incubati con una miscela di enzimi per un periodo di tempo variabile a 50 ° C. Un&#39;aliquota è tratto dal idrolisati e gli zuccheri riduttori vengono automaticamente determinata con il metodo colorimetrico MBTH.

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

High-throughput Saccharification Assay for Lignocellulosic Materials

Un metodo semplice e rapido per determinare il potenziale saccarificazione di un gran numero di campioni di biomassa vegetale è descritto. La piattaforma automatizzata per questa analisi comporta la preparazione della biomassa vegetale per l&#39;analisi in 96 pozzetti e le prestazioni successive di pretrattamento, l&#39;idrolisi e la quantificazione degli zuccheri rilasciato.

High-throughput per l&#39;analisi Saccarificazione materiali lignocellulosici

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 Views.  University of York. Un metodo semplice e rapido per determinare il potenziale saccarificazione di un gran numero di campioni di biomassa vegetale è descritto. La piattaforma automatizzata per questa analisi comporta la preparazione della biomassa vegetale per l'analisi in 96 pozzetti e le prestazioni successive di pretrattamento, l'idrolisi e la quantificazione degli zuccheri rilasciato.

Video: High-throughput Saccharification Assay for Lignocellulosic Materials

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.

RNA Secondaria previsione della struttura utilizzando la forma di alta velocità

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

Ricerca

Biologia

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.

High-throughput screening funzionale utilizzando una casalinga 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 ...

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.

Un saggio di legame High Throughput MHC II per l&#39;analisi quantitativa dei peptidi epitopi

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.

High-throughput Titolazione della luciferasi che esprimono ricombinanti virus

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.

Recupero di Zebrafish adulti cuori per applicazioni ad alta velocità effettiva

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.

Screening ad alto rendimento per i modulatori chimici di Genes post-trascrizionale regolamentati

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.

Rivelazione di bersagli miRNA in high-throughput usando il saggio 3&#39;LIFE

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.

Analisi qPCRTag - A High Throughput, Real Time PCR Assay per Sc2.0 genotipizzazione

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.

High Throughput<em&gt; Danio Rerio</em&gt; Energia spese 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 ...

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.

Metodo standardizzato per la sterilizzazione di High-throughput di semi di Arabidopsis

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