著者らは、セルロース分解酵素の贈り物をViksø -ニールセンを（ノボ）に感謝いたします。この作品は、FP7のリニューアルでとBBSRCプロジェクトBB/G016178とBB/G016194によって賄われていた。

1。サンプルの調製我々は、通常、草本や木本植物のいずれの茎で動作。草本材料の場合、我々は成熟して植物を栽培し、老化が完了すると種子の開発の後に我々は、乾燥茎を収集する。茎は、4 mmのセグメントに切断三粉砕用ボールで2mlバイアルに配置されます。木質材料の場合、サンプルはもちろんの木製のファイルと粗いおがくずに粉砕され、その後、さらなる処理のためにボールミルに投入。 試料は、粉砕/フォーマットするステーション（図1）内のラックに配置されます。この駅には順次、ミックスを磨く、および各サンプルの重量を量る。サンプルごとに必要な繰り返し回数が一定の粒​​子サイズのための本質的なばらつきが決定される予備的な一連の実験における地盤材料をテストすることによって確立されます。 2 mLバイアルは、下部に穴を開けていると材料が選択さをはるかに越えてバイアルの振動によって分配される。振動腕は粉体の正確な分注を可能にするバランスからのフィードバックによって制御されます。ロボットは、標準的な分析で4 mgの/ウェルと4回の繰り返しがほとんどのケースで必要とされる分注します。これは、20植物サンプル/プレートを分析することができます。 2。前処理サンプルがフォーマットされると、96ウェルプレートは、自動液体ハンドリングシステム（図2）によって処理されます。ここでは軽度の前処理は、酸またはアルカリ溶液350μLを追加し、適応ブロック上にプレートを加熱することにより、植物材料に実行されます。分析中に蒸発を避けるために、96ウェルプレートは、シリコンマットで密封されています。前処理の温度及び時間は、調査される材料に応じて変更することができます。最高温度は、しかしながら、100℃です。厳しい前処理をテストするための代替手順は、オフラインの材料を前処理し、プロセスからpretreatmetを排除することです。 3。加水分解<ol><li>材料を前処理したら、ロボットは自動的に前処理溶液、850μLの25 mM酢酸緩衝液で洗浄、すべてのウェル、5回を削除します。すすぎは、前処理溶液にバッファを追加し、続いてサンプル中の固形物を沈降させた後、総容積の50％を除去することによって行われている。吸引の高さは、井戸の底から固形物を吸引避けるために、液体の質量の半分に設定され、このアプローチを使用して固形物のごくわずかの損失があります。これらは洗浄後ものpHは4.5であり、材料は、酵素加水分解のための準備ができています。 </li><li>セルラーゼおよびヘミセルラーゼのソリューションを含む酵素混合物はロボットによって追加されます。各ウェルに、酵素の混合物の850μL分注し、プレートを50℃の振盪インキュベーターに移動​​されます。標準的な加水分解は、8時間のために実行されますが、この時間は、実験の目的に適合させることができる。草のために使用される標準的な酵素の負荷は、材料の7 FPU / gである。 </li></ol> 4。還元糖の検出加水分解後にリリースされた還元糖の定量は、修正された3 -メチル-2 -ベンゾチアゾリノンhydrozone（MTBH）方法3を用いて行った。それは自動化するための最も簡単で、タンパク質などの化合物からの干渉に少なくとも影響を受けていたためMBTHは、最​​も適切な方法として選択されまし​​た。我々は標準的な光学96ウェルプレートに適している250μLの最終体積で、それは正確にバイオマスの加水分解物中に存在する濃度で糖を定量化することができるように、ロボットプラットフォーム上で使用できるように、この高感度の方法を変更。以下のすべてのステップが自動的に実行され、還元糖の3つの独立した決定は、それぞれの糖化反応のために実施した。 <ol><li>加水分解物の30μlをディープウェルプレートから採取し、スカートPCRを96ウェルプレートの25 mM酢酸ナトリウム緩衝液の45μLと混合した。 </li><li> 1N NaOHを25μL、0.43 mg / mlのMBTH、0.14 mg / mlのDTTを含む溶液50μlを加えた。 </li><li>混合した後、PCRプレートは60℃に加熱させたすべての96ウェルに熱の正確に同じ量を提供クラーまたは類似の装置で20分間。 </li><li>得られた反応は、光学プレートに移した。 </li><li>酸化試薬（0.2％FeNH4（SO4）2、0.2％スルファミン酸と0.1％塩酸）100μLを加える。よく混和して、少なくとも1時間の開発を残す</li><li> 各プレートはまた、50 nmolの、100 nmolの、150 nmolのグルコースの標準的な反応が含まれている必要があります。光学プレートは620nmで読み取られます。 </li></ol> 5。代表的な結果： 分析の様々なタイプの例を図3および4に示されている。すべての結果は、自動化されたプラットフォームを用いて得られた。図3再セルロース分解酵素の量が増加すると地上のポプラから8時間のインキュベーションによって解放還元糖の増加を示す。図4は、酸とポプラの試料のアルカリ前処理の効果を示す。 NaOHの前処理は、加水分解中の糖の放出を容易に希釈H2SO4前その方が効率的です。加水分解後に測定糖の量は、前処理が1M以上の水酸化ナトリウムの濃度を使用して実行されたときに減少します。 <img src="/files/ftp_upload/3240/3240fig1.jpg" alt="図1" /> 図1研削とバイオマスの96ウェルフォーマットのためのロボットステーション<img src="/files/ftp_upload/3240/3240fig2.jpg" alt="図2" /> ハイスループットの糖化の分析については、 図2。液体ハンドリングステーション<img src="/files/ftp_upload/3240/3240fig3.jpg" alt="図3" /> 図3。ポプラのサンプルから糖当量を減らすのリリースで異なる酵素負荷量の影響<img src="/files/ftp_upload/3240/3240fig4.jpg" alt="図4" /> 酸の前処理に比べ、同じ温度と時間でのNaOH前処理の後にリリース90でH 2 SO 4の異なる割合でpretreament℃で30分間後にリリースされた図4。ポプラの試料の糖化に関するさまざまな前処理の効果。A.糖。B.糖 。

<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>試薬の名前</th><th>会社</th><th>カタログ番号</th></tr><tr><td>ロボットを研削＆計量</td><td> Labmanオートメーション</td><td></td></tr><tr><td>キャップ2 mlマイクロチューブ</td><td>ザルスタット株式会社</td><td> 72.694 </td></tr><tr><td> 5ミリメートルステンレススチール製ビーズ</td><td>キアゲン株式会社</td><td> 69989 </td></tr><tr><td> 1.2ミリリットルAbgene井戸ストレージプレート</td><td>フィッシャーサイエンティフィック株式会社</td><td> TUL - 050 - 050C </td></tr><tr><td> 96正方形ウェルプレート用ワットマンキャップマット</td><td>フィッシャーサイエンティフィック株式会社</td><td> 7704-0104 </td></tr><tr><td>液体韓陵のロボット</td><td>テカングループ株式会社</td><td>自由エボ200 </td></tr><tr><td> Sulphuric酸</td><td>フィッシャーサイエンティフィック株式会社</td><td> S/9231/PB17 </td></tr><tr><td>水酸化ナトリウム</td><td>フィッシャーサイエンティフィック株式会社</td><td> BPE359 - 500 </td></tr><tr><td>酢酸ナトリウム</td><td>シグマアルドリッチ</td><td> S8750 - 500G </td></tr><tr><td>酢酸</td><td>フィッシャーサイエンティフィック株式会社</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ウェルPCRフルスカートプレート</td><td>ザルスタット株式会社</td><td> 72.1980.202 </td></tr><tr><td> D -グルコース</td><td>フィッシャーサイエンティフィック株式会社</td><td> G/0450/53 </td></tr><tr><td> 3 - メチル-2 - ベンゾチアゾリノンヒドラゾン塩酸塩水和物</td><td>シグマアルドリッチ</td><td> 129739 - 25G </td></tr><tr><td> DL -ジチオスレイトール</td><td>シグマアルドリッチ</td><td> D9163 - 1G </td></tr><tr><td>コー​​ニングマイクロプレート96ウェル平底</td><td>フィッシャーサイエンティフィック株式会社</td><td> TKT - 521 - 050H </td></tr><tr><td>アンモニウム鉄（III）硫酸塩和物</td><td>シグマアルドリッチ</td><td> F1668 - 250G </td></tr><tr><td>スルファミン酸</td><td>シグマアルドリッチ</td><td> 242772 - 500G </td></tr><tr><td>塩酸</td><td>フィッシャーサイエンティフィック株式会社</td><td> 12462-0026 </td></tr></tbody></table>

high-throughput saccharification assay for lignocellulosic materials

植物リグノセルロース系バイオマスを構成する多糖類は、その後、バイオリファイナリーの確立に使用することができる糖の範囲を生成するために分解することができます。これらの原料が化石燃料の現在の依存関係を置き換えるために、持続可能でカーボンニュートラルでもある新たな産業プラットフォームを、構成するであろう。リグノセルロース系材料で観察された解体への反抗は、植物細胞壁のいくつかの固有の特性によって生成されます。結晶セルロースは、xylansとarabinoxylansとしてマトリックスの多糖類に埋め込 ​​まれており、全体の構造は、1を消化することも困難であるフェノール系ポリマーリグニン、によって包み込まれているさ。我々は糖化2のばらつきを評価するための主な細胞壁の糖化のためのボトルネックとも画面の変異と繁殖個体群を発見するために必要な植物材料の消化率を向上させるために。これらのタスクは、高スループットのアプローチを必要とし、ここでは、96ウェルプレートフォーマットで糖化分析を実行できる分析プラットフォームを提示する。このプラットフォームは、多様な植物種からの大集団のリグノセルロースの消化率のスクリーニングを可能にするために開発されています。我々は、大規模な数値が自動化されたシステムで迅速に評価できるようにする、穏やかな前処理、部分的酵素加水分解と糖測定のための反応容量を縮小している。 この自動化されたプラットフォームでは、再現可能な方法で標準化された粒子サイズに植物材料を減らすために制御された条件下でボールミル粉砕を行う、バイオマスのミリグラム量で動作します。サンプルが地面になったら、自動化されたフォーマットのロボットは、96ディープウェルプレート（図1）の対応するウェルに物質の指定と記録された金額を分配する。通常は、我々は4が分析のために複製持つように4ウェルに同一の材料を分注する。プレートを、所望のレイアウトで植物の物質で満たされると、それらは手動で液体ハンドリングステーション（図2）に移動されます。このステーションではサンプルは希酸またはアルカリのどちらかで軽度の前処理に供されると90℃までの温度でインキュベートし前処理溶液は、その後削除され、サンプルは、加水分解に適したpHに戻すためのバッファで洗浄されています。次いで、試料を50時間の可変長のための酵素混合℃でインキュベートする。アリコートを加水分解物から取得され、還元糖は、自動的にMBTH比色法によって決定されます。

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

High-throughput Saccharification Assay for Lignocellulosic Materials

植物バイオマスの大量のサンプルの糖化性を決定するためのシンプルな、迅速な方法が記載されている。この分析のための自動化されたプラットフォームは、96ウェルプレートおよび放出糖の前処理、加水分解と定量化のその後のパフォーマンスの分析のための植物バイオマスの準備を行います。

リグノセルロース系材料のためのハイスループット糖化アッセイ

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 ビュー.  University of York. 植物バイオマスの大量のサンプルの糖化性を決定するためのシンプルな、迅速な方法が記載されている。この分析のための自動化されたプラットフォームは、96ウェルプレートおよび放出糖の前処理、加水分解と定量化のその後のパフォーマンスの分析のための植物バイオマスの準備を行います。

ビデオ: 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.

ハイスループットSHAPEを使用したRNA二次構造予測

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

生物学

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.

自家製デュアルグロールシフェラーゼアッセイを用いて、ハイスループット機能スクリーニング

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.

ペプチドエピトープの定量分析のための高スループット、MHC II結合アッセイ

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.

ルシフェラーゼを発現する組換えウイルスの高スループット滴定

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.

ハイスループットのアプリケーションのための大人のゼブラフィッシュハーツの回復

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.

転写後調節される遺伝子の化学モジュレーターのためのハイスループットスクリーニング

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アッセイを用いた高スループットにおけるmiRNA標的の検出

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分析 - 高スループット、Sc2.0ジェノタイピングのためのリアルタイムPCRアッセイ

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.

高スループット<em&gt;ゼブラフィッシュ</em&gt;エネルギー消費アッセイ

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.

高スループット シロイヌナズナ種子の殺菌のための標準化された方法

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