Name: preparation of high-quality fermented fish product
Uploaded: 2019-08-23T15:00:00
Description: Watch this Scientific Journal Video about Preparation of High-Quality Fermented Fish Product at JoVE.com

この研究は、江蘇自然科学基金(BK20170185)、江蘇漁業管理者プロジェクト(Y2017-30)、中国国家自然科学財団(NFSC31801575)、中国農業研究基金(NFSC31801575)の資金援助を受けました。システム(CARS-45-26)、食品科学技術の国家一流規律プログラム(JUFSTR20180201)、およびイ・トン江蘇ポストドクタープログラム。

1. サンプル調製
<ol><li>冷凍チョウザメ肉を20°C以下の流水で解凍。その後、きれいに皮膚をオフにし、立方体(2センチメートル×2センチメートル×1.5センチメートル)に魚をカットします。 注:原料は、新鮮なまたは冷凍チョウザメの肉である可能性があります。</li>
	<li>1:1(魚の立方体量に対する魚の塊)の割合で6-12%(w/v)の塩溶液で魚の立方体を1〜3時間10°Cで混合します。</li>
	<li>マリネした...

<ol>
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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbiological+changes+in+naturally+fermented+cassava+fish+(Pseudotolithus+sp.)+for+lanhouin+production.">Microbiological changes in naturally fermented cassava fish (Pseudotolithus sp.) for lanhouin production.</a> International Journal of Food Microbiology. 116 (2), 287-291 (2007).</li><li>Corral, S., Salvador, A., Flores, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt+reduction+in+slow+fermented+sausages+affects+the+generation+of+aroma+active+compounds.">Salt reduction in slow fermented sausages affects the generation of aroma active compounds.</a> Meat Science. 93 (3), 776-785 (2013).</li><li>Wang, W., Xia, W., Pei, G., Xu, Y., Jiang, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+during+fermentation+of+Suanyu+as+a+traditional+fermented+fish+product+of+China.">Proteolysis during fermentation of Suanyu as a traditional fermented fish product of China.</a> International Journal of Food Properties. 20 (90), 29-38 (2017).</li><li>Coventry, M. J., Hickey, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+spices+and+manganese+on+meat+starter+culture+activity.">The effect of spices and manganese on meat starter culture activity.</a> Meat Science. 33 (3), 391-399 (1993).</li><li>Zhu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+on+quality+characteristics+of+pickled+and+fermented+sturgeon+(Acipenser+sinensis)+meat+in+retort+cooking.">Comparative study on quality characteristics of pickled and fermented sturgeon (Acipenser sinensis) meat in retort cooking.</a> International Journal of Food Science &amp; Technology. , (2019).</li><li>Mendonca, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+the+yeast+flora+in+artisanal+country+style+and+industrial;dry+cured+sausage+(yeast+in+fermented+sausage).">Dynamics of the yeast flora in artisanal country style and industrial;dry cured sausage (yeast in fermented sausage).</a> Food Control. 29 (1), 143-148 (2013).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+autochthonous+starter+cultures+on+microbiological+and+physico-chemical+characteristics+of+Suan+yu,+a+traditional+Chinese+low+salt+fermented+fish.">Effect of autochthonous starter cultures on microbiological and physico-chemical characteristics of Suan yu, a traditional Chinese low salt fermented fish.</a> Food Control. 33 (2), 344-351 (2013).</li><li>Gao, P., Jiang, Q., Xu, Y., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esterase+activities+of+autochthonous+starter+cultures+to+increase+volatile+flavor+compounds+in+Chinese+traditional+fermented+fish+(Suan+yu).">Esterase activities of autochthonous starter cultures to increase volatile flavor compounds in Chinese traditional fermented fish (Suan yu).</a> International Journal of Food Properties. 20 (1), 663-672 (2017).</li><li>Wahyuni, S., Holilah, , Asranudin, , Noviyanti, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+shelf+life+of+wikau+maombo+brownies+cake+using+Accelerated+Shelf+Life+Testing+(ASLT)+method+with+Arrhenius+model.">Estimation of shelf life of wikau maombo brownies cake using Accelerated Shelf Life Testing (ASLT) method with Arrhenius model.</a> IOP Conference Series Earth and Environmental Science. 122 (1), 12-22 (2018).</li><li>Zeng, X. F., Xia, W. S., Jiang, Q. X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+microbial+properties+of+Chinese+traditional+low-salt+fermented+whole+fish+product+Suan+yu.">Chemical and microbial properties of Chinese traditional low-salt fermented whole fish product Suan yu.</a> Food Control. 30 (2), 590-595 (2013).</li><li>Yu, D., Jiang, Q., Xu, Y., Xia, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+shelf+life+extension+of+refrigerated+grass+carp+(Ctenopharyngodon+idellus)+fillets+by+chitosan+coating+combined+with+glycerol+monolaurate.">The shelf life extension of refrigerated grass carp (Ctenopharyngodon idellus) fillets by chitosan coating combined with glycerol monolaurate.</a> International Journal of Biological Macromolecules. 101, 448-454 (2017).</li><li>Czerner, M., Tomas, M. C., Yeannes, M. 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本研究では、ワインの香りを持つ高品質な発酵魚製品を製造するための新しい技術と、感覚特性の試験を提供する。この技術の主要なプロセスは、マリネ、乾燥、発酵および殺菌です。マリネプロセスでは、塩濃度、温度、時間はすべて魚のテクスチャ特性に影響を与えます。塩濃度(0~12%、w/v)の増加とマリネ時間の延長(0-2.5 h)に伴い、製品の硬度と噛みごたえが徐々に増加します。しかし、...

中国の現在の市販のマリネ魚製品は、重い塩味、不十分なワインの香り、低い弾力性と淡い色の問題を抱えており、消費者への受け入れを低下させます。そのため、ワインの香りを持つ高品質の魚肉製品の新しい技術を作成し、最適化する必要があります。
近年、肉や魚における現代の発酵技術の応用は、ますます多くの研究者から注目を集めています 1,2,3,4.肉や魚にスターター培養を行うことで、食品の安全性が向上し、処理時間が短縮されました。製品の感覚特性が変更されました。Saithong et al.5天然プラーソムから乳酸菌(LAB)を単離し、このLABをスターター培養物として使用し、高い酸性度を誘導し、病原性菌を抑制した。Zeng et al.6は、自律的なスターター培養物を接種すると発酵時間が短縮され、サンプルの感覚特性が向上したと報告した。Casaburi et al.7は、微生物スターター培養の使用が発酵肉の香りの発達に影響を与うと主張した。これらのスターター培養では、S.セレビシエはアルコール発酵によってワインの香りを生み出し、また、製品に他の改善された官能性の資質を与えることができる。従って、S.セレビシエは、ワインアロマ製品8、9、10およびワインアロマ魚製品に適したスターター培養物であり、S.セレビシエによって作ることができる。
ワインアロマ魚製品を作る過程で、肉や魚の食感は塩分、含水量、pH、タンパク質の脱退などに影響を受ける可能性があります。したがって、マリネ、乾燥、発酵および殺菌はすべてテクスチャ特性に影響を与える可能性があります。風味および味の形成は複雑であり、主にマリネおよび発酵によって影響を受け、炭水化物、タンパク質および脂質、および軽度の脂質酸化11、12の加水分解に非常に関連している。また、スパイス13の添加によって影響を受ける可能性があります。色の発達のために、発酵および殺菌10のプロセスに関与するメイラード反応が起こる。
この記事は、水産加工産業の発展に大きな意義を持つワインの香りを持つ発酵魚製品の工業化のための技術サポートを提供することができます。この技術は、増量プロテオリシス(より自由アミノ酸およびTCA-可溶性ペプチド)によって製品の味を改善することができ、主にアルコール(エタノール、1-オクテン-3-ol、2-メチル-1-プロパノールおよび3-メチル-1-ブタノール)、エステル(酢酸エチル)およびアルデヒド(ノンアナル、3-メチルブタナルおよびベンザルデヒド)は、より高い硬度、ばね味、グミネスおよびチューインによって口当たりを増加させ、明るい表面14によってより魅力的な錆の色を与える。また、製品を室温で保存できるため、消費者の利便性を提供します。他の以前の研究15、16、17によって説明されているように、S.セレビシエによる発酵はまた、他の肉または魚製品における官能的性質を有意に改善することが証明されている。
導入されたプロトコルは、草鯉、銀鯉、黒鯉、大きな鯉、タラ、サーモンなどの魚の他の種にも適用することができることは注目に値します。魚製品の高品質のためには、新鮮な魚、氷の中の魚や冷凍魚など、1年未満保存された冷凍魚など、加工のない魚を使用する必要があります。また、軽度の脂質酸化は風味を高めることができるので、広範な脂質酸化は不快な風味をもたらすので、脂肪の少ない魚が好ましいか、またはスキミングされた魚が推奨される。

<table>
	<tbody>
		<tr>
			<td>2,4,6-trimethylpyridine</td>
			<td>Tokyo Chemical Industry Co., Ltd.</td>
			<td>Purity 98%</td>
			<td></td>
		</tr>
		<tr>
			<td>Colorimeter</td>
			<td>Hunterlab</td>
			<td>UltraScan Pro1166</td>
			<td></td>
		</tr>
		<tr>
			<td>DB-WAX column</td>
			<td>Agilent</td>
			<td>30 m &#215; 0.25 mm &#215; 0.25&#956;m</td>
			<td></td>
		</tr>
		<tr>
			<td>Digital pH meter</td>
			<td>Mettler toledo Instrument (Shanghai) Co., Ltd.</td>
			<td>DELTA-320</td>
			<td></td>
		</tr>
		<tr>
			<td>Drying oven</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>DHG-9070A</td>
			<td></td>
		</tr>
		<tr>
			<td>Frozen sturgeon</td>
			<td>Huada Marine Industry Group Co., Ltd</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Gas chromatograph-mass spectrometer</td>
			<td>Thermo Fisher Scientific</td>
			<td>TSQ Quantum XLS</td>
			<td></td>
		</tr>
		<tr>
			<td>Humidities incubator</td>
			<td>Shanghai Yiheng Scientific Instrument Co., Ltd.</td>
			<td>LHS-250HC-II</td>
			<td></td>
		</tr>
		<tr>
			<td>Saccharomyces cerevisiae</td>
			<td>Angel Yeast Co., Ltd</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Spices</td>
			<td>Auchan Supermarket</td>
			<td>-</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterilization pot</td>
			<td>Longqiang Machinery Technology Co., Ltd.</td>
			<td>RHS-03-700</td>
			<td></td>
		</tr>
		<tr>
			<td>Supelco</td>
			<td>Sigma</td>
			<td>65&#956;m, PDMS/DVB</td>
			<td></td>
		</tr>
		<tr>
			<td>Texture analyzer</td>
			<td>Stable Micro Systems, Ltd.</td>
			<td>TA-XT2i</td>
			<td></td>
		</tr>
		<tr>
			<td>Vacuum package machine</td>
			<td>Quanzhou Yiminxin Electromechanical Co., Ltd.</td>
			<td>YMX-958-10L</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of high-quality fermented fish product

このプロトコルは、チョウザメ(アキラリアシネンシス)肉製品を含む工業化発酵魚製品の調製方法を提供する。手順は:(1)切断、脱皮、スキニングオフ、クリーニング、切断を含む養殖チョウザメの前処理。(2)6-12%(w/v)塩溶液(1:1、魚立方体塊溶液量)で魚の立方体をマリネ。(3)熱風(40~60°C)または真空により50~60%の水分含有量に魚の立方体を乾燥させる。(4)魚の立方体を0.4-1.6%(w/w)で接種し、魚の立方体に対する風味溶液中のS.セレビシエを含む発酵と6-10時間の25-35 °Cで発酵する。(5)マリネと発酵溶液で真空パッケージに魚のキューブを密封;(6)115-121 °Cで10-20分間殺菌する。この方法で調製したチョウザメ肉製品は、まろやかで厚い美味しさを持ち、魚からカビや不快な臭いを隠す可能性のあるアルコールやエステルなどの揮発性風味化合物を多量に含み、適度な塩分を有しています。内容は高いバネ、グミ、チューインなどの質感が良く、明るい錆びた色と魅力的な外観を持っています。この新しい技術は、室温で保存できる便利な魚のスナック食品を提供するために、他の魚の処理にも適用することができます。それは海洋および淡水魚の両方のために適している。

適切な塩濃度、マリネ時間と温度は、製品のテクスチャ品質をより良くします。最良のマリネ条件は次の通りでした:マリネ溶液中の8%の塩濃度。2時間のマリネ時間。10 °Cのマリネ温度。図 1を参照してください。
乾燥プロセスでは、最終的な水分含有量と乾燥温度がテクスチャと感覚の品質に影響を与える可能性があります。最良の乾燥条件は以?...

Watch this Scientific Journal Video about Preparation of High-Quality Fermented Fish Product at JoVE.com

Preparation of High-Quality Fermented Fish Product

プロトコルの目的は、サッカロマイセスセレビシエの接種に基づいて工業化された魚の発酵技術を提供することです.

高品質発酵魚製品の調製

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

この手順は、ワインの香りを持つ発酵魚製品の高品質を生成し、産業生産に適用することができます。この技術は、魚製品に魅力的な外観、独特の風味、おいしい味、および良好な食感特性を与えます。そして、この製品は、少なくとも1年間室温で保存することができます。この手順を開始するには、テキストプロトコルで概説されているように、解凍、清潔、および皮膚冷凍チョウザメ肉。魚を立方体に切ります。次に、魚の立方体を摂氏10度で1対1の比率で6〜12%の塩溶液と1〜3時間混ぜます。その後、マリネした魚の立方体をステンレス製の格子板に置き、40~60°Cの温度で熱気で6~10時間乾燥させます。スパイス液を準備するには、ネギ1.2単位、ショウガ1単位、スターアニス0.6単位、フェンネル0.6単位、緑茶0.3単位、40単位の水で0.3単位のコショウを含むスパイスを混ぜます。30分間沸騰させ、濾液を回収する。フレーバー溶液を調製するには、スパイス液、黄色ワイン、白ワイン、サトウキビ砂糖、グルタミン酸ナトリウム、酢をここに示す質量分数に混ぜます。この後、乾燥した魚の立方体をフレーバー溶液と混合し、0.4〜1.6%のサッカロミセス・セレビシエを1対1の割合で接種します。容器に封じ込め、摂氏25度から35度の温度で6~10時間発酵させます。発酵させた魚の立方体をマリネおよび発酵液を含む真空パッケージに入れ、真空強度0.085メガパスカル、シール時間3.6秒、冷却時間5.5秒で真空パッケージを密封します。その後、115〜121°Cの間の殺菌温度を使用して10〜20分間、パックされた魚の立方体を殺菌します。本研究では、高品質の発酵魚製品を調製する。適切な塩濃度、マリネ時間、温度を使用すると、製品のテクスチャ品質が向上します。最高のマリネ条件は、塩濃度8%とマリネ溶液、2時間のマリネ時間であると思われる。そして、摂氏10度のマリネ温度。乾燥プロセスでは、最終的な水分含有量と乾燥温度がテクスチャとセンサーの品質に影響を与える可能性があります。最高の乾燥条件は、55%の乾燥魚キューブと摂氏50度の乾燥温度の水分含有量であると思われます。発酵プロセスにおいて、サッカロミセス・セレビシエの添加、温度、および時間はすべて魚の味、風味、色、および食感特性に影響を与える。成功した発酵魚は、味、風味、色、食感に高いスコアを持ち、調和のとれた味、発酵香り、ワイン、ラセットカラー、明るい表面、キャラメル、アルデンテの食感をもたらします。失敗した魚製品は、味、風味、色、食感が低いため、味、苦味、酸味、魚、淡い色、粗い表面、粗い口当たり、食感の悪化が生じます。適切な殺菌および強度は4.5分と思われる。そして、適切な殺菌温度は摂氏121度と思われる。次に、ストレージに関する考慮事項が計算され、テキスト プロトコルで説明されているように決定されます。そして、賞味期限の予測は摂氏25度で264日、摂氏35度で205日です。この手順を実行する際には、発酵と滅菌の両方のステップがこの技術の成功に不可欠であることに注意することが重要です。この手順を学習した後、他の魚種の発酵に使用することができる。将来的には、他の菌株との発酵を調査する可能性があります。例えば乳酸菌、ブドウ球菌、エトセトラ。非常に高い温度が害を引き起こす可能性がありますので、魚製品を殺菌する際には、必ず予防措置を講じてください。

preparation-of-high-quality-fermented-fish-product

Research

JoVE Journal

Biochemistry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

定量的なアプリケーションのための均質なMALDI試料の調製

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

生化学

A Filtration-based Method of Preparing High-quality Nuclei from Cross-linked Skeletal Muscle for Chromatin Immunoprecipitation

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a challenge for ChIP due to technical difficulty in isolation of high-quality nuclei with minimal contamination of myofibrils. Previous protocols have attempted to purify nuclei before cross-linking, which incurs the risk of altered DNA-protein interaction during the prolonged nuclei preparation process. In the current protocol, we first cross-linked the skeletal muscle tissue collected from mice, and the tissues were minced and sonicated. Since we found that ultracentrifugation was not able to separate nuclei from myofibrils using cross-linked muscle tissue, we devised a sequential filtration procedure to obtain high-quality nuclei devoid of significant myofibril contamination. We subsequently prepared chromatin by using an ultrasonicator, and ChIP assays with anti-BMAL1 antibody revealed robust circadian binding pattern of BMAL1 to target gene promoters. This filtration protocol constitutes an easily applicable method to isolate high-quality nuclei from cross-linked skeletal muscle tissue, allowing consistent sample processing for circadian and other time-sensitive studies. In combination with next-generation sequencing (NGS), our method can be deployed for various mechanistic and genomic studies focusing on skeletal muscle function.

We present a filtration-based protocol to isolate high-quality nuclei from cross-linked mouse skeletal muscle wherein we removed the need for ultracentrifugation, making it easily applicable. We show that chromatin prepared from the nuclei is suitable for chromatin immunoprecipitation and likely chromatin immunoprecipitation sequencing studies.

クロマチン免疫沈降のための架橋された骨格筋からの高品質核の調製のためのろ過に基づく方法

Chromatin immunoprecipitation (ChIP) is a powerful method to determine protein binding to chromatin DNA. Fiber-rich skeletal muscle, however, has been a ...

Biosynthesis of a Flavonol from a Flavanone by Establishing a One-pot Bienzymatic Cascade

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro enzymatic synthesis of a flavonol. In this method, Atf3h and Atfls1, two key genes in the biosynthetic pathway of the flavonols, are cloned and overexpressed in Escherichia coli. The recombinant enzymes are purified via an affinity column and then a bienzymatic cascade is established in a specific synthetic buffer. Two flavonols are synthesized in this system as examples and determined by TLC and HPLC/LC/MS analyses. The method displays obvious advantages in the derivation of flavonols over other approaches. It is time- and labor-saving and highly cost-effective. The reaction is easy to be accurately controlled and thus scaled up for mass production. The target product can be purified easily due to the simple components in the system. However, this system is usually restricted to the production of a flavonol from a flavanone.

The derivation of a flavonol is crucial for its application in healthcare and the food industry. Here, we provide a detailed protocol for the biosynthesis of a flavonol from a flavanone and discuss the crucial steps and its advantages over other approaches.

ワンポットビエンツマティックカスケードを確立することにより、フラバノンからのフラボノールの生合成

Flavonols are a major subclass of flavonoids with a variety of biological and pharmacological activities. Here, we provide a method for the in vitro ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

遺伝子発現プロファイリングのための修飾法による穀物種子からの高品質なトランスクリプトームデータの取得

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

単一分子FISHによる転写活性アレスの単一細胞解析

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

フラグメントベースのリード発見におけるスクリーニングのためのナノ差動走査フルオリメトリー

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

Preparation of High-Temperature Sample Grids for Cryo-EM

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, mostly at 4 &#176;C and occasionally at room temperature. Recently, we discovered that the protein structure solved at low temperature may not be functionally relevant, particularly for proteins from thermophilic archaea. A procedure was developed to prepare protein samples at higher temperatures (up to 70 &#176;C) for cryo-EM analysis. We showed that the structures from samples prepared at higher temperatures are functionally relevant and temperature dependent. Here we describe a detailed protocol for preparing sample grids at high temperature, using 55 &#176;C as an example. The experiment made use of a vitrification apparatus modified using an additional centrifuge tube, and samples were incubated at 55 &#176;C. The detailed procedures were fine-tuned to minimize vapor condensation and obtain a thin layer of ice on the grid. Examples of successful and unsuccessful experiments are provided.

This paper provides a detailed protocol for preparing sample grids at temperatures as high as 70 &#176;C, prior to plunge freezing for cryo-EM experiments.

クライオ電子顕微鏡用高温サンプルグリッドの作製

The sample grids for cryo-electron microscopy (cryo-EM) experiments are usually prepared at a temperature optimal for the storage of biological samples, ...

The Extraction of Liver Glycogen Molecules for Glycogen Structure Determination

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are influenced by its structure. Hence, a suitable extraction method that isolates representative samples of glycogen is crucial to the study of this macromolecule. Compared to other extraction methods, a method that employs a sucrose density gradient centrifugation step can minimize molecular damage. Based on this method, a recent publication describes how the density of the sucrose solution used during centrifugation was varied (30%, 50%, 72.5%) to find the most suitable concentration to extract glycogen particles of a wide variety of sizes, limiting the loss of smaller particles. A 10 min boiling step was introduced to test its ability to denature glycogen degrading enzymes, thus preserving glycogen. The lowest sucrose concentration (30%) and the addition of the boiling step were shown to extract the most representative samples of glycogen.

An optimal sucrose concentration was determined for the extraction of liver glycogen using sucrose density gradient centrifugation. The addition of a 10 min boiling step to inhibit glycogen-degrading enzymes proved beneficial.

グリコーゲン構造決定のための肝臓グリコーゲン分子の抽出

Liver glycogen is a hyperbranched glucose polymer that is involved in the maintenance of blood sugar levels in animals. The properties of glycogen are ...

Preparation of siRNA Mixture for Mitochondrial DNA Synthesis

To begin, prepare a 140 nano molar mix of small interfering RNA or siRNA by diluting the siRNA in optimum medium. Add the five microliters of the diluted mix to the wells of a black 384 well cell culture microplate. Prepare the appropriate amount of RNAiMAX transfection reagent solution in optimum medium and add 10 microliters to each well of the 384 well plate with a reagent dispenser.

ミトコンドリアDNA合成のためのsiRNA混合物の調製

In Situ細胞凍結トモグラフィーのための電子顕微鏡グリッドの作製

Sample Preparation for In Situ Cryotomography of Mammalian Cells

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly relevant to cellular biology and virology. The potential of combining cryotomography with other microscopy modalities makes it a perfect technique for integrative and correlative imaging. However, sample preparation for in situ cellular tomography is not straightforward, as cells do not readily attach and stretch over the electron microscopy grid. Additionally, the grids themselves are fragile and can break if handled too forcefully, resulting in the loss of imageable areas. The geometry of tissue culture dishes can also pose a challenge when manipulating the grids with tweezers. Here, we describe the tips and tricks to overcome these (and other) challenges and prepare good-quality samples for in situ cellular cryotomography and correlative imaging of adherent mammalian cells. With continued advances in cryomicroscopy technology, this technique holds enormous promise for advancing our understanding of complex biological systems.

著者スポットライト:強化されたクライオ電子線トモグラフィーのためのグリッド準備の最適化 

This method provides an accessible and flexible protocol for the preparation of electron microscopy (EM) grids for in situ cellular cryotomography and correlative light and electron microscopy (CLEM).

哺乳類細胞の in situ クライオトモグラフィーのためのサンプル調製

In situ cellular cryotomography is a powerful technique for studying complex objects in their native frozen-hydrated cellular context, making it highly ...