Name: an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis
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Description: Watch this Scientific Journal Video about An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis at JoVE.com

我们感谢功能基因组学基金和光谱法和生物成像设备，涅槃核心研究设施，以及植物型研究设施，涅槃生物资源中心的工作人员。

1.从植物种子总RNA提取<ol><li>制备缓冲套，离心柱，1.5和2.0毫升聚丙烯管，和无核酸酶的1.5mL的聚丙烯管中。 </li><li>添加1％（重量/体积）分子生物学级的聚乙烯吡咯烷酮（以下简称PVP）细胞裂解缓冲液提取RNA并涡旋大力。孵育20分钟，在25℃以完全溶解。 20分钟温育后，通过将管倒置，以防止气泡的形成轻轻混匀缓冲区。在存放使用前室温（15-25°C）。 </li><li> 拟南芥植物和发生在冰上1.5毫升聚丙烯管丰收果实。关于铝板的地方水果保持在4℃，立体显微镜下分离从果实种子。 <ol><li>放置分离的种子（大约200颗种子）转换成已经存储在铝机架在冰上1.5毫升聚丙烯管，并立即放置在液体n中的管itrogen。 </li></ol></li><li>从液氮中取出管子并将其返回到铝架上的冰。在1000 xg离心加入100微升含有1％1分钟PVP和离心机缓冲器的在4℃下。 </li><li>均质用马达粉碎机60秒，同时保持该管的铝机架上冰的不锈钢杵样品。 </li><li>添加含有1％的PVP缓冲器550微升，通过转动管倒置轻轻混匀缓冲区，并在25℃下孵育10分钟。 </li><li>离心机在8000 xg离心25℃5分钟，并转移550μL上清液至新的1.5毫升聚丙烯管中。 </li><li>离心机以10,000 xg离心，在25℃5分钟，并转移450μL上清液至新的1.5毫升聚丙烯管中。 </li><li>使用上清液作为提取RNA的细胞裂解物。以下，按照制造商的说明在市售的试剂盒中描述的方法。 </li><li>洗脱使用RNA洗脱缓冲液的最小体积，以确保RNA的足够高的浓度用于监测低水平的转录物的表达模式。存放RNA的冰柜直到使用。 </li></ol> 2. RNA质量认证<ol><li>解冻总RNA，通过轻轻敲击混合，并放置在管中于冰上的铝架上。 </li><li>使用微量分光光度计测量RNA浓度，A260 / A280比率，和A260 / A230比值。 </li><li>稀释在无RNA酶的水中的总RNA至70毫微克/微升的最终浓度。 </li></ol> 3.从种子总RNA逆转录<ol><li>从商业试剂盒解冻总RNA和缓冲器集。保持酶从轻轻敲击后在冰上套件。制备无核酸0.2mL的聚丙烯管中。 </li><li>添加5μL的总RNA，1寡μL的dT引物（50μM），和1个随机的μM6聚体（50μM）的0.2毫升polypropy冰上lene的管。 </li><li>孵育在37ºC15分钟，并放置在冰上。 </li><li>以下，按照制造商的说明在逆转录PCR试剂盒中描述的方法。 </li><li>使用前置于冰上逆转录产品。 </li></ol> 4.实时定量PCR分析<ol><li>构建质粒携带使用制造商的协议的靶基因序列。 </li><li>调整浓度100-500,000拷贝/μL为标准曲线的DNA模板。 </li><li>稀释的cDNA溶液（1:100）用蒸馏水。 </li><li>添加稀释的cDNA溶液2μL，和用于标准曲线，从定量实时PCR试剂盒的主混合物的质粒。 </li><li> 95℃30秒，然后在95℃下40个循环进行5秒和60℃35秒:使用以下的循环条件成立实时PCR。 </li><li>根据人分析拷贝数ufacturer的指示为实时PCR系统。 </li></ol>

<ol>
	<li>Hills, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+storage-product+synthesis+in+seeds.">Control of storage-product synthesis in seeds.</a> Curr Opin Plant Biol. 7 (3), 302-308 (2004).</li><li>Li-Beisson, Y., 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=Acyl-lipid+metabolism.">Acyl-lipid metabolism.</a> Arabidopsis Book. 11, e0161 (2013).</li><li>Bates, P. D., Stymne, S., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+pathways+in+seed+oil+synthesis.">Biochemical pathways in seed oil synthesis.</a> Curr Opin Plant Biol. 16 (3), 358-364 (2013).</li><li>Santos-Mendoza, M., 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=Deciphering+gene+regulatory+networks+that+control+seed+development+and+maturation+in+Arabidopsis.">Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis.</a> Plant J. 54 (4), 608-620 (2008).</li><li>Durrett, T. P., Benning, C., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+triacylglycerols+as+feedstocks+for+the+production+of+biofuels.">Plant triacylglycerols as feedstocks for the production of biofuels.</a> Plant J. 54 (4), 593-607 (2008).</li><li>Kanai, M., 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=The+Plastidic+DEAD-box+RNA+helicase+22,+HS3,+is+essential+for+plastid+functions+both+in+seed+development+and+in+seedling+growth.">The Plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.</a> Plant Cell Physiol. 54 (9), 1431-1440 (2013).</li><li>Kanai, M., 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=Extension+of+oil+biosynthesis+during+the+mid-phase+of+seed+development+enhances+oil+content+in+Arabidopsis+seeds.">Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.</a> Plant Biotechnol J. 14 (5), 1241-1250 (2016).</li><li>Dekkers, B. J., 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=Identification+of+reference+genes+for+RT-qPCR+expression+analysis+in+Arabidopsis+and+tomato+seeds.">Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds.</a> Plant Cell Physiol. 53 (1), 28-37 (2012).</li><li>Salzman, R. A., 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=An+improved+RNA+isolation+method+for+plant+tissues+containing+high+levels+of+phenolic+compounds+or+carbohydrates.">An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates.</a> Plant Mol Biol Rep. 17 (1), 11-17 (1999).</li><li>Vicient, C. M., Delseny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+total+RNA+from+Arabidopsis+thaliana+seeds.">Isolation of total RNA from Arabidopsis thaliana seeds.</a> Anal Biochem. 268 (2), 412-413 (1999).</li><li>Wang, G. F., 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=Isolation+of+high+quality+RNA+from+cereal+seeds+containing+high+levels+of+starch.">Isolation of high quality RNA from cereal seeds containing high levels of starch.</a> Phytochem Analysis. 23 (2), 159-163 (2012).</li><li>Birtic, S., Kranner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-quality+RNA+from+polyphenol-,+polysaccharide-+and+lipid-rich+seeds.">Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds.</a> Phytochem Analysis. 17 (3), 144-148 (2006).</li></ol>

The authors have nothing to disclose.

基因表达谱有助于我们植物生理学的理解;因此，特定的RNA分离方法已经开发了用于每个样品的条件9-12。我们调查了被RNA分离过程中从种子抑制，发现RNA结合硅胶膜是严重抑制的过程。油，蛋白质和多酚的大量抑制RNA分离。我们修改了RNA提取过程以除去这些化合物与的RNA结合到二氧化硅膜上的处理前的裂解溶液。重要的修改包括添加1％PVP（步骤1.2）和从离心裂解液上清液的收集（步骤1.7，1.8）。在加入1％的PVP的目的是去除多酚。我们研究的PVP的最有效的浓度使用，并且确认了1％的PVP是最有效的浓度为这一过程。从离心裂解液收集上清的目的是除去油和蛋白质。上清液应仔细收集，以避免保留在上清液的油层，并在管的沉淀。这两个简单的修改大幅提高回收率，A260 / A280的比率，并且从中间的种子和发展的后期阶段（ 表1）分离的RNA的A260 / A230比值。 我们的方法中，其表示标准协议的一个简单的修改，使用了市售的试剂盒进行RNA分离。因此，我们的方法是用于分离从种子高度纯化的RNA，而不需要麻烦的额外步骤，诸如苯酚 - 氯仿提取和乙醇沉淀使用氯化锂实用。我们的方法也有效用于与成熟油菜籽和大豆种子（ 表2）使用，这表明修饰输油富含蛋白质的作物种子工作良好。来自4 DAF种子（ 表1）和青年根RNA的回收率S，叶和茎较低使用我们的方法，这表明我们的方法不适合于从在油和蛋白质差组织的RNA分离。因此，该方法仅用于RNA的从含有油，蛋白质和多酚，如种子和果实组织的隔离是有利的。 总的来说，我们的方法是合适的，用于获取从植物种子高度纯化的RNA，无需使用酚，氯仿或其他进程的用于RNA纯化。这种方法将有利于在籽基因表达的更精确的分析。

植物产生的种子，其产生下一代。种子积累大量存储储备，如油，碳水化合物和蛋白质，对于后发芽生长。人类利用种子贮藏储备食品和动物饲料的来源，因此，植物种子是可食用的有机物全球主要供应商之一。提高种子产量是植物科学的一个重要挑战。 因为种子贮藏储量食品和工业材料的商业价值的来源，这些储备的代谢的调节的分子机制已被广泛研究1-6。进一步阐明这些机制将用于提高农作物的种子产量是有用的。发展种子植物子房受精后，他们通过一系列的发展阶段1,6,7的成熟。进一步了解的分子机制基本种子发育需要详细，要生产由一系列发育中的种子的精确基因表达谱。然而，高含量的油，蛋白质，碳水化合物，和多酚类物质在植物的种子使其难以高度纯化的RNA，这排除基因表达的精确分析隔离。 在这里，我们介绍一种从含大量油脂，蛋白质和多酚的油籽RNA提取的有效方法。使用这种方法，研究人员将能够制备高度纯化的RNA。这种RNA将成为监测的关键基因控制种子贮藏储备的开发和成熟的油籽代谢调控转录变化非常有用。

<table><tbody><tr><td>RNeasy Plant Mini Kit</td><td>QIAGEN</td><td>74904</td><td></td></tr><tr><td>polyvinylpyrrolidone</td><td>Sigma-Aldrich</td><td>P5288-100G</td><td></td></tr><tr><td>HOMOGENIZER S-303</td><td>AS ONE</td><td>1-1133-02</td><td></td></tr><tr><td>NanoDrop Lite</td><td>Thermo Scientific</td><td>ND-NDL-US-CAN</td><td></td></tr><tr><td>PrimeScript RT reagent Kit (Perfect Real Time)</td><td>TAKARA</td><td>RR037A</td><td></td></tr><tr><td>KAPA SYBR Fast qPCR kit</td><td>Kapa biosystems</td><td>KK4601</td><td></td></tr></tbody></table>

an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

我们首先调查了使用拟南芥成熟种子的PVP的最佳浓度。总RNA根据上面使用含0％，0.25％，0.5％，1.0％或2.0％的PVP细胞裂解缓冲液中描述的协议从大约1,000种子中分离。均质化和离心后，同时避免了油层和种子碎片（ 图1A）收集上清液。 <img alt="图1" src="/files/ftp_upload/55008/55008fig1.jpg" /> 图1: 在细胞裂解缓冲液的最佳聚乙烯吡咯烷酮浓度的估计。总RNA用含有不同浓度的PVP的细胞裂解缓冲液提取。 （A）均匀化退火和离心后的种子的照片。 （B）的波长的吸光度的每个样品的曲线图表示。<html> <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 分离的RNA的数量和纯度从波长吸光度的曲线图（ 图1B），这表明，1.0％的PVP是从种子中分离大量的纯化的RNA的最有效的浓度估计。 发育中的种子从拟南芥水果立体显微镜（ 图2A，2B）根据隔绝在冰上预冷的铝板。将种子用不锈钢杵和电动研磨机中的管上的冰的铝机架（ 图2C，2D）匀浆。 2.JPG"/&gt; 图2:隔离和发展 拟南芥 种子的同质化 。开展种子从果实上的立体显微镜（A）在一个寒冷的铝板隔离。心皮从椎弓根（B）的剥离，发育中的种子被收集。种子被均质化使用在管的不锈钢杵（C）中在冰上冷却（D）一种铝机架含有1％（重量/体积）聚乙烯吡咯烷酮的裂解缓冲液。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 总RNA从种子使用我们的新开发的方法或常规方法开花（以下简称为DAF）之后在4,8和12天后分离出来。的浓度，A260 / A280比值，和A260 /从200种子中分离的RNA的A230比示于表1中 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>方法</td><td> DAF </td><td> RNA浓度</td><td> A260 / A280 </td><td> A260 / A230 </td></tr><tr><td rowspan="3">常规</td><td> 4 </td><td> 72.4±7.4 </td><td> 1.90±0.02 </td><td> 2.27±0.06 </td></tr><tr><td> 8 </td><td> 10.8±3.0 </td><td> 1.50±0.08 </td><td> 1.40±0.17 </td></tr><tr><td> 12 </td><td> 4.9±1.7 </td><td> 1.57±0.10 </td><td> 0.76±0.18 </td></tr><tr><td rowspan="3">新</td><td> 4 </td><td> 63.7±2.6 </td><td> 2.10±0.08 </td><td> 2.29±0.03 </td></tr><tr><td> 8 </td><td> 46.3±4.9 </td><td> 2.15±0.05 </td><td> 2.23±0.09 </td></tr><tr><td> 12 </td><td> 41.77; 3.1 </td><td> 2.09±0.04 </td><td> 2.17±0.07 </td></tr></tbody></table> 表1:来自 拟南芥 种子中 分离的RNA的浓度，A260 / A280比值，和A260 / A230比值 。总RNA从约200颗种子（从190到206的种子）4,8分离，并用传统的方法或我们的新开发的方法12的DAF。 RNA的浓度，A260 / A280比值，和A260 / A230的比率进行了测量。值代表三个独立实验的平均值±SD。 结果表明，我们的方法，使我们能够高度纯化的RNA，从图8和12的DAF种子隔离开。总RNA逆转录成cDNA，并将cDNA溶液稀释100倍。 WRI1的拷贝数（ 皱1:AT3G54320），SDP1（ 糖 DEPENDENT1:AT5G04040）7 &lt;/ SUP&gt;和EIF3K（真核翻译起始因子3K:AT4G33250）作为内部对照8进行测定，和转录物的相对量通过定量实时PCR分析（ 图3）估计的。 <img alt="图3" src="/files/ftp_upload/55008/55008fig3.jpg" /> 图3: 在发育中的种子低水平转录物的定量。总RNA是使用我们的方法从4，8和12的DAF种子中提取，并分别制备的cDNA溶液。 WRI1（A）和SDP1（B）的转录物的相对量通过定量实时PCR测定。 EIF3K用作内部对照。值代表平均值的三个独立实验的SD。 DAF;开花后几天。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig3large.jpg" target="_blank">请点击他</a>再查看该图的放大版本。 虽然WRI1和SDP1转录物的量是在发育中的种子比较低，这是能够检测在不同发育阶段的种子之间表达变化。为了验证我们的方法可以在其他油籽被利用，总RNA来自欧洲油菜和大豆的成熟种子中分离。 RNA的浓度，A260 / A280比值，和A260 / A230的比率示于表2中 。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>方法</td><td>厂</td><td> RNA浓度</td><td> A260 / A280 </td><td> A260 / A230 </td></tr><tr><td>常规</td><td>甘蓝型油菜</td><td> 7.7±0.8 </td><td> 1.5±0.12 </td><td> 0.75±0.11 </td></tr><tr><td></td><td> G.最大</td><td&gt; 9.7±2.3 </td><td> 1.61±0.06 </td><td> 0.69±0.09 </td></tr><tr><td>新</td><td>甘蓝型油菜</td><td> 143.2±19.0 </td><td> 2.17±0.03 </td><td> 2.23±0.13 </td></tr><tr><td></td><td> G.最大</td><td> 152.3±4.4 </td><td> 2.17±0.02 </td><td> 2.30±0.02 </td></tr></tbody></table> 表2:RNA浓度，A260 / A280比值，和A260 / A230比值从 甘蓝型油菜 和 大豆 的成熟种子中分离 。总RNA从约100毫克甘蓝型油菜和使用常规方法或我们的新开发的方法， 大豆种子分离。种子以前粉碎并用研钵和杵在液氮中冷冻大致均化。该RNA浓度，A260 / A280比，和A260 / A230的比率进行了测量。值代表三个独立实验的平均值±SD。

Watch this Scientific Journal Video about An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis at JoVE.com

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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Biochemistry

9.7K 次观看.  National Institute for Basic Biology. We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

视频: An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

Isolation of Cognate RNA-protein Complexes from Cells Using Oligonucleotide-directed Elution

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes.
The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.

This manuscript describes an approach to isolate select cognate RNPs formed in eukaryotic cells via a specific oligonucleotide-directed enrichment. We demonstrate the applicability of this approach by isolating a cognate RNP bound to the retroviral 5' untranslated region that is composed of DHX9/RNA helicase A.

从细胞使用寡核苷酸定向洗脱同源RNA-蛋白质复合物的隔离

Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. ...

科研

生物化学

An Oligonucleotide-based Tandem RNA Isolation Procedure to Recover Eukaryotic mRNA-Protein Complexes

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein complexes is essential to understanding mRNA regulation inferred by interacting proteins or non-coding RNAs. Herein, we describe a tandem RNA isolation procedure (TRIP) that enables the purification of endogenously formed mRNA-protein complexes from cellular extracts. The two-step protocol involves the isolation of polyadenylated mRNAs with antisense oligo(dT) beads and subsequent capture of an mRNA of interest with 3&#39;-biotinylated 2&#39;-O-methylated antisense RNA oligonucleotides, which can then be isolated with streptavidin beads. TRIP was used to recover in vivo crosslinked mRNA-ribonucleoprotein (mRNP) complexes from yeast, nematodes and human cells for further RNA and protein analysis. Thus, TRIP is a versatile approach that can be adapted to all types of polyadenylated RNAs across organisms to study the dynamic re-arrangement of mRNPs imposed by intracellular or environmental cues.

A tandem RNA isolation procedure (TRIP) for recovery of endogenously formed mRNA-protein complexes is described. Specifically, RNA-protein complexes are crosslinked in vivo, polyadenylated RNAs are isolated from extracts with oligo(dT) beads, and particular mRNAs are captured with modified RNA antisense oligonucleotides. Proteins bound to mRNAs are detected by immunoblot analysis.

基于寡核苷酸的串联 RNA 分离法恢复真核 mRNA-蛋白复合物

RNA-binding proteins (RBPs) play key roles in the post-transcriptional control of gene expression. Therefore, biochemical characterization of mRNA-protein ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

改进小RNA-seq:减少偏差，更好地检测2'-O-甲基RNA

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

遗传学

Isolation of High Purity Tissues from Developing Barley Seeds

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of cereal seeds is challenging owing to the minute size, the liquid character of some tissues, and the tight inter-tissue connections. Here, we demonstrate a detailed protocol for dissection of the embryo, endosperm, and seed maternal tissues at early, middle, and late stages of barley seed development. The protocol is based on a manual tissue dissection using fine-pointed tools and a binocular microscope, followed by ploidy analysis-based purity control. Seed maternal tissues and embryos are diploid, while the endosperm is triploid tissue. This allows the monitoring of sample purity using flow cytometry. Additional measurements revealed the high quality of RNA isolated from such samples and their usability for high-sensitivity analysis. In conclusion, this protocol describes how to practically dissect pure tissues from developing grains of cultivated barley and potentially also other cereals.

Here we present a protocol for high purity manual isolation and quality control of embryo, endosperm and seed maternal tissues during entire barley seed development.

Understanding the mechanisms regulating the development of cereal seeds is essential for plant breeding and increasing yield. However, the analysis of ...

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.

通过基因表达分析的改良方法从谷物种子获取高质量的转录数据

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

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high concentrations of phenolic compounds and polysaccharides that interfere with RNA purification. Isolation of high-quality RNA from these species requires rigorous tissue collection procedures in the field and the employment of an RNA isolation protocol comprised of multiple organic extraction steps in order to isolate RNA of sufficient quality for microarray and other genomic analyses. The isolation of high-quality RNA from field-collected loblolly pine samples can be challenging, but several modifications to standard tissue and RNA isolation procedures greatly improve results. The extent of general RNA degradation increases if samples are not properly collected and transported from the field, especially during large-scale harvests. Total RNA yields can be increased significantly by pulverizing samples in a liquid nitrogen freezer mill prior to RNA isolation, especially when samples come from woody tissues. This is primarily due to the presence of oxidizing agents, such as phenolic compounds, and polysaccharides that are both present at high levels in extracts from the woody tissues of most conifer species. If not removed, these contaminants can carry over leading to problems, such as RNA degradation, that result in low yields and a poor quality RNA sample. Carryover of phenolic compounds, as well as polysaccharides, can also reduce or even completely eliminate the activity of reverse transcriptase or other polymerases commonly used for cDNA synthesis. In particular, RNA destined to be used as template for double-stranded cDNA synthesis in the generation of cDNA libraries, single-stranded cDNA synthesis for PCR or qPCR's, or for the synthesis of microarray target materials must be of the highest quality if researchers expect to obtain optimal results. RNA isolation techniques commonly employed for many other plant species are often insufficient in their ability to remove these contaminants from conifer samples and thus do not yield total RNA samples suitable for downstream manipulations. In this video we demonstrate methods for field collection of conifer tissues, beginning with the felling of a forty year-old tree, to the harvesting of phloem, secondary xylem, and reaction wood xylem. We also demonstrate an RNA isolation protocol that has consistently yielded high-quality RNA for subsequent enzymatic manipulations.

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Many plant tissues, including phloem and xylem from loblolly pine (Pinus taeda L.), contain high levels of phenolics and polysaccharides that interfere with RNA purification. This presentation discusses techniques for the harvest of field-grown tissues and isolation of RNA of sufficient quality for microarrays and other genomic analyses.

的RNA分离方法的改进火炬松（ P。火炬松 L）和其他针叶树种

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

生物学

Polysome Purification from Soybean Symbiotic Nodules

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper describes methods optimized to isolate plant-derived polyribosomes and their associated mRNAs to be analyzed using RNA-sequencing. First, cytoplasmic lysates are obtained through homogenization in polysome- and RNA-preserving conditions from whole, frozen soybean nodules. Then, lysates are cleared by low-speed centrifugation, and 15% of the supernatant is used for total RNA (TOTAL) isolation. The remaining cleared lysate is used to isolate polysomes by ultracentrifugation through a two-layer sucrose cushion (12% and 33.5%). Polysome-associated mRNA (PAR) is purified from polysomal pellets after resuspension. Both TOTAL and PAR are evaluated by highly sensitive capillary electrophoresis to meet the quality standards of sequencing libraries for RNA-seq. As an example of a downstream application, after sequencing, standard pipelines for gene expression analysis can be used to obtain differentially expressed genes at the transcriptome and translatome levels. In summary, this method, in combination with RNA-seq, allows the study of the translational regulation of eukaryotic mRNAs in a complex tissue such as the symbiotic nodule.

This protocol describes a method for eukaryotic polysome purification from intact soybean nodules. After sequencing, standard pipelines for gene expression analysis can be used to identify differentially expressed genes at the transcriptome and translatome levels.

大豆共生结节多聚体纯化

The aim of this protocol is to provide a strategy for studying the eukaryotic translatome of the soybean (Glycine max) symbiotic nodule. This paper ...

发育生物学

Metabolic Labeling of Newly Transcribed RNA for High Resolution Gene Expression Profiling of RNA Synthesis, Processing and Decay in Cell Culture

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene expression. Along with a better understanding of the involved molecular mechanisms, precise measurements of the underlying kinetics have become increasingly important. Here, these powerful methodologies face major limitations due to intrinsic properties of the template samples they study, i.e. total cellular RNA. In many cases changes in total cellular RNA occur either too slowly or too quickly to represent the underlying molecular events and their kinetics with sufficient resolution. In addition, the contribution of alterations in RNA synthesis, processing, and decay are not readily differentiated.
We recently developed high-resolution gene expression profiling to overcome these limitations. Our approach is based on metabolic labeling of newly transcribed RNA with 4-thiouridine (thus also referred to as 4sU-tagging) followed by rigorous purification of newly transcribed RNA using thiol-specific biotinylation and streptavidin-coated magnetic beads. It is applicable to a broad range of organisms including vertebrates, Drosophila, and yeast. We successfully applied 4sU-tagging to study real-time kinetics of transcription factor activities, provide precise measurements of RNA half-lives, and obtain novel insights into the kinetics of RNA processing. Finally, computational modeling can be employed to generate an integrated, comprehensive analysis of the underlying molecular mechanisms.

Total cellular RNA provides a poor template for studying short-term changes in RNA synthesis and decay as well as the kinetics of RNA processing. Here, we describe metabolic labeling of newly transcribed RNA with 4-thiouridine followed by thiol-specific biotinylation and purification of newly transcribed RNA allowing to overcome these limitations.

高分辨率基因表达谱RNA的合成，加工和衰变的新转录RNA在细胞培养的代谢标记

The development of whole-transcriptome microarrays and next-generation sequencing has revolutionized our understanding of the complexity of cellular gene ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

高效，快速的早期胚胎分离<em&gt;拟南芥</em&gt;种子

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

对于高度纯化RNA的种子定量转录组分析的隔离中使用的有效方法

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