Name: simultaneous dna-rna extraction from coastal sediments and quantification of 16s rrna genes and transcripts by real-time pcr
Uploaded: 2016-06-11T14:00:00.000+00:00
Duration: 11 min 37 s
Description: Watch this Scientific Journal Video about Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR at JoVE.com

本出版物已经从与自然环境研究委员会（NERC）的资助下，授权号NERC NE / JO11959 / 1和爱尔兰科学基金会及以下授权号11 / SIRG / B2159awarded的玛丽 - 居里行动COFUND到CJS进行的研究散发出和东方学术研究联合会（ARC东部）。

1.海洋海岸沉积物DNA和RNA提取<ol><li>核糖核酸（RNA酶） - 免费的材料和工作区的制备<ol><li>通过用0.1％焦碳酸二乙酯（DEPC）处理制备无RNA酶的蒸馏水（DH 2 O），在37℃CO / N孵育。通过在121℃高压灭菌的dh 2 O 15分钟，清除残留的DEPC。使用DEPC处理的dh 2 O，使所有的解决方案。 </li><li>烘烤在180°CO / N的所有玻璃器皿。取下塑料盖和轮辋，在2 M氢氧化钠O / N一溶液浸泡它们，并在使用前处理的dh 2 O DEPC冲洗。 </li><li>制备的CTAB-磷酸盐缓冲溶液按表1中 。 </li><li>制备的PEG-NaCl的沉淀溶液按表2。 </li><li>清理所有的工作表面（ 即台，离心，通风橱），并使用启动前RNA酶净化解决方案微量。在整个过程中，应戴上手套。 </li&gt; <li>使用无RNase的塑料制品用微量过滤嘴沿避免样品交叉污染。 </li></ol></li><li>环境样品采集和存储<ol><li>使用无菌50毫升猎鹰管从顶部2.5厘米收集退潮时海岸泥沙。收集约15-20摹沉积物。样品采集后小心地盖上盖子。立即传输到实验室中，在4℃的冷却器以便立即处理。 </li><li>通过用无菌刮铲和等分0.5克湿重沉积物混合到2ml珠打浆管样品均质化。等分试样额外重复，闪冻在液氮放置。商店等分在-80℃。注意:处理液氮时要戴上防护手套和护目镜。 注意:如果在野外现场不靠近实验室，等分0.5克泥沙分为2毫升无菌离心管在外地的网站。通过将它们立即投入到李闪存冻结管镑氮和运送至实验室。样品储存在-80°C，直到处理。 </li></ol></li><li> DNA的共同提取和RNA沉积物 注意:以下协议是格里菲思方法6的修改版本。 <ol><li>加入500μl的CTAB-磷酸盐缓冲液和500微升酚:氯仿:异戊醇（25:24:1），以含有0.5g沉淀物的2 mL萃取珠裂解管并倒转试管5-10倍，以均质化试样。 注意:执行在通风罩在任何时候都穿着适当的防护装备（ 如实验室外套，手套和护目镜）这一步。 </li><li>涡全速2.5分钟。离心机以16,000 xg离心在4℃下10分钟。 </li><li>在一个通风橱中提取的顶部的水层，并转移到一个新的1.5毫升无菌离心管中。保持在冰上的样品中，加入500μl冰冷的氯仿:异戊醇（24:1）。 </li><li>倒置几次直到乳液是visib勒。离心机在4℃下以16,000 xg离心10分钟。 </li><li>在一个通风橱中提取的顶部的水层，并将其放置在一个新的1.5毫升无菌试管中。加入30％的PEG-NaCl溶液两卷沉淀核酸拌匀。 </li><li>在冰上孵育2小时或在4℃CO / N离开样品。 </li><li>在孵育离心机样品以16,000×g离心20分钟结束时，在4℃。 注:沉淀可在管的底部是可见的。 </li><li>轻轻地取出用微量上清，确保不打扰颗粒。离开大约10微升PEG溶液在管和加入1毫升冰冷的70％乙醇。 </li><li>离心16,000 xg离心在4℃下20分钟。慢慢微量小心不要碰到沉淀除去乙醇。 </li><li>离心5秒，并使用微量吸管除去残余的乙醇。离开沉淀空气干燥约5分钟。 </li><li>重悬在50粒料微升DEPC处理过的H 2 O审查通过在1％琼脂糖凝胶19运行5微升琼脂糖凝胶电泳的质量和DNA / RNA的产率。划分核酸分成两等份，15微升为DNA和30微升用于RNA。 </li><li>直到需要在-80存储该DNA℃。分离RNA在下一节中介绍。 </li></ol></li></ol> 2.游离DNA-RNA的RNA和质量检查的制备<ol><li> DNA消化<ol><li>添加DNA酶I缓冲液3微升和1.5微升DNA酶Ⅰ至核酸30微升体积，孵育在37℃下30分钟。 </li><li>通过涡旋30秒混合DNA酶失活试剂。添加4.8微升溶液于样品。孵育在室温5分钟，偶尔挖掘管底部到该溶液中混合。 </li><li>离心在10,000rpm xg离心90秒在RT，将上清液（RNA）转移到新的管中，小心不要转移能够抑制下游反应沉淀物。 </LI&gt; </ol></li><li> RNA质量检查<ol><li>加入2微升RNA至18微升无菌无RNA酶-DH 2 O的稀释1:10。 <ol><li>在单独的PCR反应，加入1微升RNA整齐或1:10的稀释无菌0.5毫升PCR管。添加PCR反应组分以扩增16S rRNA基因按表3。包含一个"正"（ 例如 ，从一个纯的细菌培养物中提取的DNA）中，"PCR抑制剂"控制（ 即 ，1微升阳性纯培养物DNA的和整齐提取的RNA 1微升）和无菌无RNA酶-DH 2 O.的"负"控制</li></ol></li><li>设置进行下述扩增参数热循环:95℃5分钟，（95℃30秒，57℃30秒，72℃1分钟）×35个循环，72℃10分钟。 </li><li>在85 V代表40分钟运行在1％琼脂糖凝胶对PCR产物的10％。重复DNA酶I处理，如果在环境RN形成PCR产物一个样本。 </li></ol></li></ol>由RNA第一链cDNA的3代<ol><li>按表4添加试剂RNA酶/ DNA酶的免费0.2毫升PCR管。 </li><li>孵育在65℃5分钟的样品。在冰上放置至少1分钟，然后在25℃孵育10分钟。 </li><li>到相同的管中添加表5中列出的试剂。 </li><li>孵育在55℃的样品50分钟。灭活在72℃下进行10分钟的反应。 </li><li>在-20°C，直到使用储存的cDNA。 </li></ol> 4.定量PCR <ol><li> DNA的标准产生的qPCR <ol><li>扩增来自从纯培养用Q-PCR引物（1369F＆1492R 21）提取的基因组DNA的细菌的16S rRNA序列20（或任何其他感兴趣的序列）。 </li><li>纯化使用根据制造商的说明商业试剂盒的PCR产物。 </li><li>克隆纯化的PCR产物成3&#39;-T突出端根据制造商的说明书克隆载体系统。 </li><li>变换含有PCR根据制造商的说明插入到大肠杆菌 JM109感受态细胞的载体。 </li><li>板变换的50微升到氨苄青霉素（100微克/毫升），X-gal的（20微克/毫升）和IPTG（0.5毫摩尔）的Luria BERTANI琼脂板（胰蛋白胨10克/升，酵母提取物5g / L的，氯化钠10克/ L，琼脂15g / L）。孵育O / N在37℃。 </li><li>选择从板单个正白色转化体。接种阳性转化体在含有100μg/ ml氨苄青霉素50毫升LB肉汤和孵育O / N以250rpm振荡在37℃。 </li><li>从O / N培养，分离和使用根据制造商的说明商业试剂盒纯化质粒。 </li><li>线性用适当的限制性内切酶，其能够切割质粒一次（ 例如 ，EcoRI）消化的质粒。 </li><li<html> &gt;通过测量吸收比A260 / 280 19分析质粒的纯度。如果该比例低于1.8，再提取用苯酚-氯仿19的质粒。注:纯DNA有一个1.8的比例。 </li><li>确定在使用分光光度计19 260nm处吸光度的质粒DNA的浓度。 注意:可替换地，量化的精度可通过使用配合使用荧光​​计的荧光DNA结合染料进行改进。 </li><li>序列的质粒插入到确认目标的碱基的大小。计算的量在质粒插入DNA（ 即 ，百分比）（以一个3000 bp的载体例如 ，一个123 bp的DNA片段是总DNA的3.9％），并通过在4.1.10中得到的浓度相乘，以确定目标（插入）的DNA浓度。 </li><li>使用下面的公式来计算每微升目标的副本: 负载/ 54067 / 54067eq1.jpg"/&gt; </li><li>由平均分子量双链DNA的（双链DNA，每个碱基对660道尔顿）的靶DNA的碱基对的数量乘以计算目标分子量（TMW）。 </li><li>在稀释的范围内的目标DNA利用DNA的2微升和18微升无菌卫生署2 O的10月10日至2月10日份混合每个稀释度良好，在进行下一步之前，稀释后短暂离心。 </li></ol></li><li>对于RNA定量PCR标准曲线的产生<ol><li>从步骤4.1.5，筛选菌落PCR 19多家白色菌落（大约5）使用目标反向引物（ 即 R1492），并正向载体质粒引物M13F。 注意:引物的结合将扩增与T7启动子位点上游的正确义方向克隆的插入物。 </li><li>纯化使用依照制造商的指令的市售试剂盒的PCR产物。量化纯化的PCR pRODUCT在使用分光光度计260纳米。 </li><li>添加200纳克PCR产物中的20微升每核糖核苷酸，1 T7反应缓冲液和1 T7聚合酶酶用于体外转录反应的7.5毫的最终体积。 </li><li>孵育在37℃进行4小时的反应。我添加1个单位不含RNA酶的DNA酶的反应，在37℃下孵育15分钟以除去模板DNA。 </li><li>恢复通过乙醇沉淀19的体外转录的RNA和使用荧光或通过260nm处的吸光度定量RNA产量。 </li><li>计算每个RNA的转录微升数量。添加的碱基对的数目在靶RNA（ 例如 ，123个碱基）的T7启动子（153个碱基）的长度和由平均分子量RNA的，每基座340道尔顿繁殖。 </li><li> 500ng的量化的靶标RNA的添加逆转录酶（RT）反应在部分3串行稀释的cDNA概述如在4.1.13概述了使用作为标准曲线。 </li></ol></li><li>实时荧光定量PCR <ol><li>解冻环境的DNA / cDNA样品，标准和在冰上的qPCR试剂。避光的荧光探针。 </li><li>稀释的标准曲线的标准在4.1.14中概述。使环境的DNA的1:10稀释液/加入2微升样品，以18微升无菌卫生署2 O的cDNA的整齐〜10 -3 </li><li>作出反应的总数加上10％按表6（引物和探针）和表7（反应混合物）一个主混合物反应。充分混匀，短暂离心。 </li><li>加主混合物的19微升和1μl模板（标准，环境样品或水为阴性对照）在各孔中96孔光学定量PCR板。执行一式三份各反应（标准，环境样品和阴性对照）。 </li><li>覆盖用Q-PCR光学盖96孔板中。短暂离心板。加载板进入定量PCR机的加热块，并盖上盖子。 </li><li>打开定量PCR管理器软件。点击"协议选项卡"，点击"新建"，添加以下扩增条件:3分钟95℃（10秒95℃，30秒60℃）×40个循环。设置样本量为20微升，点击"确定"并保存协议。 </li><li>点击"板"标签。在"编辑选择"点击"选择荧光基团"中，增加报告染料合适的探头， 例如 ，荧光素。点击"确定"。 </li><li>突出显示包含标准的井，从"样品类型"菜单中选择"标准"。点击"复制系列"改变"大小复制"到3，点击"应用"。点击"稀释系列"，增加了对标准1计算起始浓度，表明加入标准曲线的稀释倍数（10），选择"减少"或根据订单"增加"到板上。点击"应用"。 </li><li>突出以及含有未知样品的位置，从"样品类型"中选择"未知"的下拉菜单。点击"复制系列"，变"复制大小"来3.单击"应用"。编辑在"样品名称"样品名称。对于无模板对照（NTC）重复井过程。 </li><li>点击编辑板中的"确定"按钮，保存文件。点击"开始→运行"。 </li><li>在运行完成后，数据分析窗口将打开。点击"量化"标签，显示标准曲线，标准曲线描述符（ 表8）和扩增曲线。 </li><li>点击的Cp值"定量数据"选项卡上，并相应启动未知样品的基因（SQ）的数量。如果需要导出表电子表格软件。 </li><li>从未知样品乘法基因的丰度通过稀释因子，沉积物为Extrac的量泰德从与总体积的核酸洗脱到每克湿重的沉积物获得基因拷贝。 注意:洗脱的DNA为0.5g沉积物在50μl，稀释1/10，加入1微升作为模板的q PCR反应;基因拷贝微升DNA×稀释系数x洗脱体积×2的:由相乘计算基因拷贝摹-1沉淀物。 </li></ol></li></ol>

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The authors have nothing to disclose.

DNA / RNA提取用定量PCR的组合提供了一种快速，准确，比较合算的基因和转录物丰度的从一系列环境样品，如海岸沉积物的敏感定量方法。 初始核酸提取是确保微生物群落本的代表性视图达到22的关键步骤。需要被考虑为提取协议的一些限制:一）总细胞裂解的成就，二）的抑制性化合物（ 例如 ，腐殖酸或多酚的共萃取），c）在该RNA部分污染DNA，四）储存时的提取核酸的快速降解。必须采取预防措施，以规避这些限制。例如，特别注意，必须注意确保核酸提取的样品类型（ 例如 ，沉淀物，土壤，废水等优化）。在核酸的产量和质量为DNA和RNA显著改进可以通过进行初步实验23来实现。为了最大限度地减少抑制化合物的对基于PCR的应用程序24中的效果，测试的范围内所提取的DNA和RNA稀释液。规避从多个冻融提取的DNA / RNA的快速降解，避免在-80℃的遗传信息可能丢失，存储多个小体积等分试样。 当精心设计，定量PCR 是一个强大的，高度重复性和敏感的方法。值得注意的是，在此协议中概述用于扩增和标准曲线结构的方法可以适用于任何感兴趣的基因靶标，包括其他的系统发育标记物（ 即 ，古细菌的16S rRNA，真菌18S rRNA基因）或涉及在环境中的重要功能的基因。在利用定量PCR的已知限制是:a）可重放高质量的S中的代为绝对定量TANDARD曲线，二）引物/探针的选择的Q-PCR测定条件的优化，c）使用低质量/剪切核酸，四）的DNA / RNA的工作稀释度的选择，以避免抑制。此外，它必须被认为是定量PCR技术提供了基因/转录丰度可能不等同于细胞计数:这是特别当16S和18S rRNA基因被定位的情况下，如微生物在它们的基因组具有核糖体基因的不同拷贝数25。 质量差的标准曲线将导致感兴趣的基因的不准确的定量。它是存储高浓度标准小等份从新鲜标准曲线可以由库存好的做法。不要储存标准曲线，总是从每一次最集中的股票做出新的稀释液。环境样品的准确定量保证斯坦的浓度范围准曲线跨越未知样品的预期的Cp值。当量化成绩单，构建从RNA不双链DNA的标准曲线。当可能时，样品可以直接进行比较的量化应单一测定内完成，以避免测定间变异20。这可能不总是可能的。因此，为了比较测定之间产生基因的丰度，最好是具有随机子集测定之间复制样品。的引物和探针集的多种选择目前可用于定量PCR针对微生物类群和官能团选择这些的时候，以确保这两个最大的覆盖面和特异性的目标群体是需要15.仔细考虑。如果一个qPCR分析的反应效率不令人满意的，解决始于测试不同的热循环条件（如退火时间和/或温度）和/或反应条件， 例如 ，不同的引物-探针的浓度。 ØNCE的Q-PCR测定法和反应条件进行了优化，始终进行一系列的DNA / cDNA的稀释物的初始测试以确定适当的模板浓度。选择产生最高拷贝数作为最优模板稀释用于进一步分析的稀释范围。 目前，新一代测序技术，有效地环境26,27,28过多阐明微生物群落结构和功能的光。然而，这些数据集通常是基于终点PCR扩增子库，因此只提供丰特定类群的半定量评估。因此，实时基于PCR的技术的能力，以针对特定分类标记（从高域到应变水平）允许通过新一代测序得到的结果的有效验证。此外，定量PCR已被成功地用于与其它微生物生态学分子的方法，例如稳定异TOPE探测（SIP）或亲缘/功能芯片。与前者的工具相结合，定量PCR可用于定量代谢活性社区29,30。当与微阵列分析相结合，定量PCR提供环境31,32进化标记为基础的功能基因调查的关键定量解释。 因此，无论是单独使用或与生态系统功能的其他（通常基于过程的）评估相结合，定量PCR是在微生物群落和生态系统功能之间的联系难以捉摸的探索微生物生态学家的重要工具。

微生物是生物圈的驾驶生态系统功能的基石。大多数微生物仍然未培养1。因此，基于分子的方法是从根本上促进我们的环境中微生物的多样性和功能的理解。中央这些方法是核酸从环境样品提取和使用聚合酶链反应（PCR）的靶基因的随后的扩增。 的DNA / RNA提取的第一步骤的目的是裂解微生物群落本的细胞壁，除去不需要的非核酸分子（ 例如 ，有机和无机物质），并在进一步的下游分析溶液保留的DNA / RNA。在文献2,3,4,5提供多种选择，包括各种商业提取试剂盒后，格里菲斯方法6，广泛采用7,8,9。它是具有成本效益和巴rticularly非常适合于沉积物，因为它使用珠跳动步骤以裂解细胞，并结合步骤以最小化PCR抑制剂，如腐殖酸这样的共同提取，而同时回收DNA和RNA。 第二步骤利用聚合酶链式反应（PCR）扩增靶基因，如16S rRNA的分类标记，从所提取的核酸。这种方法有，并继续推动未鉴定微生物黑匣子10,11的探索。然而，终点，基于PCR的方法从各种限制的影响，可以偏置微生物群落12的表征。准确量化基因/转录丰度，实时PCR，也称为定量PCR（qPCR的）必须被使用。定量PCR利用了PCR的每个循环之后跟踪扩增子积累荧光报道染料系统。这是显著因为它意味着可以在早期指数阶段发生的量化，而比终点，将PCR反应的阶段中，当扩增子产量仍然是成正比的靶基因的初始丰盈。 两个报告系统是常用的:一个嵌入核酸染色13和DNA聚合酶14的5&#39;3&#39;外切酶活性。因为前者报告系统不显结合到所有双链DNA，它可能会导致与靶序列的过高估计，如果不想要的非特异性扩增或通过产物引物二聚体发生。为了规避这一点，可能需要扩增的广泛的优化。在后者的系统中，模板的扩增是使用Taq聚合酶劈开从内部探针的荧光团的5&#39;核酸酶活性的组合跟踪。此特征增加了测定的特异性由于荧光探针的利用，只有结合于互补的靶特异性sequenc引物对两地电子。与两个化学定量是通过确定交叉点（CP），其中的PCR扩增子作为通过增加荧光测量的积累，是显著高于背景荧光来实现。 的qPCR已在微生物生态学被广泛用来确定在不同的环境中15基因的丰度。此外，为cDNA RNA的逆转录用的qPCR和RT-qPCR的组合来量化基因的表达。因此，定量PCR和RT-qPCR的代表基因和/或转录的数字环境样品中的量化快速，有效的方法。 在海岸沉积物微生物驱动各种生态系统过程，包括有机物矿化，污染物的降解和大量营养素如氮16,17,18的生物地球化学循环。这些转换的详尽的了解需要一个全面有分起作用的微生物种群，包括基因及成绩单丰度的定量数据吨。在这里，我们推出一系列全面测试，简化和标准化的协议细菌16S rRNA基因及成绩单丰度在海岸沉积物进行量化。的协议概括了样品的采集，同时DNA和RNA提取，不含DNA的RNA制备的提取的核酸的质量检查，生成的16S rRNA-DNA和-RNA标准和环境样品的定量。从这里介绍的方法得出量化的数据需要微生物群落驾驶沿海生态系统揭示。

<table><tbody><tr><td>Diethylpyrocarbonate</td><td>Sigma</td><td>40718</td><td>Toxic, open under chemical hood</td></tr><tr><td>cetrimonium bromide (CTAB)</td><td>Sigma</td><td>52365</td><td>Irritant, open under chemical hood</td></tr><tr><td>potassium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>RES20765</td><td></td></tr><tr><td>potassium phosphate monobasic&amp;nbsp;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Phenol : Chloroform : Isoamylalcohol pH 8</td><td>Sigma</td><td>P2069</td><td>Equilibrate at pH 8 before using</td></tr><tr><td>Chloroform : Isoamylalcohol</td><td>Sigma</td><td>25666</td><td></td></tr><tr><td>sodium chloride</td><td>VWR</td><td>1.06404.0500</td><td></td></tr><tr><td>polyethylenglycol 6000&amp;nbsp;</td><td>VWR</td><td>528877-100</td><td></td></tr><tr><td>Ethanol Molecular Grade</td><td>Sigma</td><td>E7148</td><td></td></tr><tr><td>Lysing Matrix E tubes</td><td>MP Biomedical</td><td>116914050&amp;nbsp;</td><td></td></tr><tr><td>Turbo DNAse&amp;nbsp;</td><td>Ambion</td><td>AM1907</td><td></td></tr><tr><td>Taq polymerase</td><td>Sigma</td><td>D1806</td><td></td></tr><tr><td>dNTPs</td><td>Bioline</td><td>BIO-39028</td><td></td></tr><tr><td>SuperScript III</td><td>Life Technologies</td><td>18080044</td><td></td></tr><tr><td>Rnase Inhibitor</td><td>Life Technologies</td><td>10777019</td><td></td></tr><tr><td>RNAse/DNAse free 0.2&amp;nbsp;ml PCR tubes</td><td>Sarstedt</td><td>72.985.002</td><td></td></tr><tr><td>SureCleanPlus</td><td>Bioline</td><td>BIO-37047</td><td></td></tr><tr><td>pGEM Easy T Vector</td><td>Promega</td><td>A1360</td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; JM109 competent cells</td><td>Promega</td><td>L2005</td><td></td></tr><tr><td>Tryptone</td><td>Sigma</td><td>T7293</td><td></td></tr><tr><td>Yeast Extract</td><td>Sigma</td><td>70161</td><td></td></tr><tr><td>Ampicillin</td><td>Sigma</td><td>59349</td><td></td></tr><tr><td>X-Gal</td><td>Bioline</td><td>BIO-37035</td><td></td></tr><tr><td>IPTG</td><td>Bioline</td><td>BIO-37036</td><td></td></tr><tr><td>Agar</td><td>VWR</td><td>20768.235</td><td></td></tr><tr><td>Plasmid Midi Kit</td><td>Qiagen</td><td>12143</td><td></td></tr><tr><td>Qubit</td><td>Life Technologies</td><td>Q33216</td><td></td></tr><tr><td>Quant-IT DNA HS Assay</td><td>Life Technologies</td><td>Q-33120</td><td></td></tr><tr><td>Quant-IT RNA HS Assay</td><td>Life Technologies</td><td>Q32855</td><td></td></tr><tr><td>MEGAshortscript kit</td><td>Ambion</td><td>AM1354</td><td></td></tr><tr><td>TaqMan SensiFast Probe Lo-ROX kit</td><td>Bioline</td><td>BIO-84002</td><td></td></tr><tr><td>qPCR machine</td><td></td><td></td><td></td></tr></tbody></table>

simultaneous dna-rna extraction from coastal sediments and quantification of 16s rrna genes and transcripts by real-time pcr

实时聚合酶链反应也称为定量PCR（Q-PCR）是在微生物生态学一种广泛使用的工具来量化环境样品中的分类和官能团的基因的丰度。组合使用逆转录酶反应（RT-Q-PCR），它也可以被用于量化基因转录。 Q-PCR利用高度敏感的荧光检测化学，允许在反应指数阶段的PCR扩增子的定量。因此，在PCR反应的平台期检测与&#39;终点&#39;的PCR关联的偏差被避免。的协议通过提供PCR实时定量沿海沉积物中的细菌16S rRNA基因和成绩单。首先，用于从海岸沉积物，包括不含DNA的RNA的制备所需的附加步骤的DNA和RNA的共提取的方法，概述。二，一步一步的指导，为16S rRNA基因和TR量化从通过Q-PCR和RT-Q-PCR提取核酸anscripts概述。这包括DNA和RNA标准曲线的构造细节。在微生物生态学采用RT-Q-PCR检测的主要考虑因素包括在内。

质量好DNA和RNA从沉积物中提取是定量基因和转录物丰度的第一步。一个成功的提取率， 如图1样品交流，其中23S锐和的16S rRNA乐队是除了高分子量的基因组DNA带可视指示清晰的DNA和RNA带。 <img alt="图1" src="/files/ftp_upload/54067/54067fig1highres.jpg" width="400" /> 图1. DNA / RNA提取。从DNA / RNA提取的典型结果从一式三份（ABC）将0.5g沿岸沉积物。 5微升的DNA / RNA的混合物在1.4％琼脂糖，在85 V代表40分钟运行。用在10,037-200基点范围内的分子标记。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54067/54067fig1highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p class="jove_content" fo:keep-together.within页="1"&gt;要准备RNA，共提取的DNA的消化是强制性的。这必须遵循由RNA的16S rRNA的终点PCR，以确保所述DNA已被成功去除。如果DNA已被完全除去仅在阳性对照的频带被观察到。它是同时使用整齐和1:10稀释的RNA，以确保抑制剂不阻止PCR产物的形成非常重要。 RNA现在可以进行所述逆转录酶反应以将其转化成cDNA。这可与基因特异性引物或随机六聚体进行。典型地，该反应在PCR机器中进行，以确保最佳的温度分布。该cDNA被用作在随后的qPCR反应模板。 DNA和RNA进行定量，以确定在每个反应中使用的模板浓度。 定量PCR反应协议概述针对的16S rRNA的DNA。对于的16S rRNA成绩单替代标准立方米RVE和模板的cDNA。由于未知样品与标准曲线量化，当务之急是确保标准曲线的质量是好的。 图2显示了（A），标准曲线和环境DNA稀释液，（B），标准曲线的扩增准备标准曲线和环境样品（C）的转化率，以基因拷贝数的线性回归和计算。当10倍稀释的范围是正确制备和扩增各标准稀释两者3.3循环差出现时（需要3.3个循环在100％扩增效率在模板十倍增加）（ 图2BI）。标准曲线的描述符，包括0.99的90至110％（ 图2C）的范围内，R 2的值和PCR效率是期望的。它从没有模板对照（NTC）如本报道的Cp值是重要的。如果发生这种情况的标准和未知样品3.3个周期（Cp为截止&lt;EM&gt;即一个日志倍），比NTC的CP值强加20（ 图2Cii）高。从沉积物中提取DNA模板未知从整齐的10 -1，10 -2和10 -3稀释液（B）量化。整洁的样品没有放大，10 -1至10 -3稀释度的Cp值分别为24.12，26.02和28.40。该NTC Cp是30.5，27.2的NCT截止被罚款。转换基因的丰度至g -1湿重沉淀导致2.5×10 7和7.1×10 7 10 -1和10 -2分别为稀释范围。 10 -3稀释的Cp是NTC截止以下，并且因此不被使用。在这种情况下，10 -2被选为最优模板稀释。 <img alt="图2" src="/files/ftp_upload/54067/54067fig2highres.jpg" width="700" /> 图2. 16S rRNA基因QPC标准- [R放大和沿海沉积物中提取DNA环境。 （A）ⅰ）的DNA的标准曲线的制备和ii）环境的DNA稀释液定量PCR扩增，（B）中的i定量PCR扩增）的DNA的标准曲线和ii）环境样品中，Cp为各样品被示出。 （ 三 ） 我 ）与标准曲线描述标准曲线的线性回归; 二 ）从环境样品中基因的丰度的计算。 NTC:负模板控件<a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54067/54067fig2highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> CTAB </td><td> 5克</td></tr><tr><td> 磷酸氢二钾X3H 2 O </td><td> 2.58克</td></tr><tr><td> KH 2 PO 4 </td><td> 0.1克</td></tr><tr><td>氯化钠</td><td> 2.05克</td></tr><tr><td>卫生署2 O </td><td>至100ml </td></tr></tbody></table> 表1制备的CTAB-磷酸盐缓冲液中。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> PEG 6000 </td><td> 30克</td></tr><tr><td>氯化钠</td><td> 9.35克</td></tr><tr><td>卫生署2 O </td><td>至100ml </td></tr><tr><td colspan="2">注意:添加PEG 6000下慢慢适度加热和为50毫升卫生署2 O的搅拌</td></tr></tbody></table>表2的PEG-NaCl的沉淀溶液的制备。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td>未稀释的RNA / 1:10稀释RNA </td><td> 1.0微升</td></tr><tr><td> 10X PCR缓冲液</td><td> 5.0微升</td></tr><tr><td>的dNTP 10毫</td><td> 1.0微升</td></tr><tr><td> 63F正向引物</td><td> 1.0微升</td></tr><tr><td> CAG GCC TAA CAC ATG CAA GTC </td><td></td></tr><tr><td> 518R反向引物</td><td> 1.0微升</td></tr><tr><td> ATT GCG ACC GCT GCT GG </td><td></td></tr><tr><td> Taq DNA聚合酶</td><td> 0.4微升</td></tr><tr><td>无RNase-DH 2 O </td><td> 40.6微升</td></tr></tbody></table>表3.不含DNA的RNA质量检测PCR反应液。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> RNA </td><td> 0.5-5微克</td></tr><tr><td>的dNTP 10毫</td><td> 1微升</td></tr><tr><td>反向引物10微米/随机六聚体50μM </td><td> 1微升</td></tr><tr><td> DEPC-H 2 O </td><td>截至13微升</td></tr></tbody></table> 表4. cDNA合成反应混合物A. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td>缓冲区5X </td><td> 4微升</td></tr><tr><td> DTT </td><td> 1微升</td><html></tr><tr><td> RNA酶抑制剂</td><td> 1微升</td></tr><tr><td>逆转录酶</td><td> 1微升</td></tr></tbody></table> 表5. cDNA合成反应混合物B. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 目标 </td><td> 底漆名 </td><td> 序列 </td><td> 退火温度（℃） </td><td> 参考 </td></tr><tr><td rowspan="3">的16S rRNA细菌</td><td> Bact1369F </td><td> CGG TGA ATA CGT TCY CGG </td><td rowspan="3"> 56 </td><td rowspan="3">铃木等人，2000 19 </td></tr><tr><td> Prok1492R </td><td> GGW TAC CTT GTT ACG ACTŤ </td></tr><tr><td> TM1389F（探头） </td><td> CTT GTA CAC ACC GCC CGTÇ </td></tr></tbody></table>ove_content"FO:保together.within页="1"&gt; 表6.引物和探针的16S rRNA Q-PCR检测。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 量 </td></tr><tr><td> 2X预混</td><td> 10微升</td></tr><tr><td>正向引物（10μM） </td><td> 0.8微升</td></tr><tr><td>反向引物（10μM） </td><td> 0.8微升</td></tr><tr><td>探针（10μM） </td><td> 0.4微升</td></tr><tr><td>水</td><td> 7.0微升</td></tr><tr><td>最终体积</td><td> 19微升</td></tr></tbody></table> 表7.实时PCR反应混合物。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 描述符 </td><td> 意义 </td></tr><tr><td>相关系数（R 2） </td><td>标准曲线的线性的度量。理想情况下，应该是R 2 = 1。 R 2 = 0.98-0.99的价值是可以接受的。 </td></tr><tr><td>斜率（α）的</td><td>反应效率的措施。理想情况下，应相当于-3.32。值在-3.58和-3.1之间的范围内。 </td></tr><tr><td>效率（= 10（-1 /斜率）-1） </td><td>如果它是100％，这些模板指数扩增过程中每个热循环后加倍。一个很好的效率范围为90〜110％之间。 </td></tr><tr><td> y轴截距（β） </td><td>检测该反应的理论极限。虽然不是用来量化的反应灵敏度，比较同一目标的不同标准曲线时，是非常重要的。 </td></tr></tbody></table> 表8. QPC- [R反应描述。

Watch this Scientific Journal Video about Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR at JoVE.com

Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction.

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

Real Time Polymerase Chain Reaction also known as quantitative PCR (q-PCR) is a widely used tool in microbial ecology to quantify gene abundances of ...

simultaneous-dna-rna-extraction-from-coastal-sediments-quantification

Universidad San Sebastian

Research

JoVE Journal

Environment

17.5K 次观看.  National University of Ireland Galway. A protocol to quantify bacterial 16S rRNA genes and transcripts from coastal sediments via real-time PCR is provided. The methodology includes the co-extraction of DNA and RNA; preparation of DNA-free RNA; and 16S rRNA gene and transcript quantification via RT-q-PCR, including standard curve construction. 

视频: Simultaneous DNA-RNA Extraction from Coastal Sediments and Quantification of 16S rRNA Genes and Transcripts by Real-time PCR

Assessment of DNA Contamination in RNA Samples Based on Ribosomal DNA

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time PCR (RT-qPCR). It provides accurate, sensitive, reliable, and reproducible results. Several factors can affect the sensitivity and specificity of RT-qPCR. Residual genomic DNA (gDNA) contaminating RNA samples is one of them. In gene expression analysis, non-specific amplification due to gDNA contamination will overestimate the abundance of transcript levels and can affect the RT-qPCR results. Generally, gDNA is detected by qRT-PCR using primer pairs annealing to intergenic regions or an intron of the gene of interest. Unfortunately, intron/exon annotations are not yet known for all genes from vertebrate, bacteria, protist, fungi, plant, and invertebrate metazoan species.
Here we present a protocol for detection of gDNA contamination in RNA samples by using ribosomal DNA (rDNA)-based primers. The method is based on the unique features of rDNA: their multigene nature, highly conserved sequences, and high frequency in the genome. Also as a case study, a unique set of primers were designed based on the conserved region of ribosomal DNA (rDNA) in the Poaceae family. The universality of these primer pairs was tested by melt curve analysis and agarose gel electrophoresis. Although our method explains how rDNA-based primers can be applied for the gDNA contamination assay in the Poaceae family,&#160;it could be easily used to other prokaryote and eukaryote species

Here, we present a protocol for tracing genomic DNA (gDNA) contamination in RNA samples. The presented method utilizes primers specific for the internal transcribed spacer region (ITS) of ribosomal DNA (rDNA) genes. The method is suited for reliable and sensitive detection of DNA contamination in most eukaryotes and prokaryotes.

基于核糖体 dna 的 RNA 样本 dna 污染评价

One method extensively used for the quantification of gene expression changes and transcript abundances is reverse-transcription quantitative real-time ...

科研

遗传学

Isolation, Propagation, and Identification of Bacterial Species with Hydrocarbon Metabolizing Properties from Aquatic Habitats

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous catalytic enzymes and are naturally capable of metabolizing hydrocarbons. Scientists harness biodiversity in aquatic ecosystems to isolate bacteria with biodegradation and bioremediation potential. Such isolates from the environment provide a rich set of metabolic pathways and enzymes, which can be further utilized to scale up the degradation process at an industrial scale. In this article, we outline the general process of isolation, propagation, and identification of bacterial species from aquatic habitats and screen their ability to utilize hydrocarbons as the sole carbon source in vitro using simple techniques. The present protocol describes the isolation of various bacterial species and their subsequent identification using the 16S rRNA analysis. The protocol also presents steps for characterizing the hydrocarbon degrading potential of bacterial isolates. This protocol will be useful for researchers trying to isolate bacterial species from environmental habitats for their biotechnological applications.

We present the process of isolating, propagating, and characterizing hydrocarbon-degrading bacteria from aquatic habitats. The protocol outlines bacterial isolation, identification by the 16S rRNA method, and testing of their hydrocarbon-degrading potential. This article would help researchers in characterizing microbial biodiversity in environmental samples, and specifically screen for microbes with bioremediation potential.

从水生栖息地分离、繁殖和鉴定具有碳氢化合物代谢特性的细菌种类

Hydrocarbon pollutants are recalcitrant to degradation and their accumulation in the environment is toxic to all life forms. Bacteria encode numerous ...

环境科学

Extraction of High Molecular Weight Genomic DNA from Soils and Sediments

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with nutrient and energy flow within terrestrial ecosystems. Cultivation-independent environmental genomic, also known as metagenomic, approaches promise unprecedented access to this genetic information with respect to pathway reconstruction and functional screening for high value therapeutic and biomass conversion processes. However, the soil microbiome still remains a challenge largely due to the difficulty in obtaining high molecular weight DNA of sufficient quality for large insert library production. Here we introduce a protocol for extracting high molecular weight, microbial community genomic DNA from soils and sediments. The quality of isolated genomic DNA is ideal for constructing large insert environmental genomic libraries for downstream sequencing and screening applications. 
The procedure starts with cell lysis. Cell walls and membranes of microbes are lysed by both mechanical (grinding) and chemical forces (&beta;-mercaptoethanol). Genomic DNA is then isolated using extraction buffer, chloroform-isoamyl alcohol and isopropyl alcohol. The buffers employed for the lysis and extraction steps include guanidine isothiocyanate and hexadecyltrimethylammonium bromide (CTAB) to preserve the integrity of the high molecular weight genomic DNA. Depending on your downstream application, the isolated genomic DNA can be further purified using cesium chloride (CsCl) gradient ultracentrifugation, which reduces impurities including humic acids. The first procedure, extraction, takes approximately 8 hours, excluding DNA quantification step. The CsCl gradient ultracentrifugation, is a two days process. During the entire procedure, genomic DNA should be treated gently to prevent shearing, avoid severe vortexing, and repetitive harsh pipetting.

A methodology to isolate high molecular weight and high quality genomic DNA from soil microbial community is described.

超高分子量基因组DNA的提取土壤和沉积物

The soil microbiome is a vast and relatively unexplored reservoir of genomic diversity and metabolic innovation that is intimately associated with ...

生物学

Visualizing Methane-Cycling Microbial Dynamics in Coastal Wetlands

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus playing a critical role in regulating the methane cycle. The wetlands in South Texas, which are subject to frequent weather events, fluctuating salinity levels, and anthropogenic activities due to climate change, influence methane cycling. Despite the ecological importance of these processes, methane cycling in South Texas coastal wetlands remains insufficiently explored. To address this gap, we developed and optimized a method for detecting genes related to methanogens and methanotrophs, including mcrA as a biomarker for methanogens and pmoA1, pmoA2, and mmoX as biomarkers for methanotrophs. Additionally, this study aimed to visualize the spatial and temporal distribution patterns of methanogen and methanotroph abundance utilizing the geographic information system (GIS) software ArcGIS Pro. The integration of these molecular techniques with advanced geospatial visualization provided critical insights into the spatial and temporal distribution of methanogen and methanotroph communities across South Texas wetlands. Thus, the methodology established in this study offers a robust framework for mapping microbial dynamics in wetlands, enhancing our understanding of methane cycling under varying environmental conditions, and supporting broader ecological and environmental change studies.

The protocol detects key methane-cycling genes in South Texas coastal wetlands and visualizes their spatial distribution to enhance understanding of methane regulation and its environmental impacts in these dynamic ecosystems.

可视化沿海湿地中的甲烷循环微生物动力学

Coastal wetlands are the largest biotic source of methane, where methanogens convert organic matter into methane and methanotrophs oxidize methane, thus ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

核糖体RNA的蚊子肠道宏基因组RNA-seq的枯竭

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

Next-generation Sequencing of 16S Ribosomal RNA Gene Amplicons

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the long-lasting gold standards is to sequence 16S ribosomal RNA (rRNA) gene amplicons generated by domain-level PCR reactions amplifying from genomic DNA. Traditionally, this was performed by cloning and Sanger (capillary electrophoresis) sequencing of PCR amplicons. The advent of next-generation sequencing has tremendously simplified and increased the sequencing depth for 16S rRNA gene sequencing. The introduction of benchtop sequencers now allows small labs to perform their 16S rRNA sequencing in-house in a matter of days. Here, an approach for 16S rRNA gene amplicon sequencing using a benchtop next-generation sequencer is detailed. The environmental DNA is first amplified by PCR using primers that contain sequencing adapters and barcodes. They are then coupled to spherical particles via emulsion PCR. The particles are loaded on a disposable chip and the chip is inserted in the sequencing machine after which the sequencing is performed. The sequences are retrieved in fastq format, filtered and the barcodes are used to establish the sample membership of the reads. The filtered and binned reads are then further analyzed using publically available tools. An example analysis where the reads were classified with a taxonomy-finding algorithm within the software package Mothur is given. The method outlined here is simple, inexpensive and straightforward and should help smaller labs to take advantage from the ongoing genomic revolution.

Characterizing microbial community has been a longstanding goal in environmental microbiology. Next-generation sequencing methods now allow for the characterization of microbial communities at an unprecedented depth with minimal cost and labor. We detail here our approach to sequence bacterial 16S ribosomal RNA genes using a benchtop sequencer.

的16S核糖体RNA基因的扩增子的新一代测序

One of the major questions in microbial ecology is &#8220;who is there?&#8221; This question can be answered using various tools, but one of the ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

高效的核酸提取和16S rRNA基因序列的细菌群落特征

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Laboratory Protocol for Genetic Gut Content Analyses of Aquatic Macroinvertebrates Using Group-specific rDNA Primers

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the invasion of non-indigenous species. However, an exact identification of field predator-prey interactions is difficult in many cases. These analyses are often based on a visual evaluation of gut content or the analysis of stable isotope ratios (&#948;15N and &#948;13C). Such methods require comprehensive knowledge about, respectively, morphologic diversity or isotopic signature from individual prey organisms, leading to obstacles in the exact identification of prey organisms. Visual gut content analyses especially underestimate soft bodied prey organisms, because maceration, ingestion and digestion of prey organisms make identification of specific species difficult. Hence, polymerase chain reaction (PCR) based strategies, for example the use of group-specific primer sets, provide a powerful tool for the investigation of food web interactions. Here, we describe detailed protocols to investigate the gut contents of macroinvertebrate consumers from the field using group-specific primer sets for nuclear ribosomal deoxyribonucleic acid (rDNA). DNA can be extracted either from whole specimens (in the case of small taxa) or out of gut contents of specimens collected in the field. Presence and functional efficiency of the DNA templates need to be confirmed directly from the tested individual using universal primer sets targeting the respective subunit of DNA. We also demonstrate that consumed prey can be determined further down to species level via PCR with unmodified group-specific primers combined with subsequent single strand conformation polymorphism (SSCP) analyses using polyacrylamide gels. Furthermore, we show that the use of different fluorescent dyes as labels enables parallel screening for DNA fragments of different prey groups from multiple gut content samples via automated fragment analysis.

Most common gut content analyses of macroinvertebrates are visual. Requiring intense knowledge about morphological diversity of prey organisms, they miss soft bodied prey and, due to strong comminution of prey, are nearly impossible for some organisms, including amphipods. We provide detailed, novel genetic approaches for macroinvertebrate prey identification in the diet of amphipods.

用基团特异 rDNA 引物对水生无脊椎动物进行遗传肠道含量分析的实验室规程

Analyzing food webs is essential for a better understanding of ecosystems. For example, food web interactions can undergo severe changes caused by the ...

使用改良试剂盒进行经济的硅藻 DNA 提取

Extraction of Diatom DNA from Water Samples and Tissues

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. Diatom testing is also an important research content in the field of the environment and plankton. The diatom molecular biology testing technology, which focuses on diatom DNA as the primary research object, is a new method of diatom testing. Diatom DNA extraction is the basis of diatom molecular testing. At present, the kits commonly used for diatom DNA extraction are expensive, which increases the cost of carrying out related research. Our laboratory improved the general whole blood genomic DNA rapid extraction kit and obtained a satisfactory diatom DNA extraction effect, thus providing an alternative economical and affordable DNA extraction solution based on glass beads for related research. The diatom DNA extracted using this protocol could satisfy many downstream applications, such as PCR and sequencing.

作者聚焦： 用于法医溺水检查的硅藻检测 

This article describes a protocol for diatom DNA extraction using a modified common DNA extraction kit.

从水样和组织中提取硅藻DNA

Diatom testing is an essential auxiliary means in forensic practice to determine whether the corpse drowned in water and to infer the drowning location. ...

从海岸沉积物和16S量化的rRNA基因和转录同时DNA-RNA提取采用Real-time PCR检测

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从水样和组织中提取硅藻DNA

从水样和组织中提取硅藻DNA