马修奥沙利文和周飞是澳大利亚国家卫生和医学研究理事会研究生医学研究奖学金的受助人。

引物和探针的设计必须给​​予认真考虑。应该利用所有可用的序列从GenBank中，如数据库的兴趣目标，以确定​​合适的目标的保护区。大量的目标是在一个单一的MPCR检测扩增的，应该是每个扩增序列的长度相近，不应超过300个碱基对，以避免竞争。该方法的另一种应用是放大一个更长的目标，使用对保守区域的引物，并使用多个探针来识别的扩增序列变异。在这种情况下，扩增的PCR产物可能会更长。引物退火温度应该都是相似的，PCR条件进行相应的调整。应避免形成强大的二级结构或引物二聚体的引物。可以使用的Sigma Aldrich公司的DNA计算器（ <a href="http://www.sigma-genosys.com/calc/DNACalc.asp">http://www.sigma-genosys.com/calc/DNACalc.asp</a> ），可靠地预测这些功能。 DNA探针的设计应具有退火温度接近60 ° C。为了最大限度地提高特异性，两个探头可以为每个感兴趣的目标包括在实验中，杂交正向相邻的反向引物结合位点的DNA链，和其他正向引物结合位点附近的反向链。在这种情况下，正向和反向引物必须在生物素修饰的。如果只有一个探头每扩增目标正在使用，那么只需要一个引物生物素修饰。 当设计MPCR / RLB检测的引物和探针，它强烈建议，在硅片的方法来预测的PCR产物与探针杂交与体外实验结果的关联。理想情况下，这是使用的全基因组序列是可用的分离。 FastPCR（http://primerdigital.com/fastpcr.html），如软件，可以用于此目的。可以预见，在硅片分析表明碱基对的不匹配，造成低探针退火温度探头信号弱或无。 DNA的提取技术，将不同取决于被测试的样本，PCR条件将依赖于引物设计。读者可参照个别检测的出版物更多关于DNA提取和PCR试 ​​剂和条件 1-19信息。 每个检测运行适当的控制，提供至少一个正极和一个细胞膜上的每个探头的负面探针的信号，以及免费的DNA控制。此外，它是有益的，包括在顶部的膜，预计将所有的样品（如：一个物种或一个微生物属特异性探针）的积极一个探头。这可作为阳性对照探针，但也允许容易取向的结果。 1。膜的制备<ol><li>准备了以下解决方案： <ul><li> 100毫升0.5M碳酸氢钠（pH值8.4） </li><li> 100毫升0.5M碳酸氢钠3 </li><li> 20毫升16％的经发会</li><li> 250毫升0.1 M的氢氧化钠</li><li> 250毫升2xSSPE </li><li> 250毫升2xSSPE/0.1％SDS - 在60 ° C水浴地方</li><li> 250毫升20 mM的EDTA。 </li></ul></li><li>预热烤箱至60 ° C。 </li><li>用70％乙醇的清洁Immunetics Miniblotter。 </li><li>稀释在0.5 M的碳酸氢钠3寡核苷酸探针的终浓度为2 pmol /μL和200μL的体积。 </li><li>剪下一个15x15厘米BiodyneC的尼龙膜。使用铅笔和直尺，规则跨越膜的顶部0.5厘米的空间，在这里写的详细信息。 </li><li>刚在20毫升的塑料袋密封膜在室温下10分钟16％EDAC的解决方案和岩石。 </li><li>膜在去离子水冲洗30秒。 </li><li>将膜miniblotter与运行的通道穿过细胞膜（铅笔线平行）。把支持地方和接近吸墨纸垫。从渠道吸液。 </li><li>填写与150μL0.5 M的碳酸氢钠3泳道1和45。使用150μL，每个探头解决方案，以填补序列2-44车道，要小心，以避免气泡。如果气泡出现在通道中，在保持吸液管，迅速吸出解决方案，让气泡浮到吸管的顶端，然后重试。在室温下孵育5分钟。 </li><li>吸探针解决方案，除去膜和250毫升0.1 M的氢氧化钠在室温下洗9分钟。 </li><li>在250毫升2xSSPE清洗膜30秒。 </li><li>在250毫升的预热2xSSPE/0.1％SDS在烤箱清洗膜在60​​℃，5分钟。 </li><li>如果不立即用于杂交，洗膜在室温为20分钟（保留余下10毫升）在240毫升20 mM的EDTA，然后密封在塑料袋中剩下的10毫升20 mM的EDTA和STOR膜e在冰箱在4 ° C </li><li> Pyroneg洗涤剂和刷子清洗miniblotter。冲洗并晾干。 </li></ol> 2。杂交及检测<ol><li>准备了以下解决方案： <ul><li> 250毫升2xSSPE/0.1％SDS - 水浴存放在60 ° C。 </li><li> 500毫升2xSSPE/0.5％SDS - 水浴存放在60 ° C </li><li> 500毫升2xSSPE/0.5％SDS - 烤箱存放在42 ° C </li><li> 500毫升2xSSPE - 保持室温</li><li> 500毫升1％的SDS - 存储在水浴60 ° C最初（步骤3） </li><li> 250毫升20 mM的EDTA - 在室温下储存（步骤3）。 </li></ul></li><li>打开一个烤箱，在60 ° C和一个烤箱42 ° C。把水烧开，在烤盘上一个大烧杯。 </li><li>用70％乙醇Miniblotter清洁Immunetics。 </li><li> 2xSSPE/0.1％SDS成一个小容器分装10毫升。每个PCR产物中加入20μL到150μL2xSSPE/0.1％SDS编号管。 </li><li>煮沸10分钟，PCR产物在100℃使用发泡胶持有人。立即置于冰上至少5分钟。 </li><li>洗膜，在其余240毫升2xSSPE/0.1％SDS在60 ° C的烤箱5分钟。 </li><li>将膜垫与支持miniblotter。定位使渠道运行垂直（垂直铅笔线）。 </li><li>多余的液体吸miniblotter渠道。 </li><li>其余150μL2xSSPE/0.1％SDS第一个和最后一个渠道。加水煮其余通道（2-44）序列的PCR产物，要小心，以避免气泡。 </li><li>广场miniblotter持平在60℃，1小时的烤箱，让杂交发生。 </li><li>吸液从每个通道，然后从miniblotter膜。 </li><li>在60℃10分钟的烤箱预热250毫升2xSSPE/0.5％SDS清洗两次。 </li><li>浸湿尼龙分离网状2xSSPE/0.5％SDS在42 ° C，并用它来卷起膜。 </li><li>地方卷起到辊筒膜，平展，以确保膜紧靠内表面。添加3μL链霉菌过氧化物酶的共轭（罗氏应用科学部）42至15毫升2xSSPE/0.5％SDS℃，辊筒。紧紧螺丝帽和孵化辊炉在42 ° 60分钟的彗星。确保卷的方向是，使膜不收紧。定期检查是否有泄漏。 </li><li> miniblotter Pyroneg洗涤剂和水清洗，冲洗并晾干。 </li><li>从辊筒膜。其余2xSSPE/0.5％SDS在42 ° 10分钟两次清洗。 </li><li>在室温下清洗两次在2xSSPE为5分钟。 </li><li>打开烤箱高达80 ° C和地方500毫升1％SDS在烤箱预温。 </li><li> 15毫升Amersham公司的ECL检测解决方案（7.5溶液A和溶液B的7.5毫升）。放弃2xSSPE和添加检测解决方案。摇滚手轻轻为2分钟，确保完全覆盖膜与解决方案。丢弃化学发光的解决方案。 </li><li>切两片塑料的透明度，以适应曝光墨盒。将膜之间的两片和地点到曝光墨盒。 </li><li>进入暗室。红灯下，加ECL检测膜盒和公开为5分钟。电影狗耳后拆除的右上角，以便正确的方向观看时。 </li><li>制定与自动化开发或生产商的方向的化学浴电影。 </li><li>如果需要，或长或短的曝光时间，重复曝光。 </li></ol> 3。膜的再生<ol><li>洗膜在250毫升1％SDS在80℃，30分钟两次。 </li><li> 15分钟，在室温下洗膜在240毫升20毫米EDTA。 </li><li>塑料袋密封膜用10 ml 20 mM的EDTA和冷藏在4 ° C为今后的再利用。 </li></ol> 4。代表结果： 结果是最好的观看电影放在印刷电网，否则扫描图像，并导入到软件，如BioNumerics（应用数学，圣马尔滕斯 - Latem，比利时）。每个探测结果的解释应该参考阳性和阴性对照探针。结果是最好的等级为阴性，弱或阳性。积极的结果，其中的信号是强或强于阳性对照探针。阴性结果是信号不存在或等于阴性对照组（背景信号的情况下）。弱的结果是信号比阳性对照探针暗，但强于阴性对照。业绩疲软可能会导致探头约束力薄弱，或由于非特异性引物二聚体的形成信号的点突变的结果。使用两个探头每利益目标可以改善的特殊性，在一个单一的薄弱结果可以安全地解释非特异性信号。如果有任何疑问仍然存在，单丛PCR反应，可以执行任何放大器的凝胶为基础的检测和测序lified决定，如果结果是真正积极的产品。一个代表性的结果如图2所示。 <img src="/files/ftp_upload/2781/2781fig1.jpg" alt="图1"/> 图1。MPCR / RLB原则（P1）的步骤1.9。胺改性探针共价结合的尼龙膜。 （P2）步骤2.10。生物素修饰的PCR产物杂交探针。 （P3）步骤2.14。过氧化物酶标记的链霉亲和，是培养与膜结合，以生物素。 （P4），步骤2.19-2.21。过氧化物酶催化ECL检测试剂的反应，产生光的光感光胶片曝光。膜，然后洗净，再利用。 <img src="/files/ftp_upload/2781/2781fig2.jpg" alt="图2"/> 图2。代表MPCR / RLB的结果。 1至6个样品，代表阳性对照 - 它们之间存在的43个探头，每年至少有一个积极的探测信号。每一个目标序列（一个通过U）是两个不同的探头，以最大限度地提高特异性检测。探头A1和A2分别代表特​​定物种，预计每个样品要积极协助电影与定向探头。探头A1是在膜的底部反复。样品7日和8负的控制。注意探头第一季度，​​第二季度，T1和T2阴性对照的信号，表明可能非特异性结合的引物或引物二聚体产品。这些探针或引物将需要重新设计。探头C1，D1和F1显示裸奔穿过细胞膜可能是由于一些非特异性的链霉菌过氧化物酶结合物或化学发光底物摄取，但是这是很容易区分真正的积极的探测信号。探头D1和D2有其他探测器相比，相对较弱的信号，这可能是由于大型扩增产物，导致效率较低的放大。探头J2是否定的是几个样品（1，3，4，6，39）探头J1的是积极的。这很可能代表了这些样品的J2的结合位点的突变。

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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+detection+and+identification+of+common+cell+culture+contaminant+and+pathogenic+mollicutes+strains+by+reverse+line+blot+hybridization.">Simultaneous detection and identification of common cell culture contaminant and pathogenic mollicutes strains by reverse line blot hybridization.</a> Appl Environ Microbiol. 70, 1483-1486 (2004).</li><li>Kong, F., Gilbert, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplex+PCR-based+reverse+line+blot+hybridization+assay+(mPCR/RLB)+-+a+practical+epidemiological+and+diagnostic+tool.">Multiplex PCR-based reverse line blot hybridization assay (mPCR/RLB) - a practical epidemiological and diagnostic tool.</a> Nat Protoc. 1, 2668-2680 (2006).</li></ol>

没有利益冲突的声明。

MPCR / RLB方法，可同时检测大量的PCR扩增。由于高灵敏度的化学发光探针为基础的检测，一个单一的大量的引物对多重PCR反应，可用于放大DNA为模板。 如果没有探测信号与一个或多个样品，任何剩余的PCR产物进行凝胶电泳，获得可以帮助区分的问题是否与PCR扩增或探针杂交。膜的所有探头信号弱或缺席，但凝胶电泳显示PCR扩增成功，可能性包括膜标签的问题，膜后，以前使用的不正确的再生，在杂交或链霉亲和孵化不正确的温度，或有缺陷的链霉过氧化物酶结合物或检测试剂。 如果控制样本表明，个别探头可产生假阴性信号，单用凝胶为基础的检测和随后的测序PCR，可以进行验证的PCR产物的扩增和探针结合位点序列变异看。薄弱或者根本没有探头的信号，可能是由于大量扩增大小：所有的扩增产物，如果可能的话应该是类似的长度小于300 bp的。扩增产物中的DNA结构也可能产生探头约束力差，可能需要重新设计引物。个人引物或探针浓度也可能是多种多样的，以优化信号强度。 假由于探头nonamplified引物或引物二聚体产品具有约束力的积极信号，可以调查由执行单一的PCR扩增与凝胶检测和随后的测序。如果发生这种情况，重新设计的探头，可能是必要的。在探针结合位点的点突变可能会导致薄弱或者根本没有信号。允许每个扩增产物的两个探头，使得这种不易察觉的。可利用这一特点，小心的引物和探针的设计，检测目标DNA的小序列变异。 综上所述，虽然有许多产品检测以下多重PCR反应方法，MPCR / RLB低安装成本高吞吐量和廉价的优势。该方法的灵活性，允许其使用了广泛的应用。

<table border="1"><tbody><tr><th>试剂</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> 5&#39;C6胺修饰的寡核苷酸探针</td><td> Sigma - Aldrich公司</td><td></td><td></td></tr><tr><td>碳酸氢钠3 </td><td> Sigma - Aldrich公司</td><td> S - 8875 </td><td> 0.5 M的溶液pH值8.4 </td></tr><tr><td>氢氧化钠</td><td> Sigma - Aldrich公司</td><td> S5881 </td><td> 0.1M的解决方案</td></tr><tr><td> 20xSSPE缓冲区</td><td> AMRESCO </td><td> 0810 - 4L </td><td></td></tr><tr><td>十二烷基硫酸钠（SDS） </td><td> Sigma - Aldrich公司</td><td> L - 4390 </td><td>同比增长10％原液，不反应釜，1每周最多使用前应保持</td></tr><tr><td>乙二胺四乙酸（EDTA） </td><td> Sigma - Aldrich公司</td><td> E9884 </td><td> 0.5米溶液调pH值8.0和反应釜</td></tr><tr><td> N -（3 - Dimethylaminopropyl）- N&#39; - ethylcarbodiimide盐酸盐（EDAC） </td><td> Sigma - Aldrich公司</td><td> E7750 </td><td>只是在使用前用3.2克经发会和微孔水18毫升化妆20毫升16％的解决方案</td></tr><tr><td> BiodyneC 0.45微米尼龙膜20X20厘米</td><td>颇尔生命科学</td><td> 74480C </td><td></td></tr><tr><td>去离子，纯净水</td><td></td><td></td><td></td></tr><tr><td>液体Pyroneg洗涤剂</td><td>约翰逊Diversey </td><td> HH12291 </td><td></td></tr><tr><td>链霉亲和过氧化物酶的共轭</td><td>罗氏公司</td><td> 11 089 153 001 </td><td></td></tr><tr><td> Amersham公司的ECL检测试剂</td><td> GE医疗集团</td><td> RPN2105 </td><td></td></tr><tr><td> Amersham公司的ECL检测试剂</td><td> GE医疗集团</td><td> RPN2105 </td><td></td></tr><tr><td> OHP透明膜</td><td>公司快递</td><td>进出口504 OHP </td><td></td></tr><tr><td> Amersham公司hyperfilm ECL高性能发光膜18x24厘米</td><td> GE医疗集团</td><td> 28906837 </td><td></td></tr><tr><td>冰</td><td></td><td></td><td></td></tr></tbody></table> MPCR / RLB检测中使用的表 1。耗材。 <table border="1"><tbody><tr><th>设备</th><th>公司</th><th>目录编号</th><th>评论</th></tr><tr><td> Hybaid Shake&#39;n&#39;Stack炉（2）滚动的瓶子和尼龙分离网状</td><td> Thermo Scientific的</td><td> HBSNSRS220 </td><td>方法可以用一个单一的烤箱，只要有设施维护解决方案，在42 ° C和60 ° C，使用前</td></tr><tr><td>肚皮舞摇摆平台</td><td>斯托维尔生命科学</td><td>欧元BDbo </td><td></td></tr><tr><td> Miniblotter </td><td> Immunetics </td><td> MN100 - 45 </td><td></td></tr><tr><td>水浴</td><td></td><td></td><td></td></tr><tr><td>吸力</td><td></td><td></td><td></td></tr><tr><td>热板</td><td></td><td></td><td></td></tr><tr><td>曝光墨盒</td><td> Sigma - Aldrich公司</td><td> Z36 ,009 - 0 </td><td></td></tr><tr><td> X射线胶片开发</td><td></td><td></td><td>也可以使用托盘在黑暗的房间，而不是自动化的开发，定影液和水开发商。 Lumino的成像也可以用来代替X射线胶片</td></tr></tbody></table> 表2。设备所需的MPCR / RLB检测。

multiplex pcr and reverse line blot hybridization assay (mpcr/rlb)

多重PCR /反向线杂交实验可以检测高达43在43个样品使用一个多重PCR反应探头上的尼龙膜，这是可重复使用的杂交分子靶点。探头5&#39;胺修改，以允许固定膜。引物5&#39;端生物素修改，允许使用的杂交，PCR链霉菌过氧化物酶和化学发光底物通过感光膜产品的检测。设置和低耗材成本，这种技术成本低廉（约每样本2美元），高吞吐量（可同时处理多个膜），有一个很短的周转时间（约10小时）。 该技术可以通过多种方式利用。设计多个探针，可在一个单一的扩增产物的序列变异检测，或多个产品可以被放大，同时，与一个（或多个）用于后续检测探头，。结合这两种方法也可以用来在一个单一的检测。包括一个单一的目标序列的多个探头的能力，使检测具有高度特异性。 MPCR / RLB发布的应用程序包括抗生素耐药性的基因1,2的检测，耐甲氧西林的金 黄色 葡萄球菌 3-5和沙门氏菌 SP 6，血清型的肺炎链球菌，无 乳链球菌 9 7,8和肠道病毒10,11，鉴定的分子打字分枝杆菌 ，检测生殖器13日至15日和 16和呼吸道等17个病原体检测和鉴定mollicutes 18 SP 12。然而，该技术的多功能性手段的应用几乎是无限的，而不是仅限于微生物的分子分析的。 MPCR / RLB的五个步骤a）引物和探针设计，B）DNA提取和PCR扩增C）膜的制备，D）杂交和检测，E）膜的再生。

Watch this Scientific Journal Video about Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB at JoVE.com

Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

描述的是一种廉价的，同时检测到43个分子靶点的高通量方法。 MPCR / RLB的应用，包括微生物打字和多种病原体从临床标本的检测。

多重PCR和反向线杂交检测（MPCR / RLB）

Multiplex PCR/Reverse Line Blot Hybridization assay allows the detection of up to 43 molecular targets in 43 samples using one multiplex PCR reaction ...

multiplex-pcr-and-reverse-line-blot-hybridization-assay-mpcrrlb

Research

JoVE Journal

Biology

Multiplex PCR and Reverse Line Blot Hybridization Assay (mPCR/RLB)

43.3K 次观看.  University of Sydney. 描述的是一种廉价的，同时检测到43个分子靶点的高通量方法。 MPCR / RLB的应用，包括微生物打字和多种病原体从临床标本的检测。

视频: Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

Cellular Toxicity of Nanogenomedicine in MCF-7 Cell Line: MTT assay

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based medicine and/or its carrier may elicit cellular toxicity. For assessment of such cytotoxicity, a common methodology is largely dependent upon utilization of the 3-(4, 5-Dimethyl-2-thiazolyl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) assay which has been widely used as a colorimetric approach based on the activity of mitochondrial dehydrogenase enzymes in cells. In this current investigation, MCF-7 cells were inoculated in 96-well plate and at 50% confluency they were treated with different nanopolyplexes and subjected to MTT assay after 24 hours. Water soluble yellow MTT is metabolized by the metabolically active cells to the water insoluble purple formazan, which is further dissolved in dimethylsulfoxide and Sornson s buffer pH 10.5. The resultant product can be quantified by spectrophotometry using a plate reader at 570 nm.

The MTT assay is an easy and reproducible colorimetric assay for evaluation of cell viability based on reduction of yellow MTT and production of water insoluble purple formazan. Here, the viability of MCF-7 cells upon treatment of nanogenomedicine has been evaluated.

Nanogenomedicine在MCF - 7细胞的细胞毒性：MTT法

Cytotoxicity of the futuristic nanogenomedicine (e.g., short interfering RNA and antisense) may hamper its clinical development. Of these, the gene-based ...

科研

生物学

Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to &gt;98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts.
Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process.
Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method.

The Solaris qPCR Gene Expression Assays are novel pre-designed qPCR primer/probe combinations designed to simplify the qPCR process without sacrificing the specificity and robustness of the assay.

实时定量PCR使用的Thermo Scientific的Solaris定量PCR检测

The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA微阵列：样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Conversion of a Capture ELISA to a Luminex xMAP Assay using a Multiplex Antibody Screening Method

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life science research and clinical diagnostics. However, ELISA has limitations. It is typically performed in a 96-well microplate, and the wells are coated with capture antibody, requiring a relatively large amount of sample to capture an antigen of interest . The large surface area of the wells and the hydrophobic binding of capture antibody can also lead to non-specific binding and increased background. Additionally, most ELISAs rely upon enzyme-mediated amplification of signal in order to achieve reasonable sensitivity. Such amplification is not always linear and can thus skew results.
In the past 15 years, a new technology has emerged that offers the benefits of the ELISA, but also enables higher throughput, increased flexibility, reduced sample volume, and lower cost, with a similar workflow 1, 2. Luminex xMAP Technology is a microsphere (bead) array platform enabling both monoplex and multiplex assays that can be applied to both protein and nucleic acid applications 3-5. The beads have the capture antibody covalently immobilized on a smaller surface area, requiring less capture antibody and smaller sample volumes, compared to ELISA, and non-specific binding is significantly reduced. Smaller sample volumes are important when working with limiting samples such as cerebrospinal fluid, synovial fluid, etc. 6. Multiplexing the assay further reduces sample volume requirements, enabling multiple results from a single sample.
Recent improvements by Luminex include: the new MAGPIX system, a smaller, less expensive, easier-to-use analyzer; Low-Concentration Magnetic MagPlex Microspheres which eliminate the need for expensive filter plates and come in a working concentration better suited for assay development and low-throughput applications; and the xMAP Antibody Coupling (AbC) Kit, which includes a protocol, reagents, and consumables necessary for coupling beads to the capture antibody of interest. (See Materials section for a detailed list of kit contents.)
In this experiment, we convert a pre-optimized ELISA assay for TNF-alpha cytokine to the xMAP platform and compare the performance of the two methods 7-11. TNF-alpha is a biomarker used in the measurement of inflammatory responses in patients with autoimmune disorders.
We begin by coupling four candidate capture antibodies to four different microsphere sets or regions. When mixed together, these four sets allow for the simultaneous testing of all four candidates with four separate detection antibodies to determine the best antibody pair, saving reagents, sample and time. Two xMAP assays are then constructed with the two most optimal antibody pairs and their performance is compared to that of the original ELISA assay in regards to signal strength, dynamic range, and sensitivity.

An ELISA can be easily converted to a Luminex xMAP assay and, through the benefits of multiplexing, several antibodies can be screened simultaneously to identify an optimum antibody pair, resulting in increased sensitivity and dynamic range, while reducing assay cost.

转换到一个点阵仪XMAP含量，使用多重抗体筛查方法的捕获ELISA

The enzyme-linked immunosorbent assay (ELISA) has long been the primary tool for detection of analytes of interest in biological samples for both life ...

An Assay for Lateral Line Regeneration in Adult Zebrafish

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al.17&#160;that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish rather than the embryo/larvae, we developed a quantitative lateral line regenerative assay that can be applied to adult zebrafish disease models. The assay involved resolution at the 1) neuromast and 2) individual hair cell levels.

对于侧线再生成年斑马鱼的含量

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line ...

qPCRTag Analysis - A High Throughput, Real Time PCR Assay for Sc2.0 Genotyping

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic chromosome, synIII1, and one synthetic chromosome arm, synIXR2, have been constructed and their in vivo function validated in the absence of the corresponding wild type chromosomes. An important design feature of Sc2.0 chromosomes is the introduction of PCRTags, which are short, re-coded sequences within open reading frames (ORFs) that enable differentiation of synthetic chromosomes from their wild type counterparts. PCRTag primers anneal selectively to either synthetic or wild type chromosomes and the presence/absence of each type of DNA can be tested using a simple PCR assay. The standard readout of the PCRTag assay is to assess presence/absence of amplicons by agarose gel electrophoresis. However, with an average PCRTag amplicon density of one per 1.5 kb and a genome size of ~12 Mb, the completed Sc2.0 genome will encode roughly 8,000 PCRTags. To improve throughput, we have developed a real time PCR-based detection assay for PCRTag genotyping that we call qPCRTag analysis. The workflow specifies 500 nl reactions in a 1,536 multiwell plate, allowing us to test up to 768 PCRTags with both synthetic and wild type primer pairs in a single experiment.

Designer chromosomes of the Synthetic Yeast Genome project, Sc2.0, can be distinguished from their native counterparts using a PCR-based genotyping assay called PCRTagging, which has a presence/absence endpoint. Here we describe a high-throughput real time PCR detection method for PCRTag genotyping.

qPCRTag分析 - 高通量，实时定量PCR检测方法的基因分型Sc2.0

The Synthetic Yeast Genome Project (Sc2.0) aims to build 16 designer yeast chromosomes and combine them into a single yeast cell. To date one synthetic ...

A Quantitative Dot Blot Assay for AAV Titration and Its Use for Functional Assessment of the Adeno-associated Virus Assembly-activating Proteins

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus biology. In the latter respect, the recent discovery of a non-structural AAV protein, termed assembly-activating protein (AAP), has shed new light on the processes involved in assembly of the viral capsid VP proteins into a capsid. Although many AAV serotypes require AAP for assembly, we have recently reported that AAV4, 5, and 11 are exceptions to this rule. Furthermore, we demonstrated that AAPs and assembled capsids of different serotypes localize to different subcellular compartments. This unexpected heterogeneity in the biological properties and functional roles of AAPs among different AAV serotypes underscores the importance of studies on AAPs derived from diverse serotypes. This manuscript details a straightforward dot blot assay for AAV quantitation and its application to assess AAP dependency and serotype specificity in capsid assembly. To demonstrate the utility of this dot blot assay, we set out to characterize capsid assembly and AAP dependency of Snake AAV, a previously uncharacterized reptile AAV, as well as AAV5 and AAV9, which have previously been shown to be AAP-independent and AAP-dependent serotypes, respectively. The assay revealed that Snake AAV capsid assembly requires Snake AAP and cannot be promoted by AAPs from AAV5 and AAV9. The assay also showed that, unlike many of the common serotype AAPs that promote heterologous capsid assembly by cross-complementation, Snake AAP does not promote assembly of AAV9 capsids. In addition, we show that the choice of nuclease significantly affects the readout of the dot blot assay, and thus, choosing an optimal enzyme is critical for successful assessment of AAV titers.

This manuscript details a straightforward dot blot assay for quantitation of adeno-associated virus (AAV) titers and its application to study the role of assembly-activating proteins (AAPs), a novel class of non-structural viral proteins found in all AAV serotypes, in promoting the assembly of capsids derived from cognate and heterologous AAV serotypes.

AAV 滴定定量斑点印迹法及其在腺相关病毒组装活化蛋白功能评价中的应用

While adeno-associated virus (AAV) is widely accepted as an attractive vector for gene therapy, it also serves as a model virus for understanding virus ...

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust technologies that measure RNA biomarkers in situ in clinical specimens. While grind-and-bind assays like RNAseq and quantitative RT-PCR enable highly sensitive gene expression measurements, they also require RNA extraction and thus prevent valuable expression analysis within the morphological tissue context. The in situ hybridization (ISH) assay described here can detect RNA target sequences as short as 50 nucleotides at single-nucleotide resolution and at the single-cell level. This assay is complementary to the previously developed commercial assay and enables sensitive and specific in situ detection of splice variants, short targets, and point mutations within the tissue. In this protocol, probes were designed to target unique exon junctions for two clinically important splice variants, EGFRvIII and MET&#916;14. The detection of short target sequences was demonstrated by the specific detection of CDR3 sequences of T-cell receptors &#945; and &#946; in the Jurkat T-cell line. Also shown is the utility of this ISH assay for the distinction of RNA target sequences at single-nucleotide resolution (point mutations) through the visualization of EGFR L858R and KRAS G12A single-nucleotide variations in cell lines using automated staining platforms. In summary, the protocol shows a specialized RNA ISH assay that enables the detection of splice variants, short sequences, and mutations in situ for manual performance and on automated stainers.

Visualizing Genetic Variants, Short Targets, and Point Mutations in the Morphological Tissue Context with an RNA In Situ Hybridization Assay

Here, we describe an in situ hybridization assay which enables sensitive and specific detection of sequences as short as 50 nucleotides with single-nucleotide resolution at the single-cell level. The assay, which can be performed manually or automatically, can enable visualization of splice variants, short sequences, and mutations within the tissue context.

用 RNA原位杂交法对形态学组织环境中的遗传变异、短靶点和突变进行可视化

Because precision medicine is highly dependent on the accurate detection of biomarkers, there is an increasing need for standardized and robust ...

Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) is the gold standard for SARS-CoV-2 diagnosis as no permanent solution is available. However effective this technique may be, research has emerged showing its limitations in detection and diagnosis especially when it comes to low abundant targets. In contrast, droplet digital PCR (ddPCR), a recent emerging technology with superior advantages over qPCR, has been shown to overcome the challenges of RT-qPCR in diagnosis of SARS-CoV-2 from low abundant target samples. Prospectively, in this article, the capabilities of RT-ddPCR are further expanded by showing steps on how to develop simplex, duplex, triplex probe mix, and quadruplex assays using a two-color detection system. Using primers and probes targeting specific sites of the SARS-CoV-2 genome (N, ORF1ab, RPP30, and RBD2), the development of these assays is shown to be possible. Additionally, step by step detailed protocols, notes, and suggestions on how to improve the assays workflow and analyze data are provided. Adapting this workflow in future works will ensure that the maximum number of targets can be sensitively detected in a small sample significantly improving on cost and sample throughput.

This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.

用于SARS-CoV-2检测和定量的两步逆转录液滴数字PCR方案

Diagnosis of the ongoing SARS-CoV-2 pandemic is a priority for all countries across the globe. Currently, reverse transcription quantitative PCR (RT-qPCR) ...