ماثيو اوسوليفان وتشو فاي يتلقون الوطنية للصحة والبحوث الطبية الاسترالية مجلس البحوث الطبية منح الدراسات العليا.

<ol>
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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=Use+of+PCR+and+reverse+line+blot+hybridization+assay+for+rapid+simultaneous+detection+and+serovar+identification+of+Chlamydia+trachomatis.">Use of PCR and reverse line blot hybridization assay for rapid simultaneous detection and serovar identification of Chlamydia trachomatis.</a> J Clin Microbiol. 44, 1413-1418 (2006).</li><li>Wang, Y., Kong, F., Yang, Y., 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=A+multiplex+PCR-based+reverse+line+blot+hybridization+(mPCR/RLB)+assay+for+detection+of+bacterial+respiratory+pathogens+in+children+with+pneumonia.">A multiplex PCR-based reverse line blot hybridization (mPCR/RLB) assay for detection of bacterial respiratory pathogens in children with pneumonia.</a> Pediatr Pulmonol. 43, 150-159 (2008).</li><li>Wang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+multiplex+PCR-based+reverse+line+blot+(mPCR/RLB)+hybridisation+assay+for+the+rapid+identification+of+bacterial+pathogens.">Use of a multiplex PCR-based reverse line blot (mPCR/RLB) hybridisation assay for the rapid identification of bacterial pathogens.</a> Clin Microbiol Infect. 14, 155-160 (2008).</li><li>Wang, H., Kong, F., Jelfs, P., James, G., Gilbert, G. 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الإعلان عن أي تضارب في المصالح.

الأسلوب mPCR / RLB تصاريح الكشف المتزامن لعدد كبير من amplicons PCR. نظرا لحساسية عالية للكشف التحقيق المستندة chemiluminescent ، يمكن استخدام واحد تفاعل PCR متعددة مع عدد كبير من أزواج التمهيدي لتضخيم قالب الحمض النووي. ويمكن الحصول عليها إذا لم إشارات التحقيق مع واحد أو أكثر من العينات ، وأداء الكهربائي للهلام مع أي منتج PCR المتبقية مساعدة على تمييز ما إذا كانت المشكلة هي مع التضخيم PCR أو التهجين التحقيق. حيث كل إشارات التحقيق على الغشاء ضعيفة أو غائبة ، لكنها تشير إلى نجاح هلام إستشراد PCR التضخيم ، وتشمل الاحتمالات مشاكل مع وسم الغشاء ، والتجديد غير صحيح من الغشاء بعد الاستخدام السابقة ، ودرجات حرارة غير صحيحة أثناء حضانة التهجين أو streptavidin ، أو معيبة ، streptavidin المتقارن البيروكسيداز أو كاشف الكشف. إذا تبين أن عينات مراقبة تحقيقات الفردية قد يكون منتجا لا يمكن أن يؤديها إشارات سلبية كاذبة ، واحد مع PCR جل المستندة إلى الكشف عن وتسلسلها لاحقة للتحقق من التضخيم من الناتج PCR والبحث عن الاختلاف في موقع تسلسل التحقيق ملزم. قد ضعف أو غياب إشارة التحقيق يكون راجعا إلى حجم كبير amplicon : إذا كان ذلك ممكنا ينبغي أن تكون جميع amplicons طول مماثلة وغليان أقل من 300. قد هياكل الحمض النووي الثانوي amplicons تنتج أيضا الفقراء ملزمة التحقيق ، وربما يستلزم إعادة تصميم أجهزة الاشعال. ويمكن أيضا التمهيدي الفردية أو تركيزات التحقيق تكون متنوعة لتحسين قوة الإشارة. ويمكن التحقيق إشارات إيجابية كاذبة بسبب التحقيق ملزم الاشعال nonamplified أو منتج التمهيدي - PCR ديمر عن طريق أداء واحد مع الجل القائم على الكشف وتسلسلها لاحقة. إذا حدث هذا ، إعادة تصميم تحقيقات قد يلزم. قد الطفرات نقطة في مواقع التحقيق ملزم يؤدي إلى إشارات ضعيفة أو غائبة. السماح تحقيقين لكل منتج تضخيم هذا يجعل من السهل اكتشافها. ويمكن استغلال هذه الخاصية مع الحذر والتصميم التمهيدي التحقيق للكشف عن تغيرات في الحمض النووي تسلسل الصغيرة المستهدفة. باختصار ، في حين أن هناك العديد من الأساليب للكشف عن المنتجات التالية متعددة تفاعلات PCR المتاحة ، mPCR / RLB لديها ميزة كونها عالية الإنتاجية وغير مكلفة مع انخفاض تكاليف الإعداد. المرونة في طريقة تسمح باستخدامه في لطائفة واسعة من التطبيقات.

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> الكاشف </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات </th></tr><tr><td> 5 &#39;أمين C6 تعديل تحقيقات قليل النوكليوتيد </td><td> سيغما الدريخ </td><td></td><td></td></tr><tr><td> NaHCO 3 </td><td> سيغما الدريخ </td><td> S - 8875 </td><td> أدلى 0.5 M الحل تصل إلى 8.4 درجة الحموضة </td></tr><tr><td> هيدروكسيد الصوديوم </td><td> سيغما الدريخ </td><td> S5881 </td><td> 0.1M الحل </td></tr><tr><td> 20xSSPE العازلة </td><td> Amresco </td><td> 0810 - 4L </td><td></td></tr><tr><td> سلفات الصوديوم دوديسيل (SDS) </td><td> سيغما الدريخ </td><td> L - 4390 </td><td> يشكلون 10 ٪ محلول المخزون ، ينبغي أن لا الأوتوكلاف ، يمكن الاحتفاظ بها لمدة أقصاها أسبوع 1 قبل استخدام </td></tr><tr><td> Ethylenediaminetetraacetic حمض (EDTA) </td><td> سيغما الدريخ </td><td> E9884 </td><td> يشكلون 0.5 M الحل لتعديل درجة الحموضة 8.0 والأوتوكلاف </td></tr><tr><td> N - (3 - Dimethylaminopropyl) - N &#39;- ethylcarbodiimide هيدروكلوريد (EDAC) </td><td> سيغما الدريخ </td><td> E7750 </td><td> تشكل حلا مل 20 16 ٪ فقط قبل استخدام باستخدام 3.2 EDAC ز و 18 مل من الماء ميليبور </td></tr><tr><td> BiodyneC النايلون 0،45 ميكرون الطول غشاء 20x20 </td><td> بال علوم الحياة </td><td> 74480C </td><td></td></tr><tr><td> Deionised ، والمياه النقية </td><td></td><td></td><td></td></tr><tr><td> المنظفات السائلة Pyroneg </td><td> جونسون دايفرسي </td><td> HH12291 </td><td></td></tr><tr><td> Streptavidin - البيروكسيداز المتقارن </td><td> روش </td><td> 11 089 153 001 </td><td></td></tr><tr><td> أمرشام ECL كشف الكواشف </td><td> جنرال إلكتريك للرعاية الصحية </td><td> RPN2105 </td><td></td></tr><tr><td> أمرشام ECL كشف الكواشف </td><td> جنرال إلكتريك للرعاية الصحية </td><td> RPN2105 </td><td></td></tr><tr><td> OHP الفيلم الشفافية </td><td> الشركات اكسبرس </td><td> EXP 504 OHP </td><td></td></tr><tr><td> أمرشام hyperfilm ECL عالية الأداء الفيلم chemiluminescent 18x24 سم </td><td> جنرال إلكتريك للرعاية الصحية </td><td> 28906837 </td><td></td></tr><tr><td> جليد </td><td></td><td></td><td></td></tr></tbody></table> الجدول 1. مستهلكات المستخدمة في فحص mPCR / RLB. <table border="1" style=";text-align:right;direction:rtl"><tbody><tr><th> معدات </th><th> شركة </th><th> فهرس العدد </th><th> تعليقات </th></tr><tr><td> أفران Shake&#39;n&#39;Stack Hybaid (2) زجاجة المتداول وشبكات النايلون فصل </td><td> الحرارية العلمية </td><td> HBSNSRS220 </td><td> لا يمكن أن يؤديها الأسلوب مع فرن واحد طالما هناك تسهيلات للحفاظ على الحلول في درجة حرارة 42 درجة مئوية و 60 درجة مئوية قبل الاستخدام </td></tr><tr><td> وتهز الراقصة منصة </td><td> Stovall علوم الحياة </td><td> اليورو BDbo </td><td></td></tr><tr><td> Miniblotter </td><td> Immunetics </td><td> MN100 - 45 </td><td></td></tr><tr><td> ماء الحمام </td><td></td><td></td><td></td></tr><tr><td> مص </td><td></td><td></td><td></td></tr><tr><td> صفيحة ساخنة </td><td></td><td></td><td></td></tr><tr><td> التعرض خرطوشة </td><td> سيغما الدريخ </td><td> Z36 ،009 - 0 </td><td></td></tr><tr><td> فيلم الأشعة السينية المطور </td><td></td><td></td><td> ويمكن أيضا استخدام مطور ، يتدخل والمياه في الصواني في غرفة مظلمة بدلا من الآليةالمطور. ويمكن أيضا Lumino - تصوير استخدامها بدلا من الأشعة السينية الفيلم </td></tr></tbody></table> الجدول 2. المعدات اللازمة لفحص mPCR / RLB.

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

PCR المتعدد / عكس طخة تهجين مقايسة الخط يسمح للكشف عن ما يصل الى 43 اهداف في 43 عينات الجزيئي باستخدام أحد تفاعل PCR متعددة تليها التهجين دقق في غشاء النايلون ، والتي يمكن إعادة استخدامها. تحقيقات والأمينات 5 &#39;تعديل للسماح لتثبيت الغشاء. الاشعال و5 &#39;تعديل البيوتين الذي يسمح الكشف عن المنتجات المهجنة باستخدام PCR - streptavidin البيروكسيداز وركيزة chemiluminescent عبر الفيلم للضوء. مع الإعداد والتكاليف المنخفضة للاستهلاك ، وهذا أسلوب غير مكلف (حوالي 2 دولار أمريكي لكل عينة) ، إنتاجية عالية (يمكن معالجتها الأغشية متعددة في وقت واحد) ، وفترة زمنية قصيرة (حوالي 10 ساعة). ويمكن استخدام هذه التقنية في عدد من الطرق. ويمكن تصميم عدة تحقيقات للكشف عن التباين تسلسل ضمن منتج واحد تضخيمها ، أو يمكن تضخيم منتجات متعددة في وقت واحد ، مع تحقيقات (أو أكثر) واحدة تستخدم للكشف عن لاحقة. ويمكن أيضا الجمع بين النهجين أن تستخدم في غضون مقايسة واحد. وتشمل القدرة على تحقيقات متعددة للتسلسل هدف واحد يجعل من الفحص محددة للغاية. تطبيقات المنشورة mPCR / RLB تشمل الكشف عن الجينات المقاومة للمضادات الحيوية 1،2 ، كتابة من المكورات العنقودية الذهبية المقاومة للميثيسيلين 3-5 والسالمونيلا س 6 ، المصلي الجزيئي لل7،8 العقدية الرئوية ، العقدية القاطعة للدر 9 و 10،11 الفيروسات المعوية ، وتحديد س 12 من المتفطرة ، والكشف عن الأعضاء التناسلية والمسالك التنفسية 13-15 16 وغيرها من مسببات الأمراض والكشف عن 17 وتحديد mollicutes 18. ومع ذلك ، فإن براعة تقنية يعني تطبيقات غير محدودة تقريبا ، وليس مقتصرا على التحليل الجزيئي للكائنات الحية الدقيقة. الخطوات الخمس في mPCR / RLB هي التمهيدي) وتصميم المسبار ، ب) استخراج الحمض النووي وPCR التضخيم ج) إعداد الغشاء ، د) التهجين والكشف ، وه) التجديد في الغشاء.

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Multiplex PCR and Reverse Line Blot Hybridization Assay mPCR/RLB

وصفت وغير مكلفة ، وطريقة إنتاجية عالية للكشف في وقت واحد لمدة تصل إلى 43 أهداف الجزيئي. تطبيقات mPCR / RLB تشمل كتابة الجراثيم واكتشاف مسببات الأمراض متعددة من العينات السريرية.

PCR متعددة وعكس الخط الفحص لطخة تهجين (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 ...

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

43.3K Views.  University of Sydney. وصفت وغير مكلفة ، وطريقة إنتاجية عالية للكشف في وقت واحد لمدة تصل إلى 43 أهداف الجزيئي. تطبيقات mPCR / RLB تشمل كتابة الجراثيم واكتشاف مسببات الأمراض متعددة من العينات السريرية.

Video: 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.

سمية Nanogenomedicine الخلوية في الخط الخليوي MCF - 7 : فحص MTT

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

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.

الكمية في الوقت الحقيقي باستخدام PCR العلمية الحرارية سولاريس qPCR الفحص

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 ...

Rapid PCR Thermocycling using Microscale Thermal Convection

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable concentration level. But the design of conventional PCR thermocycling hardware, predominantly based on massive metal heating blocks whose temperature is regulated by thermoelectric heaters, severely limits the achievable reaction speed1. Considerable electrical power is also required to repeatedly heat and cool the reagent mixture, limiting the ability to deploy these instruments in a portable format.
Thermal convection has emerged as a promising alternative thermocycling approach that has the potential to overcome these limitations2-9. Convective flows are an everyday occurrence in a diverse array of settings ranging from the Earth's atmosphere, oceans, and interior, to decorative and colorful lava lamps. Fluid motion is initiated in the same way in each case: a buoyancy driven instability arises when a confined volume of fluid is subjected to a spatial temperature gradient. These same phenomena offer an attractive way to perform PCR thermocycling. By applying a static temperature gradient across an appropriately designed reactor geometry, a continuous circulatory flow can be established that will repeatedly transport PCR reagents through temperature zones associated with the denaturing, annealing, and extension stages of the reaction (Figure 1). Thermocycling can therefore be actuated in a pseudo-isothermal manner by simply holding two opposing surfaces at fixed temperatures, completely eliminating the need to repeatedly heat and cool the instrument.
One of the main challenges facing design of convective thermocyclers is the need to precisely control the spatial velocity and temperature distributions within the reactor to ensure that the reagents sequentially occupy the correct temperature zones for a sufficient period of time10,11. Here we describe results of our efforts to probe the full 3-D velocity and temperature distributions in microscale convective thermocyclers12. Unexpectedly, we have discovered a subset of complex flow trajectories that are highly favorable for PCR due to a synergistic combination of (1) continuous exchange among flow paths that provides an enhanced opportunity for reagents to sample the full range of optimal temperature profiles, and (2) increased time spent within the extension temperature zone the rate limiting step of PCR. Extremely rapid DNA amplification times (under 10 min) are achievable in reactors designed to generate these flows.

We describe a novel method to perform DNA replication via the polymerase chain reaction (PCR). Thermal convection is harnessed to continuously shuttle reagents between denaturing, annealing, and extension conditions by maintaining opposing surfaces of the reactor at constant temperature. This inherently simple design promises to make rapid PCR more accessible.

PCR Thermocycling السريع باستخدام الحراري الحرارية الميكروسكيل

Many molecular biology assays depend in some way on the polymerase chain reaction (PCR) to amplify an initially dilute target DNA sample to a detectable ...

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.

ميكروأرس] DNA : نموذج مراقبة الجودة ، وتهجين صفيف والمسح الضوئي

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.

تحويل لالتقاط ELISA لفحص xMAP Luminex باستخدام فحص الجسم المضاد الطريقة ملتيبليكس

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.

على الفحص لتجديد الخط الجانبي في اسماك الزرد الكبار

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.

تحليل qPCRTag - إنتاجية عالية، في الوقت الحقيقي PCR الفحص لSc2.0 التنميط الجيني

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.

مقايسة دوت كمية وصمة عار لمعايرة إف واستخدامها لتقييم وظيفي للفيروسات المرتبطة بالغدة الجمعية-تفعيل البروتينات

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.

القياس الكمي المطلق من مستويات البلازما MicroRNA في القرود سينومولوغوس، استخدام النسخ العكسي في الوقت الحقيقي الكمي 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 ...

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.