وأيد هذا العمل من قبل المعاهد الوطنية للصحة منح DE018304 ، R01DE018281. معتمد من قبل KT GM07092 T32 - 34 ومنحة لجامعة ولاية كارولينا الشمالية في تشابل هيل من معهد هوارد هيوز الطبي (HHMI) من خلال مبادرة متوسطية في غراد. معتمد من قبل PC T32 CA009156.

<ol>
	<li>Bustin, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+MIQE+Guidelines:+Minimum+Information+for+Publication+of+Quantitative+Real-Time+PCR+Experiments.">The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-Time PCR Experiments.</a> Clinical Chem. 55 (4), 611-622 (2009).</li><li>Fr&#233;geau, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+Processing+of+Forensic+Casework+Samples+using+Robotic+Workstations+Equipped+with+Nondisposable+Tips+-+Contamination+Prevention.">Automated Processing of Forensic Casework Samples using Robotic Workstations Equipped with Nondisposable Tips - Contamination Prevention.</a> J. Forensic Sci. 53 (3), 53-533 (2008).</li><li>O'hara, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-micro+RNA+Signatures+Delineate+Stages+of+Endothelial+Cell+Transformation+in+Kaposi+Sarcoma.">Pre-micro RNA Signatures Delineate Stages of Endothelial Cell Transformation in Kaposi Sarcoma.</a> PLoS Pathog. 5 (4), e1000389-e1000389 (2009).</li></ol>

الإعلان عن أي تضارب في المصالح.

QPCR هو مقايسة حساسة للغاية والتي يمكن استخدامها للمقارنة بين مستويات التعبير الجيني بين العينات في الدراسة ، وكذلك لتحديد بالفيروس في حالة من الفيروسات. الفائدة من استخدام صفائف qPCR هو القدرة على تشغيل العديد من المشارع (في حالتنا ، أزواج التمهيدي 186) لكل عينة في فترة ق...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Freedom Evo 150</td>
<td>&nbsp;</td>
<td>Tecan</td>
<td>30017587</td>
<td>&nbsp;</td>
<tr>
<td>Matrix Electronic Multichannel Pipette</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>2001-MTX</td>
<td>(or any comparable Thermo Scientific multichannel that can pipette the volumes described above)</td>
</tr>
<tr>
<td>Matrix Integrity Filter Tips, Sterile</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>7435</td>
<td>(or comparable tip for multichannel used)</td>
</tr>
<tr>
<td>Lightcycler 480 SYBR Green 1 Master</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>04 707 516 001</td>
<td>&nbsp;</td>
<tr>
<td>Lightcycler 480 Instrument II</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05015243001</td>
<td>384-well version</td>
</tr>
<tr>
<td>Lightcycler 480 Multiwell Plate 384, white</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>04729749001</td>
<td>&nbsp;</td>
<tr>
<td>LightCycler 480 Sealing Foil</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>04729757001</td>
<td>&nbsp;</td>
<tr>
<td>LightCycler 480 Multiple Plate Analysis Software</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>05075122001</td>
<td>&nbsp;</td>
<tr>
<td>Eliminase Decontaminant</td>
<td>&nbsp;</td>
<td>Decon Laboratories</td>
<td>04-355-31</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

profiling of pre-micro rnas and micrornas using quantitative real-time pcr (qpcr) arrays

وقد برزت الكمي في الوقت الحقيقي PCR (QPCR) كأداة قيمة ودقيقة في توصيف مستويات التعبير الجيني. واحدة من مزاياها العديدة هو الحد الأدنى للكشف بالمقارنة مع غيرها من أساليب التعبير الجيني التنميط أثناء استخدام كميات صغيرة من المدخلات لكل مقايسة. وقد تحسنت الآلي qPCR الإعداد هذا المجال من خلال السماح لمزيد من التكاثر. الإعداد لها مريحة وسريعة تتيح للتجارب التي مرت عالية ، مما مكن من التنميط جينات كثيرة مختلفة في وقت واحد في كل تجربة. هذا الأسلوب مع لوحة التحكم الداخلية يقلل أيضا من المتغيرات التجريبية المشتركة لتقنيات أخرى. قمنا بتطويرها مؤخرا لفحص qPCR التنميط المسبق من microRNAs (ما قبل miRNAs) باستخدام مجموعة من أزواج التمهيدي 186. ظهرت MicroRNAs كطبقة رواية صغيرة ، الرناوات غير الترميز لديهم القدرة على تنظيم العديد من الأهداف مرنا في مرحلة ما بعد النسخي. وكتب أولا هذه الرنا RNA الصغيرة بوليميريز الثاني باعتباره ميرنا الابتدائي (الحزب الثوري المؤسسي ، ميرنا) نسخة ، وهو المشقوق ثم إلى ميرنا السلائف (ما قبل ميرنا). ويتم تصدير ما قبل miRNAs إلى السيتوبلازم حيث يشق المقامر حلقة منعطف حاد لانتاج miRNAs ناضجة. ويمكن ملاحظة زيادة في مستويات ميرنا على السلائف وعلى صعيد ميرنا ناضجة والتنميط من كل من هذه الأشكال يمكن أن تكون مفيدة. هناك عدة فحوصات متوفرة تجاريا للmiRNAs ناضجة ، ولكن تكلفتها العالية قد تردع هذه التقنية الباحثين من التنميط. هنا ، نحن نناقش فعالة من حيث التكلفة وموثوق بها ، وطريقة qPCR SYBR القائمة على التنميط المسبق miRNAs. التغيرات في مستويات ما قبل ميرنا غالبا ما تعكس التغييرات ميرنا ناضجة ويمكن أن تكون مؤشرا مفيدا التعبير ميرنا ناضجة. ومع ذلك ، قد التنميط المتزامن لكلا miRNAs قبل أن تنضج وmiRNAs الأمثل لأنها يمكن أن تساهم المعلومات nonredundant وتوفير نظرة ثاقبة تجهيز microRNA. وعلاوة على ذلك ، يمكن توسيع هذه التقنية وصفها هنا ليشمل مجموعات من التنميط مكتبة أخرى لمسارات محددة أو مسببات الأمراض.

Watch this Scientific Journal Video about Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays at JoVE.com

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

وسوف نظهر الإعداد وتحليل microRNA - 96 - جيدا قبل صفائف QPCR باستخدام الروبوت وكذلك جنب مع ماصة العلمية الحرارية الأقنية ماتريكس.

من التنميط المسبق الجزئي الرناوات و microRNAs باستخدام الكمية في الوقت الحقيقي PCR (qPCR) صالحة

Quantitative real-time PCR (QPCR) has emerged as an accurate and valuable tool in profiling gene expression levels.  One of its many advantages is a lower ...

profiling-pre-micro-rnas-micrornas-using-quantitative-real-time-pcr

Research

JoVE Journal

Biology

Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR (qPCR) Arrays

17.2K Views.  University of North Carolina at Chapel Hill. وسوف نظهر الإعداد وتحليل microRNA - 96 - جيدا قبل صفائف QPCR باستخدام الروبوت وكذلك جنب مع ماصة العلمية الحرارية الأقنية ماتريكس.

Video: Profiling of Pre-micro RNAs and microRNAs using Quantitative Real-time PCR qPCR Arrays

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 ...

Analysis of Gene Expression in Emerald Ash Borer (Agrilus planipennis) Using Quantitative Real Time-PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB continues to spread rapidly and attacks ash trees of different ages, from saplings to mature trees. However, to date very little or no molecular knowledge exists for EAB. We are interested in deciphering the molecular-based physiological processes at the tissue level that aid EAB in successful colonization of ash trees. In this report we show the effective use of quantitative real-time PCR (qRT-PCR) to ascertain mRNA levels in different larval tissues (including midgut, fat bodies and cuticle) and different developmental stages (including 1st-, 2nd-, 3rd-, 4th-instars, prepupae and adults) of EAB. As an example, a peritrophin gene (herein named, AP-PERI1) is exemplified as the gene of interest and a ribosomal protein (AP-RP1) as the internal control. Peritrophins are important components of the peritrophic membrane/matrix (PM), which is the lining of the insect gut. The PM has diverse functions including digestion and mechanical protection to the midgut epithelium.

Analysis of Gene Expression in Emerald Ash Borer Agrilus planipennis Using Quantitative Real Time-PCR

Quantitative real-time PCR (qRT-PCR) is an effective tool to diagnose mRNA levels in different insect tissues and developmental stages. In this report we show the use of qRT-PCR to ascertain mRNA levels in different larval tissues and developmental stages of the invasive insect species, emerald ash borer.

تحليل التعبير الجيني في الحفار الرماد الزمرد ( Agrilus planipennis) عن طريق ريال الكمية من الزمن - PCR

Emerald ash borer (EAB, Agrilus planipennis) is an exotic invasive pest, which has killed millions of ash trees (Fraxinus spp) in North America. EAB ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

تحديد ملامح التعبير الجيني في C. ايليجانس استخدام ميكروأري وPCR الوقت الحقيقي

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

كمي، في الوقت الحقيقي وتحليل قاعدة الختان في نشاط إصلاح Lysates خلية الاستفادة من الآفات محددة منارات الجزيئية

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

التنميط الفردية الخلايا الجذعية الجنينية البشرية من خلال الكمي RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

قياسات الوقت الحقيقي من البروتين غشاء: مستقبلات التفاعلات عن طريق مأكل السطح الرنين (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

يستند التحقيق، في الوقت الحقيقي PCR مقاربات للقياس الكمي من microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

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 ...

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 ...

Real-Time Monitoring of Aurora kinase A Activation using Conformational FRET Biosensors in Live Cells

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon its autophosphorylation on Thr288. AURKA abundance peaks in mitosis, where it controls the stability and the fidelity of the mitotic spindle, and the overall efficiency of mitosis. Although well characterized at the structural level, a consistent monitoring of the activation of AURKA throughout the cell cycle is lacking. A possible solution consists in using genetically-encoded F&#246;rster&#8217;s Resonance Energy Transfer (FRET) biosensors to gain insight into the autophosphorylation of AURKA with sufficient spatiotemporal resolution. Here, we describe a protocol to engineer FRET biosensors detecting Thr288 autophosphorylation, and how to follow this modification during mitosis. First, we provide an overview of possible donor/acceptor FRET pairs, and we show possible cloning and insertion methods of AURKA FRET biosensors in mammalian cells. Then, we provide a step-by-step analysis for rapid FRET measurements by fluorescence lifetime imaging microscopy (FLIM) on a custom-built setup. However, this protocol is also applicable to alternative commercial solutions available. We conclude by considering the most appropriate FRET controls for an AURKA-based biosensor, and by highlighting potential future improvements to further increase the sensitivity of this tool.

The activation of the multifunctional Ser/Thr kinase AURKA is hallmarked by its autophosphorylation on Thr288. Fluorescent probes relying on FRET can discriminate between its inactive and activated states. Here, we illustrate some strategies for probe engineering, together with a rapid FRET protocol to follow the kinase activation throughout mitosis.

المراقبة في الوقت الحقيقي لتنشيط Aurora kinase A باستخدام أجهزة الاستشعار الحيوية FRET التوافقية في الخلايا الحية

Epithelial cancers are often hallmarked by the overexpression of the Ser/Thr kinase Aurora A/AURKA. AURKA is a multifunctional protein that activates upon ...