P001Ben AyerSowersBiosearchTechnologies, Inc., UnitedStates of America;MultiplexedqPCR remains achallenging endeavor for reasons that include: 1) designing assays tocombinewithout interference, 2) resolving fluorophores using the optics ofeachreal-time instrument, and 3) optimizing and validating each assay'sperformance. Here, we address each of these issues when developingseveralpentaplexed assays that target genes from human and mouse. Each assaywasdesigned using a free, online, software program that carefullyconsidersinter-oligo interactions while simultaneously building its multiplexedset.Situations of disproportionate copy number present a particularchallenge uponmultiplexed performance; additional validation is needed to define thelimitsof a multiplexed set, as compared to individually amplified assays. P002SaraPizzamiglioand Paolo VerderioUnit ofMedical Statistics andBiometry, Fondazione IRCCS Istituto Nazionale Tumori, Milan- Italy;There are twomajor approaches ofreal-time PCR quantification: the absolute and the relative method. Thelatterevaluates the change in expression of the target gene relative to areferencegene, whereas the absolute method uses a standard curve to quantifyunknownamount of nucleic acids in a target sample. From a statistical viewpoint, thestandard curve corresponds to the simple linear regression model andthus theabsolute quantification method allows to exploit the methodologicalbackgroundof the linear regression theory. By means of a technique known asinverseregression, the fitted standard curve is used as calibrator to estimatetheunknown nucleic acid concentration in the target sample.
Severalapproacheshave been proposed for constructing confidence intervals in inverseregression.We propose an user-friendly algorithm, named BCI (Bootstrap ConfidenceInterval), specifically designed to compute bootstrap-t confidenceinterval fornucleic acid concentration by absolute real-time PCR. The algorithm hasbeenwritten in R language an open-source statistical software and providesthebootstrap estimate of the unknown concentration both in logarithmicscale andin its original scale as copy number together with the lower and upperlimitsof the 100(1-α)% bootstrap-t confidence interval of the unknownconcentration.Users can modify the number of bootstrap resampling and the confidencelevel(1- α) of the bootstrap-t confidence interval. P003Dataanalysis for gene quantification and expression profiling using GenEx.AndersBergkvistMultiDAnalyses AB, Sweden;As the datasize and complexity fromqPCR projects increase, the need for comprehensive automated orsemi-automatedsoftware tools increase rapidly. Software tools can provide supportafter datacollection by providing data pre-processing, statistical analysis andvisualization capabilities, often for hypothesis generating purposes.Alternatively they can be used prior to experimental realization byhelping todefine experimental design parameters for hypothesis validation assays.TheGenEx software from MultiD Analyses AB provides all of the capabilitiesmentioned above. Performing accurate qPCR data pre-processing is veryimportant, particularly for quantification purposes.
Many steps areusuallyimplemented and it is useful to follow protocols in order to avoidintroductionof unwarranted processing variability and bias. The protocol availablein GenExis easy to adapt to user-specific needs while at the same timecomprehensiveenough to enable users to easily perform accurate pre-processing. Asscientistswe believe observations we make are manifestations of rationalprocesses. Ourchallenge is to identify the particular rational process that we wantto studywhile minimizing contributions from rational processes that wouldobscure theunderstanding of our particular study. Contributions from unwantedprocessesare often called random although the underlying processes may not be.Tools todifferentiate between contributions from desired and unwanted processesincludeparametric and non-parametric statistical tests, scatterplots,principlecomponent analyses and neural network analyses. These tests andanalyses and moreare available in GenEx.
Validation of scientific conclusions is notabsolute,but based on reproducibility. No scientific theory is above scrutinyandpotential revision. However, based on certain assumptions, anincreasing numberof observations that supports a particular conclusion will alsoincrease ourconfidence that the particular conclusion is going to continue to besupportedby future observations. We may thus define a level of confidence bywhich wewould assign our conclusion to be “true”. A good experimental designaimed tovalidate a hypothesis should therefore include the number of necessaryobservations needed to obtain the desired level of confidence, beforerealization of the experiment. Based on a hypothesis of an observedeffect,including desired level of confidence, amplitude of desired observedeffect andvariability of confounding effects, GenEx can calculate the number ofnecessaryobservations. The presentation will focus on important considerationsforrunning qPCR experiments and ways the GenEx software may providesupport.
P004Mario Cunha,Luis Martins and Carmo OrnelasInstitutoPortugues Oncologia deLisboa Francisco Gentil, EPE, Virology Lab, Portugal;INTRODUCTION- Real Time PCR is amethodology with increasing applications in the clinical laboratory.This newand revolutionary method combines the PCR chemistry with thefluorescentprobe/dye detection of the amplified product, all in the same reactiontube.Since the equipment used can record the emission of fluorescence duringall thecycles of amplification, a significant increase of the PCR product isdirectlylinked with the initial amount of target DNA. In Real Time PCR, we candetermine a fixed fluorescent treshold, above the background. When thePCRproduct that we want to detect cross this threshold, we can determine aparameter named Cycle Threshold (Ct). All the equipment used in RealTime PCRexperiments have some kind of software to analyse the data, namely theanalysisof the expression of Ct value relatively to the logDNA. However, thissoftware doesn’t give much details regarding the linear regression: itonlycalculates the slope, y intercept and coefficient of determination(R2). AIM - Developmentof an Excel sheet that calculates several parameters regarding thelinearregression and assay validation/calibration. RESULTS - We havedeveloped anExcel sheet that uses the data from calibration data (4 differentconcentrations of Virus) in order to determine the following parameters:.
Slope. Y Intercept.
Coefficientof Determination. EfficiencyAmplification. Detectionand quantification limit(analytical and method). StandardError (RMSE). P-Valueassociated with the linearregression (validation)Relatively tothe linear regressionparameters, we have introduced the ANOVA (Analysis of Variance) inorder todetermine the Sum of Squares of: Regression, Residual, Lack of Fit andPureError. With this approach we can make an objective analyses of thegoodness offit, residuals, determine outliers and the confidence interval of thesampleswith viral load.
Since our laboratory is accredited (ISO 17025: 2005),it alsohelps in the maintenance of records relatively to batch/expire date ofDNA/RNAextraction kit, primers, probes, master mix, internal control(amplification)and the reaction plates. CONCLUSION - This Excel sheet is a very goodalternative to the data analysis of the standard software present inthevarious Real Time PCR equipments. Zimmermann 1,M. Laule 1, P.Widmayer 1, W.
Gruissem 1,M.Docquier 2and P. Descombes 21 ETHZurich & NEBION AG, Switzerland;2University of Geneva, Switzerland;Referencegenes (or “housekeepinggenes”) are often used as internal controls for transcriptquantificationassays.
Often, classical reference genes such as GAPDH or TUBB are notsuitablefor one’s own experimental condition because their expression variessignificantly. The goal of RefGenes is to identify, from a genome-wideset ofgenes, those that are most suitable for a given condition. This isachieved byscreening Genevestigator’s large expression compendium (27,000microarrays). Validation experiments on plant and animal qRT-PCRexperimentsshowed that genes found through RefGenes performed significantly betterasnormalizers than classical reference genes. P006AHigh Throughput, Quantitative Real Time PCR Method for theDetermination ofCopy Number Variation in Knockout MiceKellyWarrington 1, LisaBrackenbury 2 and Alexander Sartori 31 AppliedBiosystems, Warrington, United Kingdom; 2Wellcome TrustSangerInstitute, Cambridge, United Kingdom; 3Applied Biosystems,Darmstadt, Germany;Knockout miceare important tools instudying gene function and investigating genetic disorders as completeloss ofgene function is established.
A knockout mouse is generated byreplacing bothalleles of a target gene within an embryonic stem cell. This is doneusing agene trap vector to generate a chimera (F1), selective breeding resultsin F2progeny and subsequent knockout mice (F3). At each stage a combinationofgenotypes is possible. The European Conditional Mouse Mutagenesisprogramme(EUCOMM) and the Knockout Mouse Project (KOMP), are utilising thisapproach toknockout 20,000 genes, to provide a public resource of thousands ofknockoutmice by 2011.A majorchallenge when generatingknockout mice is genotyping the F1-F3 progeny.
The current method ofscreeningthese mice involves sequencing and long range PCR. The former techniqueis timeconsuming and laborious where as the latter technique is unable toaccuratelydistinguish between the different genotypes. We have developed a rapidandaccurate high throughput quantitative real time PCR method to determineaccurately genotype transgenic mice and their progeny.
Furthermore,this highthroughput approach can also be used to study human copy numbervariation.Genetic variation and in the human genome can cause susceptibility orresistance to disease. Human copy number variation has been associatedwith avariety of diseases such as cancer, HIV infection, inflammatoryautoimmunedisorders, autism and schizophrenia. P007TaqMan®OpenArrayTM: A Breakthrough System For Nano-Well High ThroughputGenotypingAlexanderSartori 1, AstridFerlinz 1 and David Merrill 21 AppliedBiosystems, part of Life Technologies, Germany;2Applied Biosystems, part of Life Technologies, Foster City, USA;Molecularresearch and analysis inthe field of plant and animal genomics is rapidly developing andexpanding intonew application areas, requiring the development and introduction ofnewtechnologies, assays and tools to support his research. Within thisbroad fieldof research, marker-assisted selection, QTL mapping and backcrossanalysis arewidely used techniques to identify genes, loci and polymorphismsencoded intothe genomes of all living organisms. Here we present the TaqMan®OpenArrayTMsystem, a breakthrough technology uniquely positioned for these kindsofapplications. By combining gold standard TaqMan® SNP Genotypingassays with theefficient nano-reaction OpenArray™ platform, researchers are able togenotypelarge numbers of samples over any customizable set of markers with asingleplatform instrument. The TaqMan® OpenArray™ system utilizes 3072nano-wellplates, innovative fluidic properties and a one day protocol togenerate highquality, robust genotype profiles.
This platform is especially enablingforresearchers who have continuous streams of incoming samples thatrequire aquick and cost-efficient workflow. To demonstrate the capabilities ofthisplatform, we have genotyped several hundred single nucleotidepolymorphismsacross a panel of genomic DNA samples and report the performance interms ofassay pass rate, sample call rate and concordance to conventionalTaqMan® SNPGenotyping. In addition, innovative solutions for research projectswith samplequantity limitations have recently been developed and will be presentedherefor review.
In summary, we will discuss how this system differentiatesitselffrom the current genotyping technologies. P008SamuelArvidsson 1,2,Miroslaw Kwasniewski 1,2,3, Diego Mauricio Riano-Pachon 2and Bernd Mueller-Roeber 1,21 PotsdamUniversity, Germany; 2Max-Planck Institute for MolecularPlantPhysiology, Potsdam, Germany; 3University of Silesia,Katowice,Poland;Medium- tolarge-scale expressionprofiling using quantitative polymerase chain reaction (qPCR) assaysarebecoming increasingly important in genomics research. A majorbottleneck inexperiment preparation is the design of specific primer pairs, whereresearchers have to make several informed choices, often outside theirarea ofexpertise. Using currently available primer design tools, severalinteractivedecisions have to be made, resulting in lengthy design processes withvaryingqualities of the assays. Here we present QuantPrime, an intuitive anduser-friendly, fully automated tool for primer pair design in small- tolarge-scale qPCR analyses.
QuantPrime can be used online through theinternet(or on a local computer after download; itoffersdesign and specificity checking with highly customizable parameters andisready to use with many publicly available transcriptomes of importanthighereukaryotic model organisms and plant crops (currently 295 species intotal),while benefiting from exon-intron border and alternative splice variantinformation in available genome annotations. Experimental results withthemodel plant Arabidopsis thaliana, the crop Hordeum vulgare and themodel greenalga Chlamydomonas reinhardtii show success rates of designed primerpairsexceeding 96%. QuantPrime constitutes a flexible, fully automated webapplication for reliable primer design for use in larger qPCRexperiments, asproven by experimental data. The flexible framework is also open forsimple usein other quantification applications, such as hydrolyzation probedesign forqPCR and oligonucleotide probe design for quantitative in situhybridization.Future suggestions made by users can be easily implemented, thusallowingQuantPrime to be developed into a broad-range platform for the designof expressionassays. P009Evaluationof Digital PCR for Absolute QuantificationAlisonDevonshire,Ramnath Elaswarapu and Carole FoyLGC, United Kingdom;Digital PCRinvolves diluting andpartitioning a sample between many hundreds or thousands of individualPCRreactions such that a single molecule or less on average is present ineachreaction. Determination of the number of positive amplifications isindicativeof the number of targets present in the sample. As such, digital PCRdoes notsuffer from inaccuracies that often arise from standard real-time PCRapproaches at trace levels using calibration curves, for examplethroughextrapolation, and it affords the potential for absolutequantification.
Recentadvances in microfluidics have facilitated the development of digitalPCR bycombining microfluidics with single molecule, nanolitre volume PCR toincreaselevels of replication, throughput and cost efficiency.In this studywe have evaluated theperformance of digital PCR for absolute quantification using theFluidigmBioMark Integrated Microfluidics System as compared to standardreal-time PCRusing the ABI 7900HT system. The performance of the systems wasevaluated usinga DNA standard of known molecular weight.
The ability of the twoplatforms todiscriminate changes in copy number was assessed using different ratiosof RNAstandards in a background of human total RNA. Sensitivity wasinvestigatedusing different starting amounts of the RNA standards.The resultsof this study arepresented here and demonstrate the potential of digital PCR approachesforabsolute quantification. They also highlight some of the areas ofuncertaintyand variability that are inherent with this approach.Acknowledgements:This work wassupported by a grantfrom the Department of Innovation, Universities and Skills (U.K.) undertheNational Measurement System Chemical and Biological Metrology Programme. P012Anovel digital technology for non-enzymatic direct multiplexedmeasurement ofgene expressionChaybani,Ramin, GaryK. Geiss 1, Roger Bumgarner 2, Brian Birditt 1,Timothy Dahl 1, Naeem Dowidar 1, Dwayne L. Dunaway 1,Perry Fell 1, Sean Ferree 1, Renee D. George 1,Tammy Grogan 1, Jeffrey J.
James 1, Malini Maysuria 1,Jeffrey D. Mitton 1, Paola Oliveri 4, Jennifer L.Osborn 3,Tao Peng 2, Amber L.
Ratcliffe 1, Philippa J.Webster 1,Eric H. Davidson 4 and Leroy Hood 51 NanoStringTechnologies Inc., 201 Elliott Ave West, Suite 300, Seattle, WA 98119; 2TheDepartment of Microbiology, Box 358070, University of Washington,Seattle WA98195; 3Current address, Department of Bioengineering, Box355061,University of Washington, Seattle WA 98195; 4Division ofBiology156-29, California Institute of Technology, Pasadena CA 91125; 5TheInstitute of Systems Biology, 1441 N.
34th St., Seattle WA 98103;We describe anovel technology, thenCounter system, for highly multiplexed analysis of gene expressionlevels.Then nCounter system detects individual mRNA molecules using anassigned codesequence of fluorescent molecules, and counts the number of times thatcodeappears in a sample. No enzymes are used in our system; rather, thecollectionof probes is hybridized in solution to RNA in a sample. Experimentsperformedin a single multiplex analysis of 550 human genes revealed acorrelationcoefficient of 0.999 between replicate measurements, a detection limitbetween0.1fM (0.2 copies/cell) and 0.5fM (1 copy/cell), and a linear 500-folddynamicrange.
The nCounter system can detect a 1.5-fold increase or decreaseinexpression across a broad range of expression, and as little as 20%changes inexpression for genes present between 1fM and 10fM. We demonstrate agoodcorrelation between nCounter system and Affymetrix GeneChip technology,andbetter correlation with TaqMan, for –fold change measurements using twodifferent experimental paradigms. Furthermore, a comparison oftranscriptlevels measured by the nCounter system with SYBR green RT-PCRdemonstrated ahigh correlation in the gene expression pattern at all transcriptlevels. Weshow that a whole cell lysate can be used as starting material withequivalentresults to purified total RNA. Finally, we show that RNA extracted fromformalin-fixed paraffin embedded (FFPE) tissues can be used in thenCountersystem to analyze expression levels in archived samples.
Our uniquedirectdetection and digital quantification approach results in unprecedentedsensitivity,precision and reproducibility in gene expression analyses. Materials&Methods - nCounter hybridization reactions were performed in triplicatewithtotal RNA samples isolated from mock and polio virus infected humanA549 cells.nCounter reactions were set up as follows: 100ng of total RNA Reporterandcapture probes for 509 human mRNAs and controls made to non-humansequences (6positive, 2 negative) DNA control targets spiked in at 0.1, 0.5, 1, 5,10 and50 fM Hybridizations were carried out for 20h at 65°C. Excessreporters werethen removed by using magnetic bead based purification. The samesamples andamount of RNA were also analyzed with Affymetrix® U133Plus2 arrays,using thetwo-cycle amplification/labeling protocol recommended by themanufacturer. Weselected a subset of 14 genes in which the measured log2 fold-changewassignificant in one platform but not the other for further analysis byTaqManReal-Time PCR.
In a second experiment, nCounter hybridization reactionswereperformed in triplicate as described above with total RNA samplesisolated fromsea urchin embryos collected at seven different development timepoints. A setof 21 genes were selected for comparison with existing SYBR GreenReal-Time PCRdata generated in the Davidson Lab. P015Ben AyerSowersBiosearchTechnologies, Inc., UnitedStates of America;Fluorescence-quenchedprobes areroutinely used to gauge gene copy number. We describe a bioinformaticenginefor the design of such oligos, and used to generate five thousandTaqMan assaysfor the NIH Knockout Mouse Project (KOMP). Here, we demonstrate theperformanceof a subset when amplified upon wild-type mouse gDNA.
Analysis of thisdata-setuncovers important trends in amplification performance and emphasizesthe needto screen assay specificity using both bioinformatic and empiricalapproaches.Redundancy and accessibility are considerations that become pronouncedinlarge-volume sequence design. Based on this experience as well as userfeedback, new software functionality is introduced to improve uponthesequalities. P018Importanceof RNA integrity assessment in a qRT-PCR workflowRuedigerSalowskyand Steffen MuellerAgilentTechnologies, Germany;Real-timequantitative PCR (QPCR) isa highly sensitive method to assess gene expression changes inbiologicalsystems. As for all experimental designs, high quality startingmaterial isessential for the success of the experiment.
There are variousmechanisms bywhich RNA can be degraded either at the 5’ or 3’ end. Not knowing theextent ofpossible degradation can lead to false negative results ormisinterpretation ofdata if the amplicon falls into a degraded region. Therefore, thedegradationlevel of RNA samples is an important parameter to monitor whendesigningprimers and probes for QPCR. Here, it's shown how on-chipelectrophoresiscombined with a special RNA Integrity Number (RIN) algorithm can beused toassess the level of degradation of the RNA starting material.
Theresults ofthe experiments indicate that the amount and directionality ofdegradation arehighly gene-dependent and that the most pronounced effects appearsbelow a RINof 4.6. P019RichardMauerer 1, Yana Walczak 2,Jon Sherlock 3, AstridFerlinz 4 and Thomas Langmann 21 SynlabMedical Care Service, Germany; 2Institute of HumanGenetics,University of Regensburg, Germany; 3Applied Biosystems,part of LifeTechnologies, Foster City, USA; 4Applied Biosystems, partof LifeTechnologies, Germany;Cellularlipidomics is defined asthe analysis of metabolism, transport, and localization of lipidsspecieswithin cells. The quantitation of different lipid species from variousbiochemical pathways and biochemical analysis of lipid metabolismenzymes is anintegral part of this concept. Recent progress in the field oftranscriptomics,mainly the cost reduction of DNA-microarrays and the development ofhigh-throughput real-time reverse-transcription (RT)-PCR systems havealsoenabled researchers to perform a comprehensive transcriptomic analysisof alllipid-related genes.Here wedescribe the quantitativeanalysis of 41 selected lipid-related transcripts using a novel“Lipidomic”TaqMan Array. The TaqMan Array is based on an Applied Biosystems 7900HTmicrofluidic card. This method allows simultaneous analysis of 41lipid-relatedgenes and 7 controls in 2 replicates of 4 different samples per run. P020VladimirDenisov 1, William Strong 1, Mark Walder 1,JeffGingrich 1, Teresa Rubio 1 and Arnaud Remy 21 Bio-RadLaboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547; 2Bio-RadBioRecherche, 3 Blvd R.
Poincare, 92430 Marnes la Coquette, France;RNA qualityplays a major role inthe generation of accurate quantitative results from gene expressionanalysisexperiments. CDNA made from RNA that has been degraded will not becomeamplified to the same degree as cDNA made from intact, undegraded RNA.This canlead to erroneous conclusions regarding levels of gene expression whencomparing samples that are degraded to different extents. To examinetheeffects of RNA degradation on quantitation of specific genetranscripts, qPCRwas performed on equivalent amounts of RNA that had been degraded tovariousextents. The detection of amplified product was seen at successivelylatercycles as the RNA was degraded over time. The CT values of the qPCRreactionsfrom five gene transcripts (18S rRNA, β-actin, β-tubulin, HPRT andGADPH)showed different degradation rates. Comparing qPCR results derived fromRNA indifferent states of degradation will generate very differentquantitativeconclusions. This can be as great as 1000 fold, with samples subjectedto 7 hrof heat degradation.
The Experion automated electrophoresis system(Bio-RadLaboratories, Inc.) provides an effective method for determining boththequality and quantity of RNA in gene expression analysis experimentsusing aslittle as 200 pg of total RNA - several thousand times less materialthan thatrequired for gel electrophoresis. The calculation of the RQI uses analgorithmthat compares three regions of an electrophoretic profile, withdifferentialweighting, to a series of degradation RNA standards scale from 10(intact) to 1(fully degraded). The very simple concept behind the RQI gives resultsthat arecomparable to the RIN. The RQI is accurately calculated over a widerange ofRNA concentrations (200 pg to 500 ng), is very reproducible (%CV. P021Thomas BealsThorneDiagnostics, United States of America;The use ofmodel-based amplificationkinetics parameter estimates may improve the accuracy and productivityofreal-time amplification by extracting more information from eachexperiment.Algorithms implemented for PCR analysis can be usefully applied to2-primerramified single-stranded circle amplification (RAM.)Although RAMand PCR kinetics aresufficiently similar to be analyzed with implementations of the samealgorithmsthere are essential differences between the two technologies. Onesignificantdifference for kinetic analysis is that the isothermal RAM reaction canbesampled at a higher frequency than the PCR, as PCR is limited to onedata pointper cycle (a cycle can be sampled multiple times but those samples aimtoward asingle point-estimate.) By contrast, RAM kinetic data can be collectedcontinuously (limited only by instrumentation.) Greater samplingdensity allowsmore precise identification of kinetic phase transition (e.g. Baselinetoexponential phase, exponential to linear phase.)Here theapplication of kineticparameter identification to RAM amplification is shown, and compared toanalogous PCR analysis.
While fitting parameterized models to RAMkinetics isdone as for PCR, the interpretation of a RAM amplification fitted modelisanalogous but distinct from the interpretation of a PCR model. Forexample, PCRefficiency (signal increase per cycle) doubles at its theoreticalmaximum; inRAM the analogous interpretation is signal increase per time unit, andmeasuresthe rate of the reaction.RAMamplification, like the PCR, canbe used in high-throughput diagnostic assays. It is hoped that a morequantitative understanding of the RAM reaction will encourage broaderapplication of the technology. P022Afif MichelAbdelnour 1,Diana Ringot 1, Elie K Barbour 2 and Abalo Chango 11 Laboratoryof Nutritional Genomics, Institut Polytechnique LaSalle Beauvais,France; 2AnimalScience Department, Faculty of Agricultural and Food Sciences, AmericanUniversity of Beirut, Lebanon;We havedeveloped and validated analternative method of the absolute quantitative real-time PCR based onthe useof plasmid.
Our method uses a Bacterial Artificial Chromosome vectorpBeloBAC11. In contrast of plasmid, pBeloBAC11 is present in a singlecopynumber in the bacterium E. Taking benefit of that weconstructed areliable standards curve based on initial input amount of BAC vectorharboringa single copy of the human Reduced Folate Carrier transcript (hrfc) andtheFolate Binding Protein transcript (fbp). Standard curves for each assaywerehighly reproducible with no significant difference in slopes betweenthreedifferent runs of the three different assays. The dynamic ranges werewide,ranging from 1x102 to 1x107 copies. The linearity R2 coefficient of Ctwas 0.99for the recombinant BAC. Q-PCR efficiencies were 0.991 (CV=0.09%) and0.992(CV=0.06%) for hrfc and fbp, respectively.
The method has been appliedforsimultaneous quantification of the hrfc and fbp transcripts in tumortissuesand in their matched adjacent normal tissues. The method is sensitiveandproduces quantitative data with a good efficiency. It may be usedroutinely formeasuring multiple gene expression in diseases evolution. P023Nicky Quispe,Saima Nayab and IanKavanaghThermo FisherScientific, ABgeneHouse, Blenheim Road,Epsom KT199AP United Kingdom;DNAcontamination can often occur inquantitative reverse transcription – polymerase chain reactions(QRT-PCR), andshould be removed in order to avoid false positive results. DNase I iscommonlyused for removing DNA contamination, but this has a relatively long andharshprotocol which introduces an extra step between the isolation of RNAand theQRT-PCR reaction itself, as well as increasing the risk of RNAdegradation dueto the harsh inactivation conditions.
A nuclease from the arctic shrimpPandalusborealis, has properties that make it useful for the removal ofcontaminating DNA. The nuclease activity of the enzyme is specific todoublestranded DNA, which therefore allows the enzyme to be added directlyinto thereverse transcription step. Unlike DNase I, the shrimp nuclease iseasilyinactivated at high temperatures, such as those used for the RTdeactivation/hot start incubation step of a QRT-PCR. Here we show howtheaddition of shrimp nuclease during the reverse transcriptase step canremovecontaminating DNA from the reaction. Human genomic DNA (100ng -10pg)wasincubated with or without shrimp nuclease (10 – 0.1 units) beforeamplificationof a 74bp fragment of the Apolipoprotein B gene was carried out. Thepercentageremoval of genomic DNA was calculated by comparing the delta Ct valuesbetweenreactions where the DNA was pre-incubated with shrimp nuclease toreactionsincubated without shrimp nuclease (control). In order to determinewhether theCt shift was caused by inhibition of QPCR by shrimp nuclease, the sameunits ofpre-inactivated shrimp nuclease was added to a separate reaction.Whilst largeCt shifts were observed at all DNA concentrations for 10-1 units ofshrimpnuclease, there was also significant inhibition caused by the additionofinactivated enzyme at these concentrations.
Rotor Gene 6000 Real-time Pcr
Therefore, not all of theCt shiftcan be attributed to removal of contaminating DNA. However, atconcentrationsof between 0.8 – 0.4 units, there was no inhibition observed byinactivatedshrimp nuclease and between 99.4% - 100% removal of the DNA wasobserveddepending on the concentration. Therefore, at 0.8 – 0.4 units thisenzymeeffectively removes any contaminating DNA without adding an extra stepto theQRT-PCR protocol - and completely eliminates the need for DNase Itreatment.
Inso doing, it increases the accuracy and reproducibility of QRT-PCRreactions,especially when using crudely purified samples. P024Gerwyn Jones,Srujana Kapavarapu,Saima Nayab and Ian KavanaghThermo FisherScientific, ABgeneHouse, Blenheim Road,Epsom KT199AP United Kingdom;Adapting QPCRexperiments to runusing fast cycling conditions is a simple method of increasingexperimentalthroughput by reducing run duration times by up to 50%.
QPCR resultsand thequality thereof can be greatly affected by the characteristics of theproductbeing amplified. This is especially true when employing fast thermalcyclingprotocols, which have been shown to reduce the sensitivity and increasevariability of some assays. Speeding up thermal cycling protocols canlead toreaction failure if attempting to amplify a non-optimal assay ordifficulttarget. Therefore we have investigated the effects of amplicon length,GCcontent and secondary structure, on the performance of fast QPCRexperimentscompared to when a standard thermal cycling protocol is used. A panelof humanspecific assays were employed in QPCR experiments using fast andstandardcycling protocols. The assays were designed so that the resultingamplicons hada broad range of length, GC content and secondary structure (minimum ΔGvaluescalculated at 60C). Experiments were carried out on the RocheLightCycler 480and assay sensitivity was assessed by comparing delta Cp values,standarddeviations and differences in QPCR efficiency (calculated by thestandard curvemethod).
The results demonstrate that amplicons that are of excessivelength,high in secondary structure or high in GC content can all be causes forpoorfast QPCR results. Therefore, we recommend that assays be designed withtheseamplicon characteristics taken into account, in order to ensuresuccessful fastQPCR experiments. P025Simon Baker 2,PhillipHarries 2 and Ian Kavanagh 11 ThermoFisher Scientific, ABgene House, Blenheim Road, Epsom KT19 9AP.
UnitedKingdom; 2School of Life Sciences, Oxford BrookesUniversity,Gypsy Lane,Oxford OX3 0BP. United Kingdom;QPCRinstruments are able to monitoramplicon quantity in real-time during PCR reactions by detectingfluorescencesignals and recording fluorescence data. The majority of fluorescenceisreflected out of the PCR tube either by the polypropylene itself or bythewalls of the thermal cycler block, when clear polypropylene is used. Aims: To investigate if white 96-well platesallow better fluorescence detection of PCR products during QPCR assaysthanclear 96-well plates. Methods: QPCR experiments were performed by amplifying targetsegments ofthe genes sigB, dnaK, srfAA, and argBfrom thegenome of Bacillus subtilis BBK006. SYBR Green I was used tomonitorproduct accumulation via fluorescence. To examine the effect ofpigmentation onCT, 96-well plates were custom-manufactured with elevated pigment,reducedpigment or differently-compounded white polypropylene.Results: In generalQPCR reactions on whiteplates have lower CT values compared to QPCR reactions on clear plates.Statistical analysis revealed there was no significant differencebetween CTvalues of sets dnaK and argB when using clear andwhite plates.However, analysis of melt curves suggested this was due to poor primerhybridisation.
Further analysis revealed that CT values of sets srfAAand argB were significantly lower when using whiteplatescompared to clearplates. There was also a significant difference between melting curvesof lowtemplate DNA reactions when using clear and white plates. Conclusions:It was observed that signal noise was more prevalent in the meltingcurves ofreactions using clear plates than white plates. There were nosignificantdifferences between CT values and amplification plots of low templateDNAreactions when using the standard white plate compared to plates withalteredpigmentation. This, coupled to examination of the plates by scanningfluorimetry, suggests that the effect of the pigment is complex and notnecessarily linked to reflection of signal alone. P026Alignmentof the heat shock protein gene (hsp) sequences and development ofmultiplex PCRmethod for the simultaneous detection of bovine mastitis pathogensincludingStaphylococcus aureus, and Streptococcus spp.Hau-Yang Tsen,Yu-Hsin Chang, Wan-Yu Pai and Yu-Cheng ChiangHung KuangUniversity, Taiwan;Bovinemastitis is a multifactorialdisease caused by many different bacteria species. Of these bacteriaspecies,Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis,andStreptococcus bovis are the major pathogens.
Since the heat shockprotein (hsp)genes, especially hsp60, has been shown to have more discriminatorypower than16S rRNA gene and inter-transcriptional spacer (ITS) region, in thisstudy, wetried to develop a multiplex PCR method based on the hsp (heat shockprotein)genes for the specific detection of S. Agalactiae, S.uberis, and S.bovis. Molecular weight of the PCR products amplified were 406 bp,350bp,119bp, and 247bp, respectively, for S. Agalactiae, S.uberis, and S.bovis. Using this multiplex PCR method, all the selected target strainscouldbe specifically detected in food samples. As the multiplex PCR methodwas usedfor direct detection of mastitis pathogens in milk samples, thedetection limitwas N (N= 1–9) ×103 CFU/ml of milk samples.
If a 10 hpre-enrichment step wasperformed, the detection limit was N×100 CFU/ml. Thus, themultiplex PCR methodcould be used for the specific and sensitive detection of thesepathogenicbacteria in food and milk samples. P027qPCRas a method to estimate synchronization of immortal Hepa 1-6 cells –problemswith inhibitors and low abundant genesUrsula Prosenc,Klementina Fon Tacer and Damjana RozmanUniversityof Ljubljana, Medicalfaculty, Centerfor functional genomics and bio-chips, Ljubljana,Slovenija;The aim ofour experiment was tosynchronize the mouse immortal hepatoma cells Hepa 1-6 and to prove thesuccessof synchronization by measuring the expression of representative genesfromcholesterol synthesis and circadian regulation. These genes are knownto beexpressed in a circadian manner in the mouse liver. However, many ofthem areexpressed at low levels in immortal cell lines which represents achallenge fortheir quantification.Hepa 1-6cells were grown in 12-wellplates until confluency.
Cells were treated with 10 uM forskolin,washed twicewith ice cold PBS, lysed with TRI reagent (Invitogen) and stored at-80ºC untilRNA isolation was performed according to TRI reagent protocol. Peletwas washedonce with 75% etanol and resuspended in RNAse free water.
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Because oflow RNAyield cDNA was synthesized starting from 200ng of total RNA which is a5 timeslower concentration as usual. QPCR reaction efficiency was evaluatedwithserial dilutions of cDNA with mCyp51 (a gene from cholesterolsynthesis) and18sRNA primers on Light Cycler 480 (Roche) using the cyber greenapproach. Theresults showed that several dilutions result in the same Cp value,while atsome dilutions there was no amplification at all. After testingdifferentserial dilutions the expected results were obtained only if cDNA wasdiluted toa specific (narrow range) concentration. To increase RNA amount sampleswiththe same treatments were pooled, dried and precipitated with sodiumacetate.The qPCR has been repeated with serial dilutions and the expectedamplificationpattern was observed (different Cp values according to dilutions).It wasconcluded that the originalsamples contained an unidentified inhibitor of either qPCR or cDNAsynthesisthat has been removed by drying RNAs followed by another precipitation.Aftersolving this problem we measured the timely expression of Cyp51,Dbp and Bmal1 in a time-series experiment spanning 48hours. Inthe first 8h-12h forskolin provokes an immediate early response of the 3 measuredgenes.According to the expression of Dbp that is abundant also inimmortalcells, we conclude that synchronization of Hepa 1-6 cells line has beensuccessfulwhile the results for Cyp51 and Bmal1 are notconclusive.Additional optimization procedures are required to enable quantitativetime-series measurements of low expressed genes in immortal cells.