Sep 29, 2017. Google Engineer Dismissed the Importance of Stolen Self Driving Car Documents. Googles self driving car company Waymo sued Uber in February, claiming that Anthony Levandowski, a former Waymo engineer, stole 1. Be As Wise As Serpents Pdf. Waymos lidar systems before accepting a job with Ubers. We knew it wouldn’t be long before Congress demanded action in response to the Equifax data breach—particularly since several of its members are among the 143.

P001 Ben Ayer Sowers Biosearch Technologies, Inc., United States of America; Multiplexed qPCR remains a challenging endeavor for reasons that include: 1) designing assays to combine without interference, 2) resolving fluorophores using the optics of each real-time instrument, and 3) optimizing and validating each assay's performance. Here, we address each of these issues when developing several pentaplexed assays that target genes from human and mouse. Each assay was designed using a free, online, software program that carefully considers inter-oligo interactions while simultaneously building its multiplexed set. Situations of disproportionate copy number present a particular challenge upon multiplexed performance; additional validation is needed to define the limits of a multiplexed set, as compared to individually amplified assays. P002 Sara Pizzamiglio and Paolo Verderio Unit of Medical Statistics and Biometry, Fondazione IRCCS Istituto Nazionale Tumori, Milan - Italy; There are two major approaches of real-time PCR quantification: the absolute and the relative method. The latter evaluates the change in expression of the target gene relative to a reference gene, whereas the absolute method uses a standard curve to quantify unknown amount of nucleic acids in a target sample.

From a statistical view point, the standard curve corresponds to the simple linear regression model and thus the absolute quantification method allows to exploit the methodological background of the linear regression theory. By means of a technique known as inverse regression, the fitted standard curve is used as calibrator to estimate the unknown nucleic acid concentration in the target sample. Several approaches have been proposed for constructing confidence intervals in inverse regression. We propose an user-friendly algorithm, named BCI (Bootstrap Confidence Interval), specifically designed to compute bootstrap-t confidence interval for nucleic acid concentration by absolute real-time PCR. The algorithm has been written in R language an open-source statistical software and provides the bootstrap estimate of the unknown concentration both in logarithmic scale and in its original scale as copy number together with the lower and upper limits of the 100(1-α)% bootstrap-t confidence interval of the unknown concentration. Users can modify the number of bootstrap resampling and the confidence level (1- α) of the bootstrap-t confidence interval.

P003 Data analysis for gene quantification and expression profiling using GenEx. Anders Bergkvist MultiD Analyses AB, Sweden; As the data size and complexity from qPCR projects increase, the need for comprehensive automated or semi-automated software tools increase rapidly. Software tools can provide support after data collection by providing data pre-processing, statistical analysis and visualization capabilities, often for hypothesis generating purposes. Alternatively they can be used prior to experimental realization by helping to define experimental design parameters for hypothesis validation assays. The GenEx software from MultiD Analyses AB provides all of the capabilities mentioned above. Performing accurate qPCR data pre-processing is very important, particularly for quantification purposes.

Many steps are usually implemented and it is useful to follow protocols in order to avoid introduction of unwarranted processing variability and bias. The protocol available in GenEx is easy to adapt to user-specific needs while at the same time comprehensive enough to enable users to easily perform accurate pre-processing. As scientists we believe observations we make are manifestations of rational processes. Our challenge is to identify the particular rational process that we want to study while minimizing contributions from rational processes that would obscure the understanding of our particular study. Contributions from unwanted processes are often called random although the underlying processes may not be. Tools to differentiate between contributions from desired and unwanted processes include parametric and non-parametric statistical tests, scatterplots, principle component analyses and neural network analyses.

These tests and analyses and more are available in GenEx. Validation of scientific conclusions is not absolute, but based on reproducibility. No scientific theory is above scrutiny and potential revision. However, based on certain assumptions, an increasing number of observations that supports a particular conclusion will also increase our confidence that the particular conclusion is going to continue to be supported by future observations. We may thus define a level of confidence by which we would assign our conclusion to be “true”. A good experimental design aimed to validate a hypothesis should therefore include the number of necessary observations needed to obtain the desired level of confidence, before realization of the experiment. Based on a hypothesis of an observed effect, including desired level of confidence, amplitude of desired observed effect and variability of confounding effects, GenEx can calculate the number of necessary observations.

The presentation will focus on important considerations for running qPCR experiments and ways the GenEx software may provide support. P004 Mario Cunha, Luis Martins and Carmo Ornelas Instituto Portugues Oncologia de Lisboa Francisco Gentil, EPE, Virology Lab, Portugal; INTRODUCTION - Real Time PCR is a methodology with increasing applications in the clinical laboratory.

This new and revolutionary method combines the PCR chemistry with the fluorescent probe/dye detection of the amplified product, all in the same reaction tube. Since the equipment used can record the emission of fluorescence during all the cycles of amplification, a significant increase of the PCR product is directly linked with the initial amount of target DNA. In Real Time PCR, we can determine a fixed fluorescent treshold, above the background. When the PCR product that we want to detect cross this threshold, we can determine a parameter named Cycle Threshold (Ct).

All the equipment used in Real Time PCR experiments have some kind of software to analyse the data, namely the analysis of the expression of Ct value relatively to the log[DNA]. However, this software doesn’t give much details regarding the linear regression: it only calculates the slope, y intercept and coefficient of determination (R2). AIM - Development of an Excel sheet that calculates several parameters regarding the linear regression and assay validation/calibration. RESULTS - We have developed an Excel sheet that uses the data from calibration data (4 different concentrations of Virus) in order to determine the following parameters: • Slope • Y Intercept • Coefficient of Determination • Efficiency Amplification • Detection and quantification limit (analytical and method) • Standard Error (RMSE) • P-Value associated with the linear regression (validation) Relatively to the linear regression parameters, we have introduced the ANOVA (Analysis of Variance) in order to determine the Sum of Squares of: Regression, Residual, Lack of Fit and Pure Error. With this approach we can make an objective analyses of the goodness of fit, residuals, determine outliers and the confidence interval of the samples with viral load. Since our laboratory is accredited (ISO 17025: 2005), it also helps in the maintenance of records relatively to batch/expire date of DNA/RNA extraction kit, primers, probes, master mix, internal control (amplification) and the reaction plates.

CONCLUSION - This Excel sheet is a very good alternative to the data analysis of the standard software present in the various Real Time PCR equipments. Zimmermann 1, M. Widmayer 1, W. Gruissem 1, M.

Docquier 2 and P. Descombes 2 1 ETH Zurich & NEBION AG, Switzerland; 2University of Geneva, Switzerland; Reference genes (or “housekeeping genes”) are often used as internal controls for transcript quantification assays.

Often, classical reference genes such as GAPDH or TUBB are not suitable for one’s own experimental condition because their expression varies significantly. The goal of RefGenes is to identify, from a genome-wide set of genes, those that are most suitable for a given condition.

This is achieved by screening Genevestigator’s large expression compendium (>27,000 microarrays). Validation experiments on plant and animal qRT-PCR experiments showed that genes found through RefGenes performed significantly better as normalizers than classical reference genes. P006 A High Throughput, Quantitative Real Time PCR Method for the Determination of Copy Number Variation in Knockout Mice Kelly Warrington 1, Lisa Brackenbury 2 and Alexander Sartori 3 1 Applied Biosystems, Warrington, United Kingdom; 2Wellcome Trust Sanger Institute, Cambridge, United Kingdom; 3Applied Biosystems, Darmstadt, Germany; Knockout mice are important tools in studying gene function and investigating genetic disorders as complete loss of gene function is established. A knockout mouse is generated by replacing both alleles of a target gene within an embryonic stem cell. This is done using a gene trap vector to generate a chimera (F1), selective breeding results in F2 progeny and subsequent knockout mice (F3). At each stage a combination of genotypes is possible.

The European Conditional Mouse Mutagenesis programme (EUCOMM) and the Knockout Mouse Project (KOMP), are utilising this approach to knockout ~ 20,000 genes, to provide a public resource of thousands of knockout mice by 2011. A major challenge when generating knockout mice is genotyping the F1-F3 progeny.

The current method of screening these mice involves sequencing and long range PCR. The former technique is time consuming and laborious where as the latter technique is unable to accurately distinguish between the different genotypes. We have developed a rapid and accurate high throughput quantitative real time PCR method to determine accurately genotype transgenic mice and their progeny. Furthermore, this high throughput approach can also be used to study human copy number variation.

Genetic variation and in the human genome can cause susceptibility or resistance to disease. Human copy number variation has been associated with a variety of diseases such as cancer, HIV infection, inflammatory autoimmune disorders, autism and schizophrenia. P007 TaqMan® OpenArrayTM: A Breakthrough System For Nano-Well High Throughput Genotyping Alexander Sartori 1, Astrid Ferlinz 1 and David Merrill 2 1 Applied Biosystems, part of Life Technologies, Germany; 2Applied Biosystems, part of Life Technologies, Foster City, USA; Molecular research and analysis in the field of plant and animal genomics is rapidly developing and expanding into new application areas, requiring the development and introduction of new technologies, assays and tools to support his research.

Within this broad field of research, marker-assisted selection, QTL mapping and backcross analysis are widely used techniques to identify genes, loci and polymorphisms encoded into the genomes of all living organisms. Here we present the TaqMan® OpenArrayTM system, a breakthrough technology uniquely positioned for these kinds of applications. By combining gold standard TaqMan® SNP Genotyping assays with the efficient nano-reaction OpenArray™ platform, researchers are able to genotype large numbers of samples over any customizable set of markers with a single platform instrument. The TaqMan® OpenArray™ system utilizes 3072 nano-well plates, innovative fluidic properties and a one day protocol to generate high quality, robust genotype profiles. This platform is especially enabling for researchers who have continuous streams of incoming samples that require a quick and cost-efficient workflow. To demonstrate the capabilities of this platform, we have genotyped several hundred single nucleotide polymorphisms across a panel of genomic DNA samples and report the performance in terms of assay pass rate, sample call rate and concordance to conventional TaqMan® SNP Genotyping. In addition, innovative solutions for research projects with sample quantity limitations have recently been developed and will be presented here for review.

In summary, we will discuss how this system differentiates itself from the current genotyping technologies. P008 Samuel Arvidsson 1,2, Miroslaw Kwasniewski 1,2,3, Diego Mauricio Riano-Pachon 2 and Bernd Mueller-Roeber 1,2 1 Potsdam University, Germany; 2Max-Planck Institute for Molecular Plant Physiology, Potsdam, Germany; 3University of Silesia, Katowice, Poland; Medium- to large-scale expression profiling using quantitative polymerase chain reaction (qPCR) assays are becoming increasingly important in genomics research. A major bottleneck in experiment preparation is the design of specific primer pairs, where researchers have to make several informed choices, often outside their area of expertise. Using currently available primer design tools, several interactive decisions have to be made, resulting in lengthy design processes with varying qualities of the assays. Here we present QuantPrime, an intuitive and user-friendly, fully automated tool for primer pair design in small- to large-scale qPCR analyses.

QuantPrime can be used online through the internet (or on a local computer after download; it offers design and specificity checking with highly customizable parameters and is ready to use with many publicly available transcriptomes of important higher eukaryotic model organisms and plant crops (currently 295 species in total), while benefiting from exon-intron border and alternative splice variant information in available genome annotations. Experimental results with the model plant Arabidopsis thaliana, the crop Hordeum vulgare and the model green alga Chlamydomonas reinhardtii show success rates of designed primer pairs exceeding 96%.

QuantPrime constitutes a flexible, fully automated web application for reliable primer design for use in larger qPCR experiments, as proven by experimental data. The flexible framework is also open for simple use in other quantification applications, such as hydrolyzation probe design for qPCR and oligonucleotide probe design for quantitative in situ hybridization. Future suggestions made by users can be easily implemented, thus allowing QuantPrime to be developed into a broad-range platform for the design of expression assays. P009 Evaluation of Digital PCR for Absolute Quantification Alison Devonshire, Ramnath Elaswarapu and Carole Foy LGC, United Kingdom; Digital PCR involves diluting and partitioning a sample between many hundreds or thousands of individual PCR reactions such that a single molecule or less on average is present in each reaction.

Determination of the number of positive amplifications is indicative of the number of targets present in the sample. As such, digital PCR does not suffer from inaccuracies that often arise from standard real-time PCR approaches at trace levels using calibration curves, for example through extrapolation, and it affords the potential for absolute quantification. Recent advances in microfluidics have facilitated the development of digital PCR by combining microfluidics with single molecule, nanolitre volume PCR to increase levels of replication, throughput and cost efficiency. In this study we have evaluated the performance of digital PCR for absolute quantification using the Fluidigm BioMark Integrated Microfluidics System as compared to standard real-time PCR using the ABI 7900HT system.

The performance of the systems was evaluated using a DNA standard of known molecular weight. The ability of the two platforms to discriminate changes in copy number was assessed using different ratios of RNA standards in a background of human total RNA. Sensitivity was investigated using different starting amounts of the RNA standards. The results of this study are presented here and demonstrate the potential of digital PCR approaches for absolute quantification.

They also highlight some of the areas of uncertainty and variability that are inherent with this approach. Acknowledgements: This work was supported by a grant from the Department of Innovation, Universities and Skills (U.K.) under the National Measurement System Chemical and Biological Metrology Programme. P012 A novel digital technology for non-enzymatic direct multiplexed measurement of gene expression Chaybani, Ramin, Gary K. 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 5 1 NanoString Technologies Inc., 201 Elliott Ave West, Suite 300, Seattle, WA 98119; 2The Department of Microbiology, Box 358070, University of Washington, Seattle WA 98195; 3Current address, Department of Bioengineering, Box 355061, University of Washington, Seattle WA 98195; 4Division of Biology 156-29, California Institute of Technology, Pasadena CA 91125; 5The Institute of Systems Biology, 1441 N. 34th St., Seattle WA 98103; We describe a novel technology, the nCounter system, for highly multiplexed analysis of gene expression levels.

Then nCounter system detects individual mRNA molecules using an assigned code sequence of fluorescent molecules, and counts the number of times that code appears in a sample. No enzymes are used in our system; rather, the collection of probes is hybridized in solution to RNA in a sample.

Experiments performed in a single multiplex analysis of 550 human genes revealed a correlation coefficient of 0.999 between replicate measurements, a detection limit between 0.1fM (0.2 copies/cell) and 0.5fM (1 copy/cell), and a linear 500-fold dynamic range. The nCounter system can detect a 1.5-fold increase or decrease in expression across a broad range of expression, and as little as 20% changes in expression for genes present between 1fM and 10fM. We demonstrate a good correlation between nCounter system and Affymetrix GeneChip technology, and better correlation with TaqMan, for –fold change measurements using two different experimental paradigms. Furthermore, a comparison of transcript levels measured by the nCounter system with SYBR green RT-PCR demonstrated a high correlation in the gene expression pattern at all transcript levels. We show that a whole cell lysate can be used as starting material with equivalent results to purified total RNA. Finally, we show that RNA extracted from formalin-fixed paraffin embedded (FFPE) tissues can be used in the nCounter system to analyze expression levels in archived samples. Our unique direct detection and digital quantification approach results in unprecedented sensitivity, precision and reproducibility in gene expression analyses.

Materials & Methods - nCounter hybridization reactions were performed in triplicate with total RNA samples isolated from mock and polio virus infected human A549 cells. NCounter reactions were set up as follows: 100ng of total RNA Reporter and capture probes for 509 human mRNAs and controls made to non-human sequences (6 positive, 2 negative) DNA control targets spiked in at 0.1, 0.5, 1, 5, 10 and 50 fM Hybridizations were carried out for 20h at 65°C.

Excess reporters were then removed by using magnetic bead based purification. The same samples and amount of RNA were also analyzed with Affymetrix® U133Plus2 arrays, using the two-cycle amplification/labeling protocol recommended by the manufacturer. We selected a subset of 14 genes in which the measured log2 fold-change was significant in one platform but not the other for further analysis by TaqMan Real-Time PCR.

In a second experiment, nCounter hybridization reactions were performed in triplicate as described above with total RNA samples isolated from sea urchin embryos collected at seven different development time points. A set of 21 genes were selected for comparison with existing SYBR Green Real-Time PCR data generated in the Davidson Lab. P015 Ben Ayer Sowers Biosearch Technologies, Inc., United States of America; Fluorescence-quenched probes are routinely used to gauge gene copy number. We describe a bioinformatic engine for the design of such oligos, and used to generate five thousand TaqMan assays for the NIH Knockout Mouse Project (KOMP). Here, we demonstrate the performance of a subset when amplified upon wild-type mouse gDNA.

Analysis of this data-set uncovers important trends in amplification performance and emphasizes the need to screen assay specificity using both bioinformatic and empirical approaches. Redundancy and accessibility are considerations that become pronounced in large-volume sequence design. Based on this experience as well as user feedback, new software functionality is introduced to improve upon these qualities. P018 Importance of RNA integrity assessment in a qRT-PCR workflow Ruediger Salowsky and Steffen Mueller Agilent Technologies, Germany; Real-time quantitative PCR (QPCR) is a highly sensitive method to assess gene expression changes in biological systems. As for all experimental designs, high quality starting material is essential for the success of the experiment. There are various mechanisms by which RNA can be degraded either at the 5’ or 3’ end. Not knowing the extent of possible degradation can lead to false negative results or misinterpretation of data if the amplicon falls into a degraded region.

Therefore, the degradation level of RNA samples is an important parameter to monitor when designing primers and probes for QPCR. Here, it's shown how on-chip electrophoresis combined with a special RNA Integrity Number (RIN) algorithm can be used to assess the level of degradation of the RNA starting material. The results of the experiments indicate that the amount and directionality of degradation are highly gene-dependent and that the most pronounced effects appears below a RIN of 4.6. P019 Richard Mauerer 1, Yana Walczak 2, Jon Sherlock 3, Astrid Ferlinz 4 and Thomas Langmann 2 1 Synlab Medical Care Service, Germany; 2Institute of Human Genetics, University of Regensburg, Germany; 3Applied Biosystems, part of Life Technologies, Foster City, USA; 4Applied Biosystems, part of Life Technologies, Germany; Cellular lipidomics is defined as the analysis of metabolism, transport, and localization of lipids species within cells. The quantitation of different lipid species from various biochemical pathways and biochemical analysis of lipid metabolism enzymes is an integral part of this concept. Recent progress in the field of transcriptomics, mainly the cost reduction of DNA-microarrays and the development of high-throughput real-time reverse-transcription (RT)-PCR systems have also enabled researchers to perform a comprehensive transcriptomic analysis of all lipid-related genes.

Here we describe the quantitative analysis of 41 selected lipid-related transcripts using a novel “Lipidomic” TaqMan Array. The TaqMan Array is based on an Applied Biosystems 7900HT microfluidic card. This method allows simultaneous analysis of 41 lipid-related genes and 7 controls in 2 replicates of 4 different samples per run. How To Install A Jensen In Dash Dvd Player here. In addition, we analyzed the identical “Lipidomic” gene set and identical RNA samples on the recently launched “TaqMan Express Plates” – customizable 96-well plates with pre-spotted TaqMan Gene Expression Assays. Our special interest was to study the expression of “lipidomic” genes in macrophages and microglia under conditions mimicking sterol loading and pro-inflammatory activation. The TaqMan Array results show that (i) stimulation with the liver-X-receptor (LXR) and retinoid-X-receptor (RXR) ligands T0901317 and 9-cis retinoic acid (RA) induces several genes of lipid metabolism, (ii) lipopolysaccharide (LPS) and interferon-g (Ifn-g) strongly repress lipid-related genes, and (iii) co-incubation with docosahexaenoic acid (DHA) dampens the repressing effect of LPS.

Our results were confirmed by the data obtained with the TaqMan Express Plates. The method described here can be used to rapidly and accurate quantify transcriptionally dynamic “lipid” genes in any cell type. The “lipidomic” TaqMan Assay Set may be applied to study lipid disorders or to quantify the transcriptional effects of pharmacological treatments on lipid-related genes. P020 Vladimir Denisov 1, William Strong 1, Mark Walder 1, Jeff Gingrich 1, Teresa Rubio 1 and Arnaud Remy 2 1 Bio-Rad Laboratories, Inc., 6000 James Watson Drive, Hercules, CA 94547; 2Bio-Rad BioRecherche, 3 Blvd R. Poincare, 92430 Marnes la Coquette, France; RNA quality plays a major role in the generation of accurate quantitative results from gene expression analysis experiments. CDNA made from RNA that has been degraded will not become amplified to the same degree as cDNA made from intact, undegraded RNA.

This can lead to erroneous conclusions regarding levels of gene expression when comparing samples that are degraded to different extents. To examine the effects of RNA degradation on quantitation of specific gene transcripts, qPCR was performed on equivalent amounts of RNA that had been degraded to various extents. The detection of amplified product was seen at successively later cycles as the RNA was degraded over time. The CT values of the qPCR reactions from five gene transcripts (18S rRNA, β-actin, β-tubulin, HPRT and GADPH) showed different degradation rates.

Comparing qPCR results derived from RNA in different states of degradation will generate very different quantitative conclusions. This can be as great as 1000 fold, with samples subjected to 7 hr of heat degradation. The Experion automated electrophoresis system (Bio-Rad Laboratories, Inc.) provides an effective method for determining both the quality and quantity of RNA in gene expression analysis experiments using as little as 200 pg of total RNA - several thousand times less material than that required for gel electrophoresis.

The calculation of the RQI uses an algorithm that compares three regions of an electrophoretic profile, with differential weighting, to a series of degradation RNA standards scale from 10 (intact) to 1 (fully degraded). The very simple concept behind the RQI gives results that are comparable to the RIN. The RQI is accurately calculated over a wide range of RNA concentrations (200 pg to 500 ng), is very reproducible (%CV. P021 Thomas Beals Thorne Diagnostics, United States of America; The use of model-based amplification kinetics parameter estimates may improve the accuracy and productivity of real-time amplification by extracting more information from each experiment.

Algorithms implemented for PCR analysis can be usefully applied to 2-primer ramified single-stranded circle amplification (RAM.) Although RAM and PCR kinetics are sufficiently similar to be analyzed with implementations of the same algorithms there are essential differences between the two technologies. One significant difference for kinetic analysis is that the isothermal RAM reaction can be sampled at a higher frequency than the PCR, as PCR is limited to one data point per cycle (a cycle can be sampled multiple times but those samples aim toward a single point-estimate.) By contrast, RAM kinetic data can be collected continuously (limited only by instrumentation.) Greater sampling density allows more precise identification of kinetic phase transition (e.g. Baseline to exponential phase, exponential to linear phase.) Here the application of kinetic parameter identification to RAM amplification is shown, and compared to analogous PCR analysis. While fitting parameterized models to RAM kinetics is done as for PCR, the interpretation of a RAM amplification fitted model is analogous but distinct from the interpretation of a PCR model.

For example, PCR efficiency (signal increase per cycle) doubles at its theoretical maximum; in RAM the analogous interpretation is signal increase per time unit, and measures the rate of the reaction. RAM amplification, like the PCR, can be used in high-throughput diagnostic assays. It is hoped that a more quantitative understanding of the RAM reaction will encourage broader application of the technology.

P022 Afif Michel Abdelnour 1, Diana Ringot 1, Elie K Barbour 2 and Abalo Chango 1 1 Laboratory of Nutritional Genomics, Institut Polytechnique LaSalle Beauvais, France; 2Animal Science Department, Faculty of Agricultural and Food Sciences, American University of Beirut, Lebanon; We have developed and validated an alternative method of the absolute quantitative real-time PCR based on the use of plasmid. Our method uses a Bacterial Artificial Chromosome vector pBeloBAC11. In contrast of plasmid, pBeloBAC11 is present in a single copy number in the bacterium E. Taking benefit of that we constructed a reliable standards curve based on initial input amount of BAC vector harboring a single copy of the human Reduced Folate Carrier transcript (hrfc) and the Folate Binding Protein transcript (fbp). Standard curves for each assay were highly reproducible with no significant difference in slopes between three different runs of the three different assays.

The dynamic ranges were wide, ranging from 1x102 to 1x107 copies. The linearity R2 coefficient of Ct was 0.99 for the recombinant BAC. Q-PCR efficiencies were 0.991 (CV=0.09%) and 0.992 (CV=0.06%) for hrfc and fbp, respectively. The method has been applied for simultaneous quantification of the hrfc and fbp transcripts in tumor tissues and in their matched adjacent normal tissues.

The method is sensitive and produces quantitative data with a good efficiency. It may be used routinely for measuring multiple gene expression in diseases evolution.

P023 Nicky Quispe, Saima Nayab and Ian Kavanagh Thermo Fisher Scientific, ABgene House, Blenheim Road, Epsom KT19 9AP United Kingdom; DNA contamination can often occur in quantitative reverse transcription – polymerase chain reactions (QRT-PCR), and should be removed in order to avoid false positive results. DNase I is commonly used for removing DNA contamination, but this has a relatively long and harsh protocol which introduces an extra step between the isolation of RNA and the QRT-PCR reaction itself, as well as increasing the risk of RNA degradation due to the harsh inactivation conditions. A nuclease from the arctic shrimp Pandalus borealis, has properties that make it useful for the removal of contaminating DNA.

The nuclease activity of the enzyme is specific to double stranded DNA, which therefore allows the enzyme to be added directly into the reverse transcription step. Unlike DNase I, the shrimp nuclease is easily inactivated at high temperatures, such as those used for the RT deactivation/hot start incubation step of a QRT-PCR. Here we show how the addition of shrimp nuclease during the reverse transcriptase step can remove contaminating DNA from the reaction. Human genomic DNA (100ng -10pg) was incubated with or without shrimp nuclease (10 – 0.1 units) before amplification of a 74bp fragment of the Apolipoprotein B gene was carried out. The percentage removal of genomic DNA was calculated by comparing the delta Ct values between reactions where the DNA was pre-incubated with shrimp nuclease to reactions incubated without shrimp nuclease (control).

In order to determine whether the Ct shift was caused by inhibition of QPCR by shrimp nuclease, the same units of pre-inactivated shrimp nuclease was added to a separate reaction. Whilst large Ct shifts were observed at all DNA concentrations for 10-1 units of shrimp nuclease, there was also significant inhibition caused by the addition of inactivated enzyme at these concentrations. Therefore, not all of the Ct shift can be attributed to removal of contaminating DNA. However, at concentrations of between 0.8 – 0.4 units, there was no inhibition observed by inactivated shrimp nuclease and between 99.4% - 100% removal of the DNA was observed depending on the concentration. Therefore, at 0.8 – 0.4 units this enzyme effectively removes any contaminating DNA without adding an extra step to the QRT-PCR protocol - and completely eliminates the need for DNase I treatment. In so doing, it increases the accuracy and reproducibility of QRT-PCR reactions, especially when using crudely purified samples. P024 Gerwyn Jones, Srujana Kapavarapu, Saima Nayab and Ian Kavanagh Thermo Fisher Scientific, ABgene House, Blenheim Road, Epsom KT19 9AP United Kingdom; Adapting QPCR experiments to run using fast cycling conditions is a simple method of increasing experimental throughput by reducing run duration times by up to 50%.

QPCR results and the quality thereof can be greatly affected by the characteristics of the product being amplified. This is especially true when employing fast thermal cycling protocols, which have been shown to reduce the sensitivity and increase variability of some assays. Speeding up thermal cycling protocols can lead to reaction failure if attempting to amplify a non-optimal assay or difficult target.

Therefore we have investigated the effects of amplicon length, GC content and secondary structure, on the performance of fast QPCR experiments compared to when a standard thermal cycling protocol is used. A panel of human specific assays were employed in QPCR experiments using fast and standard cycling protocols.

The assays were designed so that the resulting amplicons had a broad range of length, GC content and secondary structure (minimum ΔG values calculated at 60C). Experiments were carried out on the Roche LightCycler 480 and assay sensitivity was assessed by comparing delta Cp values, standard deviations and differences in QPCR efficiency (calculated by the standard curve method). The results demonstrate that amplicons that are of excessive length, high in secondary structure or high in GC content can all be causes for poor fast QPCR results. Therefore, we recommend that assays be designed with these amplicon characteristics taken into account, in order to ensure successful fast QPCR experiments. P025 Simon Baker 2, Phillip Harries 2 and Ian Kavanagh 1 1 Thermo Fisher Scientific, ABgene House, Blenheim Road, Epsom KT19 9AP. United Kingdom; 2School of Life Sciences, Oxford Brookes University, Gypsy Lane, Oxford OX3 0BP.

United Kingdom; QPCR instruments are able to monitor amplicon quantity in real-time during PCR reactions by detecting fluorescence signals and recording fluorescence data. The majority of fluorescence is reflected out of the PCR tube either by the polypropylene itself or by the walls of the thermal cycler block, when clear polypropylene is used. Aims: To investigate if white 96-well plates allow better fluorescence detection of PCR products during QPCR assays than clear 96-well plates. Methods: QPCR experiments were performed by amplifying target segments of the genes sigB, dnaK, srfAA, and argB from the genome of Bacillus subtilis BBK006.

SYBR Green I was used to monitor product accumulation via fluorescence. To examine the effect of pigmentation on CT, 96-well plates were custom-manufactured with elevated pigment, reduced pigment or differently-compounded white polypropylene. Results: In general QPCR reactions on white plates have lower CT values compared to QPCR reactions on clear plates.

Statistical analysis revealed there was no significant difference between CT values of sets dnaK and argB when using clear and white plates. However, analysis of melt curves suggested this was due to poor primer hybridisation. Further analysis revealed that CT values of sets srfAA and argB were significantly lower when using white plates compared to clear plates. There was also a significant difference between melting curves of low template DNA reactions when using clear and white plates. Conclusions: It was observed that signal noise was more prevalent in the melting curves of reactions using clear plates than white plates. There were no significant differences between CT values and amplification plots of low template DNA reactions when using the standard white plate compared to plates with altered pigmentation. This, coupled to examination of the plates by scanning fluorimetry, suggests that the effect of the pigment is complex and not necessarily linked to reflection of signal alone.

P026 Alignment of the heat shock protein gene (hsp) sequences and development of multiplex PCR method for the simultaneous detection of bovine mastitis pathogens including Staphylococcus aureus, and Streptococcus spp. Hau-Yang Tsen, Yu-Hsin Chang, Wan-Yu Pai and Yu-Cheng Chiang Hung Kuang University, Taiwan; Bovine mastitis is a multifactorial disease caused by many different bacteria species. Solidworks Software Free Download For Windows 7 64 Bit With Crack.

Of these bacteria species, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, and Streptococcus bovis are the major pathogens. Since the heat shock protein (hsp) genes, especially hsp60, has been shown to have more discriminatory power than 16S rRNA gene and inter-transcriptional spacer (ITS) region, in this study, we tried to develop a multiplex PCR method based on the hsp (heat shock protein) genes for the specific detection of S. Agalactiae, S. Uberis, and S. Molecular weight of the PCR products amplified were 406 bp, 350bp, 119bp, and 247bp, respectively, for S. Agalactiae, S.

Uberis, and S. Using this multiplex PCR method, all the selected target strains could be specifically detected in food samples. As the multiplex PCR method was used for direct detection of mastitis pathogens in milk samples, the detection limit was N (N= 1–9) ×103 CFU/ml of milk samples. If a 10 h pre-enrichment step was performed, the detection limit was N×100 CFU/ml. Thus, the multiplex PCR method could be used for the specific and sensitive detection of these pathogenic bacteria in food and milk samples.

P027 qPCR as a method to estimate synchronization of immortal Hepa 1-6 cells – problems with inhibitors and low abundant genes Ursula Prosenc, Klementina Fon Tacer and Damjana Rozman University of Ljubljana, Medical faculty, Center for functional genomics and bio-chips, Ljubljana, Slovenija; The aim of our experiment was to synchronize the mouse immortal hepatoma cells Hepa 1-6 and to prove the success of synchronization by measuring the expression of representative genes from cholesterol synthesis and circadian regulation. These genes are known to be expressed in a circadian manner in the mouse liver. However, many of them are expressed at low levels in immortal cell lines which represents a challenge for their quantification. Hepa 1-6 cells were grown in 12-well plates until confluency. Cells were treated with 10 uM forskolin, washed twice with ice cold PBS, lysed with TRI reagent (Invitogen) and stored at -80ºC until RNA isolation was performed according to TRI reagent protocol.

Pelet was washed once with 75% etanol and resuspended in RNAse free water. Because of low RNA yield cDNA was synthesized starting from 200ng of total RNA which is a 5 times lower concentration as usual. QPCR reaction efficiency was evaluated with serial dilutions of cDNA with mCyp51 (a gene from cholesterol synthesis) and 18sRNA primers on Light Cycler 480 (Roche) using the cyber green approach. The results showed that several dilutions result in the same Cp value, while at some dilutions there was no amplification at all.

After testing different serial dilutions the expected results were obtained only if cDNA was diluted to a specific (narrow range) concentration. To increase RNA amount samples with the same treatments were pooled, dried and precipitated with sodium acetate.

The qPCR has been repeated with serial dilutions and the expected amplification pattern was observed (different Cp values according to dilutions). It was concluded that the original samples contained an unidentified inhibitor of either qPCR or cDNA synthesis that has been removed by drying RNAs followed by another precipitation. After solving this problem we measured the timely expression of Cyp51, Dbp and Bmal1 in a time-series experiment spanning 48 hours. In the first 8h -12h forskolin provokes an immediate early response of the 3 measured genes. According to the expression of Dbp that is abundant also in immortal cells, we conclude that synchronization of Hepa 1-6 cells line has been successful while the results for Cyp51 and Bmal1 are not conclusive. Additional optimization procedures are required to enable quantitative time-series measurements of low expressed genes in immortal cells.

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