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Droplet Digital PCR Enables Reproducible Quantification of microRNA Biomarkers

A study published online in Nature Methods recently demonstrated that Droplet Digital PCR (ddPCR™) technology can be used to precisely and reproducibly quantify microRNA (miRNA) in plasma and serum across different days, paving the way for further development of miRNA and other nucleic acids as circulating biomarkers.

“In the field of circulating microRNA diagnostics, droplet digital PCR enables us to finally perform biomarker studies in which the measurements are directly comparable across days within a laboratory and even among different laboratories,” said Dr. Muneesh Tewari, Associate Member in the Human Biology Division at the Fred Hutchinson Cancer Research Center and lead author of the study.

Challenges in miRNA quantification

miRNAs are small regulatory RNA molecules with diverse cellular functions. The human genome may encode over 1,000 miRNAs, which could target about 60 percent of mammalian genes. Because they are abundant in many cell types, exist in highly stable extracellular forms, and may provide direct information about disease processes, they are being actively studied as blood-based biomarkers for cancer and other diseases.

Quantitative real-time PCR (qPCR) has been used for the analytical measurement of miRNAs in blood samples; however, researchers have found that qPCR measurements of miRNAs in serum or plasma display unacceptably high interday variability, undermining the use of miRNAs as reliable blood-based biomarkers. An approach that yields more dependable results has therefore been sought by researchers in this field.

Advantages of ddPCR for miRNA detection

Digital PCR has many advantages over qPCR including the ability to provide absolute quantification without a standard curve and robustness to variations in PCR efficiency across different samples and assays. These and other advantages are embodied in Bio-Rad Laboratories’ QX100™ Droplet Digital PCR (ddPCR™) system, which was introduced in 2011.

“We chose to use Bio-Rad’s QX100 Droplet Digital PCR system because it was the first system on the market that could make digital PCR practical from a cost and throughput standpoint for routine use in the lab,” said Dr. Tewari.

To assess the imprecision introduced by each workflow step — serial dilution preparation, reverse transcription (RT), and PCR technical replicates — Dr. Tewari and his team conducted nested analyses of ddPCR vs. qPCR on cDNA from a dilution series of six different synthetic miRNAs in both water and plasma on three separate days. In comparison to qPCR, the researchers found that ddPCR demonstrated greater precision (48–72% lower coefficients of variation) with respect to PCR-specific variation

Next, the team performed a side-by-side comparison of qPCR to ddPCR for detecting miRNAs in serum. They collected sera samples from 20 patients with advanced prostate cancer and 20 age-matched male controls and measured the abundance of miR-141, which has been shown to be a biomarker for advanced prostate cancer. Samples were analyzed by qPCR and ddPCR with individual dilution series replicates prepared on three different days. They found that ddPCR improved day-to-day reproducibility seven-fold relative to qPCR. It was also able to demonstrate differences between case vs. control specimens with much higher confidence than qPCR (p=0.0036 vs. p=0.1199).

“Droplet digital PCR will allow us to accurately follow serum microRNA biomarker concentrations over time during a patient’s treatment course, something that has been nearly impossible to achieve with real-time PCR,” he said.

Study: Absolute quantification by droplet digital PCR versus analog real-time PCR [Nature Methods]

Source: EurekAlert! 

Fluidigm and Olink Bioscience Bring a New Level of Protein Biomarker Discovery to the Life Science Research Market

Fluidigm and Olink Bioscience have teamed up to enable interrogation of 96 samples across 92 proteins in a single run from one microliter of sample in less than a day.

The two companies will co-market the combination of their respective products — Fluidigm’s BioMarkTM HD System and Olink Bioscience’s Proseek® Multiplex technology — bringing protein research to the Fluidigm platform and Olink Bioscience’s protein detection assays to the high-throughput, high reproducibility and unparalleled sensitivity realm of Fluidigm’s real-time PCR system.

Fluidigm’s BioMark HD System and Olink Bioscience’s Proseek Multiplex technologies provide researchers with the highest throughput multiplexing solution for protein biomarker discovery in the market today. Typically, researchers are limited to working with a few tens-of-protein biomarkers at a time. Using the BioMark HD System with the Proseek Multiplex Oncology I 96×96 Kit, a researcher can simultaneously analyze 96 human samples against a panel of 92 analytes, such as growth factors, inflammatory markers, soluble receptors, or cancer antigens. With the addition of four control analytes (two incubation controls, and extension and detection controls), researchers can now obtain results for up to 9,216 reactions in just a few hours.

The first 92-plex Olink panel, available now, is focused on biomarker discovery for cancer. Panels addressing cardiovascular disease and inflammation are expected to be offered later this year.

“Protein research is so important because these biomarkers are used to monitor health states and predict treatment outcomes in patients. One of the biggest trends in the life sciences industry today is research to uncover biomarkers that can lead to companion diagnostics,” said Simon Fredriksson, Olink Bioscience president and chief executive officer. “Conventional immunoassays have been unable to scale due to increasing antibody cross-reactivity when more and more assays are run simultaneously. Olink’s Proseek Multiplex generates high quality data even in highly multiplexed formats, and using these assays in conjunction with Fluidigm’s BioMark HD System gives protein researchers easy access to unprecedented volume and quality of data.”

The Olink Bioscience assay provides a signal when pairs of antibodies equipped with DNA reporter molecules bind to their targets to create new DNA amplicons. The amplicons are subsequently quantified by high throughput real-time PCR. With PCR’s large dynamic range and superb sensitivity, in combination with Olink Bioscience’s unique protein detection assays, the opportunities are enormous for powerful analysis of multivariate biomarker patterns.

“Analyzing 92 proteins from one microliter of sample enables new biomarker discovery and validation,” said Gajus Worthington, Fluidigm president and chief executive officer. “Many sample sources, including those from biorepositories or model organisms, are limited, and researchers can simply run out before they are able to find useful biomarker panels. The combination of Fluidigm’s BioMark HD System and Olink’s Proseek Multiplex assay represents a robust new tool for the protein research community.”

Fluidigm’s BioMark HD System is a multi-application genomics and proteomics platform that provides results equivalent to the gold standards for every respective experimental approach. The system produces high-quality data from RNA, miRNA and DNA from sample sizes down to the single cell level — and now extends to proteins. The BioMark HD System performs analysis of protein expression, gene expression, copy number variation, SNP genotyping, and digital PCR.

Olink Bioscience’s Proseek Multiplex is a multivariate protein biomarker detection kit based on Olink’s proprietary Proximity Extension Assay (PEA). It uniquely allows multiplexing of immunoassays without compromising assay performance. PEA uses pairs of oligonucleotide-labeled antibodies equipped with DNA reporter molecules to bind to proteins of interest in a highly specific manner, solving the antibody cross-reactivity dilemma that plagues and limits conventional protein assays.

Source: Business Wire

Life Technologies Signs Five-Year Agreement with the FDA

Life Technologies Corporation (NASDAQ: LIFE) recently announced that it has signed a five-year agreement with the Food and Drug Administration (FDA) to accelerate and advance food safety testing of E. coli and Salmonella, two foodborne contaminants commonly associated with outbreaks and/or recalls.

The collaboration consists of three distinctive projects:

  • Life Technologies will design and validate new food safety tests for the detection and identification of foodborne pathogens, with pathogen strains provided by the FDA.
  • Life Technologies will design and validate a complete workflow for food pathogen detection on the Ion PGM™ platform, while optimizing sample preparation methods.
  • The FDA will have the opportunity to evaluate new disruptive technology platforms by Life Technologies for the use in pathogen detection.

Life Technologies will use its bioinformatics resources to independently develop real-time PCR (polymerase chain reaction) assays against unique E. coli and Salmonella targets in collaboration with the FDA. It will then provide the test results for these targets to the FDA for further validation.

The FDA will use Life Technologies’ Ion PGM™ Sequencer to generate whole genome sequence information from defined bacteria, and for strains that will be excluded from detection. Life Technologies’ bioinformatics team will then analyze the genomic information and provide assays or tests to the FDA for further validation and analysis. Whole genome sequences generated under the collaboration will be added to the National Institutes of Health Genbank so they can be accessed by the food safety research community.

“We are excited to be entering this cooperative research and development agreement with the FDA as we have been working alongside them in one capacity or the other for over 10 years,” said Nir Nimrodi, Vice President and General Manager for Food Safety and Animal Health at Life Technologies. “The FDA will call on us, particularly when it comes to developing rapid detection kits. This agreement allows them to have new rapid track and trace products for rapid identification of foodborne contaminants.”

Lastly, the FDA will validate and test Life Technologies’ next-generation sequencing technologies for Salmonella investigations as part of its effort to develop new rapid detection tools that can improve the public health response to future outbreaks.

Life Technologies has a strong history of combating pathogen outbreaks. In 2011, Life Technologies developed a custom assay to accurately detect the highly pathogenic E. coli 0104:H4 bacterium that spread through Europe, killing hundreds of people.

“The assay was developed using samples supplied by scientists at University Hospital Munster, who completed the DNA sequencing and analysis work on the deadly E. coli strain using the Ion PGM™ Sequencer,” said Maneesh Jain, Ion Torrent Vice President of Marketing for Life Technologies. “Now the FDA will validate and test this same technology to understand the molecular relationship within the Salmonella pathogen in the hopes to prevent future outbreaks from occurring.”

The PGM™ offers scientists the simplest and fastest workflow, lowest cost-per-sample, and highest sensitivity for multiple basic and clinical research applications.

Source: Life Technologies

Exosome Diagnostics Presents Data Demonstrating Utility of Urine Exosome Technology to Predict Prostate Biopsy Outcome at American Urological Association Annual Meeting

Exosome Diagnostics, a leading developer of biofluid-based molecular diagnostic products for use in personalized medicine research and clinical diagnostics, recently presented data at the American Urological Association Annual Meeting in San Diego demonstrating the performance of urine exosome technology in accurately predicting the outcome of a prostate biopsy.

In the study, a urine sample was collected from patients scheduled to undergo prostate biopsy or radical prostatectomy. Exosomes containing ribonucleic acids (RNA) shed into the urine from the prostate were analyzed using Exosome Diagnostics’s proprietary EXO70 Urine RNA Isolation Kit in conjunction with real-time PCR. Notably, patients did not undergo a digital rectal exam or a prostate massage prior to the urine collection.

“There is clearly an unmet need in the field of prostate cancer detection and prognosis today,” said James McKiernan, M.D., professor of urology and the director of urologic oncology at NewYork-Presbyterian Hospital/Columbia University Medical Center in New York City. “A noninvasive method to improve upon PSA and its potential to predict who does or does not have cancer would immediately benefit patients and help address the controversy surrounding prostate cancer detection. A potential future application—the ability to use exosome technology to determine who has significant prostate cancer prior to performing a biopsy—would be revolutionary. In these preliminary studies exosome technology has shown promise to deliver on both of these challenges.”

James McCullough, chief executive officer of Exosome added, “These results are part of a multi-center clinical study program for EXO106, the first in vitro diagnostic in our exosome genitourinary (GU) oncology program. EXO106 is being developed to provide patients and clinicians with a non-invasive diagnostic that can deliver real-time information following the finding of an elevated PSA. We have now completed testing on over 1,000 urine samples from patient to characterize diagnostic performance.”

In the study, biopsy patients were divided into two cohorts. The first cohort was tested using a known prostate cancer biomarker, while a novel four-gene prostate cancer signature was tested in the second cohort. Both groups were stratified based on whether their biopsies were positive or negative. Besides predicting positive biopsy outcome, exosome testing was able to distinguish histologically less aggressive, lower Gleason scores (≤7) cancers from those with higher, more aggressive Gleason scores (≥8). The results demonstrate that urinary exosome-derived RNA can be used to non-invasively evaluate gene expression in the prostate and accurately predict the likelihood of a positive or negative needle biopsy in addition to distinguishing more aggressive cancers.

The study was funded by the Prostate Cancer Foundation.

Source: PR Newswire

QIAGEN Acquires Ingenuity Systems, Adding Leading Solution for Analysis and Interpretation of Complex Biological Data

QIAGEN N.V. (NASDAQ: QGEN; Frankfurt Prime Standard: QIA) today announced the acquisition of Ingenuity Systems, Inc., the leading provider of software solutions to efficiently and accurately analyze and interpret the biological meaning of genomic data.

Ingenuity, a privately-held U.S. company based in California’s Silicon Valley, has created the market leading expertly curated knowledge system of biomedical information and analysis solutions for the exploration, interpretation and analysis of complex biological systems.

New technologies such as next-generation sequencing (NGS) are now generating more data in a single year than was created in all prior history, making the analysis and interpretation of this extensive and very complex biological data a critical success factor.

The foundation of Ingenuity’s product portfolio is the Ingenuity Knowledge Base, a 14-year effort to accurately, manually curate, model and computationally structure the vast amount of biomedical literature, including genomic variations implicated in human disease and thousands of disease models. The Ingenuity Knowledge Base and software applications enable users to accurately interpret the meaning of increasingly large amounts of biological data to better guide scientific experiments and medical treatment decisions.

“The interpretation of biological information is becoming a cornerstone of QIAGEN’s ecosystem of Sample & Assay Technologies for molecular testing – both in life sciences research and in diagnostics. We are establishing a leading role in this field and intend to further expand the value proposition and scope of our offering,” said Peer M. Schatz , Chief Executive Officer of QIAGEN N.V. “Ingenuity has created unparalleled leadership with its Knowledge Base and interpretation solutions to unlock the value of complex genomic and other biological information. Combining the highest-quality knowledge content with powerful search capabilities and easy-to-use interfaces, the Ingenuity suite provides customers with scientifically and clinically relevant insights into diseases. We are looking forward to expanding the seamless integration of leading biomedical information solutions into our full range of molecular testing solutions, thereby providing our customers a unique experience from sample to interpreted result and recommendations for next steps. Integration of Ingenuity’s solutions into a powerful, full-range ecosystem of QIAGEN’s molecular testing solutions, such as PCR and next-generation sequencing, promises to offer significant value to QIAGEN customers and shareholders.”

“Today we can sequence an entire human genome in just a couple of days for less than a few thousand dollars, but the data analysis can take from months to years. The Ingenuity suite of products enables this rapid and accurate interpretation in a matter of minutes, and this is fundamentally impacting scientific research and the ability to diagnose and manage patient care,” saidJake Leschly , President and CEO of Ingenuity Systems. “The Ingenuity mission from day one has been to accelerate scientific discovery and enhance clinical decision-making through a rich understanding of biological systems. As part of QIAGEN, we will offer our biological knowledge content and software in the context of a best-in-class ecosystem of molecular testing solutions and to a greatly expanded range of customers around the world.”

Ingenuity’s Knowledge Base is built on algorithms that pair all human gene variants with biological interpretations based on known outcomes and findings. These pairings are collected and reviewed by experts (curated) in a standardized way from a wide range of sources including clinical and scientific publications with the support of advanced technology and refined processes. This process and the associated learning algorithms translate sourced data into interoperable and linkable collections of relevant information. The leadership of Ingenuity’s products grows out of a long-standing commitment to ensure the highest standards, particularly in the use of expert teams, supported by the most advanced technology.

Ingenuity product offerings include:

  • Ingenuity® Pathways Analysis™(IPA) is the industry gold standard web-based software application that enables researchers to model, analyze and understand the complex biological systems at the core of life sciences research. IPA is deployed in most major pharmaceutical companies and hundreds of biotechnology companies and academic institutions globally. It supports data analysis and interpretation from all high-throughput analysis platforms and is used in virtually all areas of drug discovery and development from target identification and validation to biomarkers, predictive toxicology and pharmacogenomics. IPA has been cited in thousands of peer-reviewed journal articles.
  • Ingenuity®Variant Analysis™ is a web-based NGS data analysis application that answers a critical need for researchers trying to rapidly identify relevant causal variants in human diseases in a matter of hours. It enables customers to rapidly identify and prioritize variants by drilling down to a small, targeted subset of compelling variants based upon published biological evidence and their own knowledge of disease biology. Customers can also interrogate variants from multiple biological perspectives, explore different biological hypotheses and identify the most promising variants for follow-up analysis. Thousands of users from leading institutions around the world have already uploaded genomic data using Ingenuity Variant Analysis, which has been used to analyze more than 45,000 biological samples so far. Ingenuity is fully committed to meeting the highest standards for its Data Center operations and Ingenuity Variant Analysis, and announced in February 2013 that it had passed an audit under the U.S. Health Insurance Portability and Accountability Act (HIPAA) and is compliant with the U.S.-EU Safe Harbor framework.
  • Ingenuity®iReport™ is an interactive web-based report optimized for gene expression experiments from RNA-Seq, microarray and real-time PCR platforms. It provides fast and accurate biological and statistical analysis of a customer’s biological data.

Ingenuity is also developing a new product to enable broader adoption of NGS in molecular diagnostics by offering an optimized and scalable solution for interpreting and scoring clinical variants identified by sequencing-based molecular diagnostic tests. The company has been actively forming relationships with reference laboratories to bring the power of this technology to these customers, including through cloud-based applications.

Ingenuity will significantly expand and strengthen QIAGEN’s own curated database, which is commercialized embedded in wet lab assays sold through QIAGEN’s highly successful GeneGlobe content portal. GeneGlobe offers hundreds of assay panels for PCR and NGS as well as more than 60,000 fully annotated molecular assays for the most sought-after diseases and pathways, curated from the biomedical literature.

Ingenuity’s solutions are used by tens of thousands of researchers and clinicians at hundreds of leading pharmaceutical, biotechnology, academic, diagnostic and clinical institutions worldwide. Many members of Ingenuity’s senior management team, including Mr. Leschly, will continue with QIAGEN, and the offices will remain in Redwood City, California. Ingenuity Systems was selected as a 2013 Technology Pioneer by the World Economic Forum For more information on Ingenuity’s technology platform and products, go to http://www.ingenuity.com/.

Source: Ingenuity Systems