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Personalized Medicine Roadmap: Definiens Releases Five Steps to Utilize Data Mining with Image Analysis

Definiens, a healthcare company that advances personalized medicine through image analysis and digital pathology solutions, recently released tips for integrating data mining with image analysis. As pathologists, researchers and clinicians seek to advance personalized medicine through the development and prescription of targeted therapies, data generated through image analysis of digitized tissue sections is becoming essential to stratifying patients and providing personalized care.

Definiens and Advanced Cell Diagnostics Launch Software for Quantitative RNA In Situ Hybridization

Definiens AG, a healthcare company that advances personalized medicine through image analysis and digital pathology solutions, and Advanced Cell Diagnostics (ACD) of Hayward California, a leader in molecular pathology, announced recently the commercial launch of RNAscope® SpotStudio™, a custom-designed image analysis software application for ACD’s RNAscope®Assays to detect and quantify RNA biomarkers. By combining state-of-the-art image analysis and advanced in situ hybridization technologies, gene expression can be measured quantitatively at single cell resolution and interpreted by pathologists within context.

AB SCIEX Proteomics Scientist Wins HUPO 2013 Science and Technology Award

The Human Proteome Organization (HUPO) recently awarded Christie Hunter, Ph.D, director of proteomics applications at AB SCIEX, its 2013 Science and Technology Award at an award ceremony during last week’s HUPO 2013 conference in Japan. Dr. Hunter was recognized for her contributions to the development and commercialization of a breakthrough approach for targeted proteomics. The analytical strategy of targeted proteomics was recently named “Method of the Year” by Nature Methods.

Targeted proteomics is a standardized, biological research workflow that focuses on reproducibly quantifying a specific subset of proteins within a sample. It generates data that is vital for biologists to answer hypothesis-driven, biological questions.

A decade ago, proteomics research was dominated by discovery workflows, which provided valuable information on a single sample but lacked the reproducibility to generate robust quantitation across a larger sample set. New innovation was needed at the time to move the field beyond simply producing large lists of identified proteins and toward providing highly quantitative answers.

This led to the development of a multiple reaction monitoring (MRM)-triggered, tandem mass spectrometry (MS/MS) workflow at AB SCIEX to rapidly create high sensitivity MRM assays to target peptides that are unique to their associated proteins. This workflow was made possible by the combination of triple quadrupole and linear ion trap functionality in a single system called the AB SCIEX QTRAP® System.

Dr. Hunter ‒ in collaboration with researcher Dr. Leigh Anderson, the founder of the Plasma Proteome Institute and head of SISCAPA Assay Technologies ‒ pioneered a workflow that applied MRM to the targeted quantification of proteins and peptides in plasma by mass spectrometry. In their initial publication[1], Dr. Hunter and Dr. Anderson demonstrated that a targeted workflow could be applied to multiplexed quantitation of proteins in human plasma with high reproducibility and high confidence in the results.

The impact of the paper resulted in broad adoption of the MRM technique around the world to accelerate the verification and validation of putative protein biomarkers, generating more than 800 citations, according to Google Scholar. Less than a decade after this important work, most proteomics laboratories today use a triple quadrupole-based mass spectrometer to perform MRM analysis.

“We congratulate Dr. Christie Hunter on receiving such a prestigious award from HUPO in recognition of her significant contributions to the rise of targeted proteomics as a viable technique to advance biomarker research,” said Dave Hicks, Vice President and General Manager of the Pharmaceutical and Academic Business at AB SCIEX.

“Dr. Hunter and her AB SCIEX colleagues continue to participate in exciting collaborations with leading proteomics researchers around the world to drive new innovations in software, chemistries and instrumentation that further expand quantitative proteomics workflows for the growing community of mass spectrometry users at large,” added Hicks.

Currently, Dr. Hunter is playing a pivotal role in the development of higher specificity workflows for targeted protein quantitation to overcome situations where sensitivity is limited by interferences or background. She is involved in the investigation of the utility of differential mobility separations for added selectivity of quantitation of peptides in complex mixtures. She is also working to enhance data-independent acquisition strategies, such as SWATHTM Acquisition, for quantitative proteomics to increase the multiplexing and reproducibility that can be achieved in a single experiment.

Source: AB SCIEX

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! 

Agilent Technologies Launches New AssayMAP Peptide Sample-Prep Solution

Agilent Technologies Inc. (NYSE: A) recently announced the launch of its new AssayMAP peptide sample-prep solution, which offers automated protein digestion, peptide cleanup and fractionation in preparation for mass-spectrometry analysis.

As more scientists adopt LC/MS analysis for biomarker discovery, the bottleneck shifts to sample preparation. As these types of studies involve much larger numbers of samples, traditional manual methods are proving untenable in terms of hands-on time and unreliable in terms of reproducibility.

“Using the combination of extremely consistent, parallelized digestion with automated reverse-phase cleanup via AssayMAP, at a scale appropriate for ultrasensitive proteomics applications, has enabled us to contemplate collaborative studies of previously unheard-of scales and throughput,” said Jacob Jaffe, Ph.D., assistant director of proteomics platforms at the Broad Institute in Cambridge, Mass.

“The AssayMAP peptide sample-preparation solution is one of the ways Agilent is addressing the need for high-throughput, reproducible sample-preparation methods in front of precision analytical instruments,” said Kathleen Shelton, senior director of automation marketing at Agilent. “Agilent’s unique ability to deliver high-quality solutions for automating sample preparation through analysis enables scientists to maximize workflow productivity and be more successful.”

AssayMAP helps address the bottleneck created with traditional sample preparation for mass-spec analysis and enables unprecedented precision and throughput. This easy-to-use solution includes validated automation protocols driven through intuitive software, the AssayMAP Bravo liquid-handling instrument, and a suite of AssayMAP cartridges optimized for peptide cleanup and fractionation. The AssayMAP peptide sample-prep solution makes large campaigns possible for mass-spec analysis, leveraging the strength of this analytical method to unambiguously identify peptides.

Source: Agilent Technologies