Posts Tagged ‘droplet digital pcr’

Bio-Rad’s New ddPCR Library Quantification Kit Optimizes Performance of Ion Torrent NGS Systems

 :: Posted by American Biotechnologist on 09-24-2013

Bio-Rad Laboratories, Inc., today announced the availability of its new ddPCR™ library quantification kit for Ion Torrent library preparation. Used with Bio-Rad’s QX200™ Droplet Digital™ PCR system, the new kit provides researchers with the ability to precisely and directly measure amplifiable library concentrations.

The Ion AmpliSeq library kit is used to prepare libraries for Ion Torrent next-generation sequencing (NGS) systems. Using the ddPCR library quantification kit to quantify Ion AmpliSeq gDNA and RNA libraries maximizes the number of useable reads, enables consistent loading, and optimizes the utilization of every sequencing run. The resulting data provide additional measures of library quality not provided by other methods, including the percentage of nonamplifiable species such as adapter dimers and the size range of library inserts.

Additional key benefits of the ddPCR library quantification kit for Ion Torrent systems include:

  • Superior performance — produces highly precise measurements of amplifiable library concentrations without the use of standards
  • Visualization of library quality — ddPCR fluorescence amplitude plots highlight well formed and poorly formed libraries
  • Efficient utilization of sequencing runs — enables consistent loading and maximum efficiency of the Ion Torrent sequencing platforms

Kits for other NGS platforms are also in development. For more information on the ddPCR library quantification kit, please visit: www.bio-rad.com/ion-torrent.

Ion Torrent and Ion AmpliSeq are trademarks of Life Technologies Corporation.

Breaking Leukemia’s Limits of Detection with Droplet Digital™ PCR

 :: Posted by American Biotechnologist on 09-17-2013

Extremely Rare Mitochondrial DNA Deletions Associated with Aging Can Be Accurately Detected with Droplet Digital™ PCR

 :: Posted by American Biotechnologist on 09-12-2013

jason bielasA study published recently in Aging Cell identifies a new tool to accurately analyze extremely rare mitochondrial DNA (mtDNA) deletions associated with a range of diseases and disorders as well as aging. This approach, which relies on Droplet Digital PCR (ddPCR™) technology, will help researchers explore mtDNA deletions as potential disease biomarkers.

The accumulation of mtDNA mutations is associated with aging, neuromuscular disorders, and cancer. However, methods to probe the underlying mechanisms behind this mutagenesis have been limited by their inability to accurately quantify and characterize new deletion events, which may occur at a frequency as low as one deletion event per 100 million mitochondrial genomes in normal tissue. To address these limitations, researchers at the Seattle, Washington–based Fred Hutchinson Cancer Research Center developed a ddPCR-based assay known as “Digital Deletion Detection” (3D) that allows for the high-resolution analysis of these rare deletions.

“It is incredibly difficult to study mtDNA mutations, let alone deletions, within the genome,” said Dr. Jason Bielas, assistant member of the Public Health Sciences Division at Fred Hutchinson Cancer Research Center and lead author of the study. “Our 3D assay shows significant improvement in specificity, sensitivity, and accuracy over conventional methods such as those that rely on real-time PCR.”

Bielas added, “The increase in throughput afforded by Droplet Digital PCR shortened the analysis of deletion events to days compared to months using previous digital PCR methods. Without the technology, we could not have made this discovery.”

At the center of the study was Bio-Rad Laboratories’ QX100™ ddPCR system. Using the QX100 system, Bielas and his team analyzed eight billion human brain mtDNA genomes and identified more than 100,000 genomes with a deletion. They discovered that, contrary to popular belief, the majority of the increase in mtDNA deletions was not caused by new deletions but rather by the expansion of previous deletions. They hypothesized that the expansion of existing mutations should be considered the primary factor contributing to age-related accumulation of mtDNA deletions.

How the 3D Assay Works
3D is a novel three-step process that includes enrichment for deletion-bearing molecules, single-molecule partitioning of genomes into droplets for direct quantification via ddPCR, and breakpoint characterization using next-generation sequencing.

Once the enrichment process is completed using methods previously developed by Bielas and colleagues, the concentration of molecules within the droplets is adjusted with the QX100 system so that the majority of droplets contain no mutant genomes, while a small fraction contain only one. This process allows each deletion to be amplified without bias and without introducing the artifacts that are common in qPCR.

Following amplification, deletions can be analyzed using ddPCR to determine the absolute concentration of mutated molecules. Using the relationship between droplet fluorescence and amplicon size, Bielas and his team were able to characterize the size and complexity (whether they were a result of a few clonal expansions or a large collection of random deletions) of rare mitochondrial deletions in human brain samples.

The 3D assay provides an important new tool that will allow researchers to better study the mechanisms of deletion formation and expansion, and their role in aging. Droplet Digital PCR’s high throughput and increased sensitivity will also allow Bielas’ lab to target other low-level disease-causing mtDNA deletions in skeletal muscle, brain tissue, and blood.

What is droplet digital PCR?

 :: Posted by American Biotechnologist on 09-04-2013

Yesterday we told you how scientists at the Fred Hutchinson Cancer Research Center were using droplet digital PCR to accurately quantify microRNA biomarkers in an accurate and reproducible fashion.

As a courtesy to those readers who may have missed it previously, we are reposting a video that was produced a couple of years ago with a comprehensive technical introduction to digital PCR. Please let us know if you have any questions regarding this video or droplet digital PCR in general.

Droplet Digital™ PCR Enables Reproducible Quantification of microRNA Biomarkers

 :: Posted by American Biotechnologist on 09-03-2013

Muneesh Tewari

A study published online in Nature Methods today 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 by 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, reverse transcription (RT), and the preparation of 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. The team found that ddPCR improved day-to-day reproducibility sevenfold 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,” Dr. Tewari said.